nitrophenols and 4-hydroxy-5-nitrophenyl-acetic-acid

B lymphocytes and their differentiated daughter cells are charged with responding to invading pathogens and producing protective antibodies against these pathogens. The physiology of B cells is intimately connected with the function of the B cell antigen receptor (BCR). Upon activation of BCR, transmembrane signals are generated, and several downstream pathways are activated, which provide a primary directive for the cell's subsequent response. mTOR is a serine/threonine kinase that controls cell proliferation and metabolism in response to a diverse range of extracellular stimuli. The activation of mTOR signaling downstream of PI3K/Akt activity by B cell receptor (BCR) engagement has been generally assumed to be essential for B cell responses. This chapter seeks to present two protocols to evaluate mTOR activity in B cells bearing BCR specific to 4-hydroxy-3-nitrophenylacetyl (NP)-hapten.

Topics: Animals; B-Lymphocytes; Flow Cytometry; Haptens; Mice; Nitrophenols; Phenylacetates; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Antigen, B-Cell; Signal Transduction; TOR Serine-Threonine Kinases

Topics: Adoptive Transfer; Animals; Antibody Formation; B-Lymphocyte Subsets; beta-Galactosidase; Cell Lineage; Chickens; gamma-Globulins; Gene Rearrangement, B-Lymphocyte; Genes, Reporter; Germinal Center; Haptens; Immunization; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Immunologic Memory; Integrases; Lac Operon; Mice; Mice, Congenic; Mice, Inbred C57BL; Mice, Transgenic; Nitrophenols; Phenylacetates; Somatic Hypermutation, Immunoglobulin; Spleen

Antibodies can have a medicinal role because of their high specificity in molecular recognition. Although monoclonal antibodies specific for an antigen can be obtained relatively easily, it is difficult to acquire ones with high affinity. Recently, antibody engineering using an in vitro evolutionary process has been put to this task. However, little progress seems to have been made. Our previous study revealed that antibodies have low or high evolvability. An antibody with a poor ability to evolve would also not attain high affinity in in vitro maturation. In this review, we provide information for the improved design of the antigen-combining site and that this will contribute to the engineering of antibodies using an in vitro evolutionary process.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Complementarity Determining Regions; Drug Design; Evolution, Molecular; Humans; Hybridomas; Mice; Nitrophenols; Phenylacetates; Protein Engineering

Topics: Animals; Chromatography, High Pressure Liquid; Humans; Nitrophenols; Phenylacetates; Protein Binding; Proteins; Tyrosine

Topics: Animals; Antibody Formation; B-Lymphocytes; Cell Separation; Haptens; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes

Topics: Animals; Antigens; B-Lymphocytes; Gene Rearrangement; Genes, Immunoglobulin; Haptens; Hybrid Cells; Mice; Mice, Inbred C57BL; Mutation; Nitrophenols; Phenylacetates

Topics: Animals; Antibody Diversity; Antibody Formation; B-Lymphocytes; Immunologic Memory; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Stem Cells

We have described a model system of immunoregulation in which gene products associated with both the H-2 and Igh complexes guide a series of cellular interactions. The Igh restriction represents the requirement for T cell recognition of Igh-linked NPb-related idiotypic determinants. The data provide additional evidence that the T and B cell Igh products are distinct. Immunoglobulin products are involved in the selection of T cell receptors; Igh genes are not expressed in T suppressor cell. The H-2 restrictions generally involve the I-J subregion. These restrictions are imposed by the presentation of antigen or suppressor factor on a specialized population of I-J bearing, antigen-presenting cells, thereby functioning in a manner analogous to that proposed for the induction of helper T cells. The NP suppressor cell pathway consists of multiple cellular elements, including at least 3 distinct T cell populations and 2 or more distinct antigen-presenting populations. Generally, specific soluble suppressor factors produced by each Ts cell subset are involved in the cellular communication process. The terminal phases of the suppressor cell cascade involve an antigen dependent non-specific bystander mechanism. Tables IX and X summarize our present view of the NP suppressor cell cascade. It is still not possible to include all the other experimental models involving suppressor cell interactions into the above scheme. However, the disparities between the various systems have in some cases narrowed and in other instances suggested that multiple mechanisms of immune regulation may occur concurrently.

Topics: Animals; B-Lymphocytes; H-2 Antigens; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Idiotypes; Mice; Nitrophenols; Phenylacetates; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

Topics: Animals; Cell Fusion; Epitopes; Histocompatibility Antigens; Hybridomas; Hypersensitivity, Delayed; Immune Tolerance; Killer Cells, Natural; Mice; Nitrophenols; Phenylacetates; Rats; T-Lymphocytes; T-Lymphocytes, Regulatory

Topics: Animals; Epitopes; Genes, MHC Class II; Guinea Pigs; Haptens; Histocompatibility Antigens; HLA Antigens; Humans; Hypersensitivity, Delayed; Lymphocyte Culture Test, Mixed; Lymphokines; Mice; Nitrophenols; Phenylacetates; Rats; Receptors, Antigen, T-Cell; Suppressor Factors, Immunologic; T-Lymphocytes

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody-Producing Cells; B-Lymphocytes; Binding Sites, Antibody; Binding, Competitive; Cell Differentiation; Cross Reactions; Immunoglobulin Idiotypes; Lymphocyte Activation; Mice; Nitrophenols; p-Azobenzenearsonate; Phenylacetates; Phosphorylcholine; Polysaccharides, Bacterial; T-Lymphocytes

Topics: Animals; Antibodies, Anti-Idiotypic; Female; Haptens; Immunoglobulin Idiotypes; Immunoglobulin lambda-Chains; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

Ideally, a vaccine should provide life-long protection following a single administered dose. In our previous study, the immunopotentiator CVC1302, which contains pattern- recognition receptor (PRR) agonists, was demonstrated to prolong the lifetime of the humoral immune response induced by killed foot-and-mouth disease virus (FMDV) vaccine. To elucidate the mechanism by which CVC1302 induces long-term humoral immunity, we used 4-hydroxy-3-nitrophenylacetyl (NP)-OVA as a pattern antigen and administered it to mice along with CVC1302, emulsified together with Marcol 52 mineral oil (NP-CVC1302). From the results of NP-specific antibody levels, we found that CVC1302 could induce not only higher levels of NP-specific antibodies but also high-affinity NP-specific antibody levels. To detect the resulting NP-specific immune cells, samples were taken from the injection sites, draining lymph nodes (LNs), and bone marrow of mice injected with NP-CVC1302. The results of these experiments show that, compared with mice injected with NP alone, those injected with NP-CVC1302 had higher percentages of NP+ antigen-presenting cells (APCs) at the injection sites and draining LNs, higher percentages of follicular helper T cells (TFH), germinal center (GC) B cells, and NP+ plasma-blasts in the draining LNs, as well as higher percentages of NP+ long-lived plasma cells (LLPCs) in the bone marrow. Additionally, we observed that the inclusion of CVC1302 in the immunization prolonged the lifetime of LLPCs in the bone marrow by improving the transcription expression of anti-apoptotic transcription factors such as Mcl-1, Bcl-2, BAFF, BCMA, Bax, and IRF-4. This research provides a blueprint for designing new generations of immunopotentiators.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Female; Immunity, Humoral; Immunoglobulin G; Mice, Inbred BALB C; Nitrophenols; Ovalbumin; Phenylacetates; Receptors, Pattern Recognition; T-Lymphocytes

Ageing is a complex multifactorial process associated with a plethora of disorders, which contribute significantly to morbidity worldwide. One of the organs significantly affected by age is the gut. Age-dependent changes of the gut-associated microbiome have been linked to increased frailty and systemic inflammation. This change in microbial composition with age occurs in parallel with a decline in function of the gut immune system; however, it is not clear whether there is a causal link between the two. Here we report that the defective germinal centre reaction in Peyer's patches of aged mice can be rescued by faecal transfers from younger adults into aged mice and by immunisations with cholera toxin, without affecting germinal centre reactions in peripheral lymph nodes. This demonstrates that the poor germinal centre reaction in aged animals is not irreversible, and that it is possible to improve this response in older individuals by providing appropriate stimuli.

Topics: Aging; Animals; Cholera Toxin; Dysbiosis; Fecal Microbiota Transplantation; Female; Gastrointestinal Microbiome; Germinal Center; Immunization; Immunoglobulin A; Mice; Nitrophenols; Peyer's Patches; Phenylacetates

Topics: Antigens; Biosensing Techniques; Glucuronidase; Immunoassay; Immunoglobulin Variable Region; Models, Molecular; Nitrophenols; Phenylacetates; Protein Structure, Quaternary; Protein Structure, Tertiary

In this study, a novel noncompetitive homogeneous immunoassay for antigen detection was developed. We utilized β-glucuronidase (GUS), a homotetrameric enzyme, the assembly of all of whose subunits is necessary to attain its activity. By using a mutant GUS (GUSm), wherein the dimerization of dimers, which is a rate-limiting step, can be effectively inhibited by a set of interface mutations, we attempted to create a biosensor for detecting various molecules. Usually, the affinity between the two variable region domains (VH and VL) of an antibody, especially for a small molecule, is relatively low. However, in the presence of an antigen, the affinity increases so that they bind tighter to each other. A pair of fusion proteins, comprising the VH and VL regions of the antibody as the detector tethered to a GUSm subunit as the reporter, was constructed to detect antigen 4-hydroxy-3-nitrophenylacetyl (NP) and bone Gla protein (BGP) through GUS activity measurement. Colorimetric and fluorescence assays could detect NP, 5-iodo-NP, and BGP within 1 h without separation steps and with a higher signal/background ratio than conventional ELISA. The instantaneous response after simple mixing of the components makes this system convenient and high-throughput. The system could be effective for the analyses of various small molecules in environmental and clinical settings.

Topics: Antibodies; Antigens; Biosensing Techniques; Enzyme-Linked Immunosorbent Assay; Glucuronidase; Humans; Immunoassay; Nitrophenols; Osteocalcin; Phenylacetates

Specific IgG antibodies, passively administered together with erythrocytes, suppress antibody responses against the erythrocytes. Although used to prevent alloimmunization in Rhesus (Rh)D-negative women carrying RhD-positive fetuses, the mechanism behind is not understood. In mice, IgG suppresses efficiently in the absence of Fcγ-receptors and complement, suggesting an Fc-independent mechanism. In line with this, suppression is frequently restricted to the epitopes to which IgG binds. However, suppression of responses against epitopes not recognized by IgG has also been observed thus arguing against Fc-independence. Here, we explored the possibility that non-epitope specific suppression can be explained by steric hindrance when the suppressive IgG binds to an epitope present at high density. Mice were transfused with IgG anti-4-hydroxy-3-nitrophenylacetyl (NP) together with NP-conjugated sheep red blood cells (SRBC) with high, intermediate, or low NP-density. Antibody titers and the number of single antibody-forming cells were determined. As a rule, IgG suppressed NP- but not SRBC-specific responses (epitope specific suppression). However, there was one exception: suppression of both IgM anti-SRBC and IgM anti-NP responses occurred when high density SRBC-NP was administered (non-epitope specific suppression). These findings answer a longstanding question in antibody feedback regulation and are compatible with the hypothesis that epitope masking explains IgG-mediated immune suppression.

Topics: Animals; Epitopes; Erythrocytes; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin M; Immunosuppression Therapy; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, IgG; Sheep

Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response.

Topics: Animals; Antibody Formation; B-Lymphocytes; Biomarkers; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Rearrangement, B-Lymphocyte; Germinal Center; High-Throughput Nucleotide Sequencing; Immunization; Immunoglobulin Variable Region; Immunologic Memory; Immunophenotyping; Mice; Nitrophenols; Phenylacetates; Somatic Hypermutation, Immunoglobulin; T-Lymphocytes

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells,

Topics: Animals; Antibodies, Antinuclear; Autoimmunity; B-Lymphocytes; CD40 Ligand; Gene Expression Regulation; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Nitrophenols; Phenylacetates; Receptors, Fc; Signal Transduction; T-Lymphocytes

Rapidly evolving pathogens such as HIV or influenza can quickly mutate their antigenic profiles, reducing the efficacy of conventional vaccines. Despite this challenge, functionally required epitopes are highly conserved among heterologous viral strains and represent a key vulnerability that could be targeted during vaccine development. As the antigenicity of these conserved epitopes is frequently subdominant, there is a critical need for innovative vaccination strategies designed to target these neutralizing epitopes. Here, we immunized mice with antigens containing discrete immunodominant and subdominant moieties and show that treatment with soluble heterologous antigen bearing only the immunodominant epitope selectively suppresses these germinal center (GC) B cells. By exploiting this intrinsic tolerance mechanism, we promote the expansion of subdominant B cells in the GC and the subsequent long-lived components of the humoral response. We propose that this strategy may be applied to elicit preferential expansion of subdominant B cells that recognize weakly immunogenic epitopes on microbial pathogens.

Topics: Animals; Antibody Formation; B-Lymphocytes; Cell Count; Clone Cells; Germinal Center; Immunodominant Epitopes; Mice, Inbred C57BL; Nitrophenols; Ovalbumin; Phenylacetates; Plasma Cells; Solubility

Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains. We examined whether any biophysical properties of antigen-combing sites relate to this shift in B-cell clones by preparing single-chain Fv (scFv). Thermodynamic and kinetic parameters of the interaction of scFv with various haptens are in accordance with those of intact antibodies, indicating that scFvs are appropriate models for the study on structure and function of antibodies. Next, we measured thermal stability of scFvs using differential scanning calorimetry and found that the apparent melting temperature of free Tyr95-type was 64-66°C,while that of Gly95-type was 47-48°C, indicating that the latter was highly unstable. However, Gly95-type greatly gained thermal stability because of hapten binding. We discussed the relationship between thermal stability resulted by hapten binding and dynamism of antibody response during immunization.

Topics: Animals; Antibody Affinity; Binding Sites, Antibody; Calorimetry, Differential Scanning; Circular Dichroism; Glycine; Humans; Immunoglobulin Heavy Chains; Kinetics; Nitrophenols; Phenylacetates; Protein Stability; Receptors, Antigen, B-Cell; Single-Chain Antibodies; Surface Plasmon Resonance; Thermodynamics

Caspase-1 is required for nephritis and robust autoantibody development in the pristane model of murine lupus. The objective of this study was to evaluate the immune response and to study the splenic B and T cell populations in wild-type (WT) and caspase-1-/- mice following pristane injection in order to develop an understanding of why absence of caspase-1 is protective in pristane-induced lupus.. Immunization responses to NP-Ficoll and NP-ovalbumin were assessed in WT and caspase-1-/- mice. In vitro IgM and IgG responses to R848 were measured by ELISA. Serum IgM anti-dsDNA and IL-1β were also measured by ELISA. B and T cell populations 2 weeks and 6 months following pristane injection were measured by flow cytometry in WT and caspase-1-/- mice.. Caspase-1-/- mice generate equivalent IgG responses to NP-Ficoll and NP-ova antigens when compared to wild-type mice. Additionally, they secrete IgM and IgG in response to TLR7 activation. Pristane injected WT and caspase-1-/- mice generate robust IgM anti-dsDNA responses. Caspase-1-/- mice have a significant reduction in marginal zone B cell populations compared to WT 6 months after pristane exposure whereas T cell responses are intact in these mice.. Caspase-1-/- mice have intact immune responses but do not develop an expanded marginal zone B cell population in response to pristane-induced lupus. This may be one explanation for reduced IgG autoantibody production in these mice.

Topics: Animals; Antibodies, Antinuclear; B-Lymphocytes; Caspase 1; Cells, Cultured; Disease Models, Animal; Ficoll; Genetic Predisposition to Disease; Imidazoles; Immunization; Immunoglobulin G; Immunoglobulin M; Lupus Erythematosus, Systemic; Mice, Inbred BALB C; Mice, Knockout; Nitrophenols; Ovalbumin; Phenotype; Phenylacetates; Spleen; T-Lymphocytes; Terpenes; Time Factors

Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP1-, NIP1-, or NNP1-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation.

Topics: Animals; Antibody Affinity; Antibody Specificity; Biosensing Techniques; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Somatic Hypermutation, Immunoglobulin; Surface Plasmon Resonance

Polysaccharopeptide (PSP), from Coriolus versicolor, has been used widely as an adjuvant to chemotherapy with demonstrated anti-tumor and broad immunomodulating effects. While PSP's mechanism of action still remains unknown, its enhanced immunomodulatory potential with acacia gum is of great interest. Acacia gum, which also contains polysaccharides and glycoproteins, has been demonstrated to be immunopotentiating. To elucidate whether PSP directly activates T-cell-dependent B-cell responses in vivo, we used a well-established hapten carrier system (Nitrophenyl-chicken gamma globulin (NP-CGG)). 6-week C57BL/6 male mice were immunised with 50 μg of NP25-CGG alum precipitate intraperitoneally. Mice were gavaged daily with 50 mg/kg PSP in a vehicle containing acacia gum and sacrificed at days 0, 4, 7, 10, 14 and 21. ELISA was used to measure the total and relative hapten-specific anti-NP IgA, IgM and IgG titre levels compared to the controls. It was found that PSP, combined with acacia gum, significantly increased total IgG titre levels at day 4 (P< 0.05), decreased IgM titre levels at days 4 and 21 (P< 0.05) with no alterations observed in the IgA or IgE titre levels at any of the time points measured. Our results suggest that while PSP combined with acacia gum appears to exert weak immunological effects through specific T-cell dependent B-cell responses, they are likely to be broad and non-specific which supports the current literature on PSP. We report for the first time the application of a well-established hapten-carrier system that can be used to characterise and delineate specific T-cell dependent B-cell responses of potential immunomodulatory glycoprotein-based herbal medicines combinations in vivo.

Topics: Adjuvants, Immunologic; Animals; Antibodies; B-Lymphocytes; gamma-Globins; Gum Arabic; Haptens; Immunization; Immunoglobulin G; Immunoglobulin M; Immunotherapy; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Neoplasms; Nitrophenols; Phenylacetates; Proteoglycans; T-Lymphocytes; Trametes

Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

Topics: Models, Molecular; Nitrophenols; Phenylacetates; Protein Domains; Protein Stability; Protein Structure, Secondary; Single-Chain Antibodies; Temperature

Reactive nitrogen species (RNS) can modify proteins at tyrosine and tryptophan residues, and they are involved in the pathogenesis of various human diseases. In this study, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method that enables the simultaneous measurement of urinary 3-nitrotyrosine (3-NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA). After the addition of stable isotope-labeled internal standards, urine samples were purified and enriched using manual solid-phase extraction (SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis. The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYR and NHPA, respectively. Inter- and intraday imprecision was <15%. The mean relative recoveries of 3-NTYR and NHPA in urine were 89-98% and 90-98%, respectively. We further applied this method to 65 urinary samples from healthy subjects. Urinary samples were also analyzed for N-nitrosodimethylamine (NDMA) as well as oxidative and methylated DNA lesions, namely, 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), N7-methylguanine (N7-MeG), and N3-methyladenine (N3-MeA), using reported LC-MS/MS methods. Urinary 3-NTYR and NHPA levels were measured at concentrations of 63.2 ± 51.5 and 77.4 ± 60.8 pg/mL, respectively. Urinary 3-NTYR and NHPA levels were highly correlated with each other and with 8-oxoGua and 8-oxodGuo. Our findings demonstrated that a relationship exists between oxidative and nitrative stress. However, 3-NTYR and NHPA were correlated with N7-MeG and N3-MeA but not with NDMA, suggesting that NDMA may not be a representative biomarker of N-nitroso compounds that are induced by RNS.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenine; Adult; Chromatography, High Pressure Liquid; Chromatography, Liquid; Deoxyguanosine; Dimethylnitrosamine; DNA Methylation; Guanine; Humans; Limit of Detection; Middle Aged; Nitrophenols; Oxidation-Reduction; Phenylacetates; Solid Phase Extraction; Tandem Mass Spectrometry; Tyrosine; Young Adult

Homeostatic B Cell-Attracting chemokine 1 (BCA-1) otherwise known as CXCL13 is constitutively expressed in secondary lymphoid organs by follicular dendritic cells (FDC) and macrophages. It is the only known ligand for the CXCR5 receptor, which is expressed on mature B cells, follicular helper T cells (Tfh), Th17 cells and regulatory T (Treg) cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune disorders (e.g. Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosis). We, therefore, hypothesized that antibody-mediated disruption of the CXCL13 signaling pathway would interfere with the formation of ectopic lymphoid follicles in the target organs and inhibit autoimmune disease progression. This work describes pre-clinical development of human anti-CXCL13 antibody MAb 5261 and includes therapeutic efficacy data of its mouse counterpart in murine models of autoimmunity.. We developed a human IgG1 monoclonal antibody, MAb 5261 that specifically binds to human, rodent and primate CXCL13 with an affinity of approximately 5 nM and is capable of neutralizing the activity of CXCL13 from these various species in in vitro functional assays. For in vivo studies we have engineered a chimeric antibody to contain the same human heavy and light chain variable genes along with mouse constant regions. Treatment with this antibody led to a reduction in the number of germinal centers in mice immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) and, in adoptive transfer studies, interfered with the trafficking of B cells to the B cell areas of mouse spleen. Furthermore, this mouse anti-CXCL13 antibody demonstrated efficacy in a mouse model of Rheumatoid arthritis (Collagen-Induced Arthritis (CIA)) and Th17-mediated murine model of Multiple Sclerosis (passively-induced Experimental Autoimmune Encephalomyelitis (EAE)).. We developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.

Topics: Animals; Antibodies, Blocking; Arthritis, Experimental; Arthritis, Rheumatoid; B-Lymphocytes; Cell Movement; Cells, Cultured; Chemokine CXCL13; Dendritic Cells, Follicular; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Genetic Engineering; Germinal Center; Hemocyanins; Humans; Immunoglobulin G; Immunotherapy; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Multiple Sclerosis; Nitrophenols; Phenylacetates; Receptors, CXCR5; Recombinant Fusion Proteins; Signal Transduction; Th17 Cells

We developed a method to detect and isolate plasma cells that produce antigen-specific antibodies. An affinity matrix of hapten was constructed on a cell surface, and subsequent incubation allowed cells to secrete antibodies. Anti-hapten antibodies preferentially bound to the affinity matrix on the cells from which they were secreted. We showed that the combination of surface biotinylation and streptavidin which was conjugated with a high valence of hapten was suitable for sensitive detection of antibody binding. Using this protocol, anti-hapten plasma cells from immunized mouse spleen were detected and enriched by flow cytometry. This method allows for isolation of intact plasma cells according to the antibody specificity and may be useful for highly efficient and precise analysis of an antibody repertoire.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody Affinity; Antibody Specificity; Cells, Cultured; Flow Cytometry; Haptens; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Plasma Cells; Spleen

Studies on the structural basis of antibody affinity maturation have been carried out by measuring the affinity of secreted antibodies, and information on structures has often been obtained from nucleotide sequences of BCRs of memory B cells. We considered it important to establish whether the repertoire of secreted antibodies from plasma cells is really in accord with that of BCRs on memory B cells at the same time points post-immunization. We isolated plasma cells secreting antibodies specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten by affinity matrix technology using biotin-anti-CD138 and streptavidin-NP-allophycocyanin, to which anti-NP antibodies secreted by autologous plasma cells bound preferentially. We found that plasmablasts occupied >90% of the antibody-secreting cell compartment in the primary response and that they secreted antibodies whose VH regions were encoded by V186.2(+)Tyr95(+) sequences, which provided an increase in the medium level of affinity by somatic hypermutation (SHM) of heavy chains at position 33. After secondary immunization, a further increase in antibody affinity was observed, which was explained by the appearance of a number of plasma cells secreting V186.2(+)Gly95(+) antibodies that acquired high affinity by multiple SHMs as well as plasmablasts secreting V186.2(+)Tyr95(+) antibodies. However, we did not detect any plasmablasts secreting V186.2(+)Gly95(+) antibodies, showing that plasmablasts and plasma cells have a different antibody repertoire, i.e. their respective repertoires are asymmetric. On the basis of these findings, we discussed the relationship between the BCR affinity of memory B cells and plasmablasts as well as plasma cells as pertaining to their ontogeny.

Topics: Animals; Antibodies; Antibody Diversity; Antibody-Producing Cells; B-Lymphocytes; Cell Differentiation; Cells, Cultured; Chickens; gamma-Globulins; Immunization, Secondary; Immunologic Memory; Lymphocyte Activation; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Somatic Hypermutation, Immunoglobulin

Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response.

Topics: Animals; Antibody Affinity; Antibody-Dependent Cell Cytotoxicity; B-Lymphocytes; Cytidine Deaminase; Haptens; Hybridomas; Immunization, Secondary; Immunoglobulin M; Immunologic Memory; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Plasma Cells; Protein Binding; Single-Domain Antibodies; Somatic Hypermutation, Immunoglobulin

The effect of a quinoline-3-carboxamide on the T cell-dependent B cell response was investigated in C57BL/6 mice after NP-CGG immunization. The primary serum response to the hapten was slightly inhibited by treatment with a quinoline-3-carboxamide. This inhibition was paralleled by reduced numbers of germinal centre (GC) B cells and follicular T cells in the spleen up to 21 days after immunization. Also, both the number of GCs formed and their size were reduced by quinoline-3-carboxamide treatment. In contrast to the observation in the primary immune response, there was no inhibitory effect on the secondary immune response. These data could help to explain how quinoline-3-carboxamides can modulate immune function in autoimmune diseases without being immunosuppressive.

Topics: Animals; B-Lymphocytes; Cells, Cultured; Chickens; gamma-Globulins; Germinal Center; Haptens; Immunity, Humoral; Immunization; Immunoglobulin G; Immunomodulation; Lymphocyte Count; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Quinolines; Spleen; T-Lymphocytes

B cells are required for follicular Th (Tfh) cell development, as is the ICOS ligand (ICOS-L); however, the separable contributions of Ag and ICOS-L delivery by cognate B cells to Tfh cell development and function are unknown. We find that Tfh cell and germinal center differentiation are dependent on cognate B cell display of ICOS-L, but only when Ag presentation by the latter is limiting, with the requirement for B cell expression of ICOS-L overcome by robust Ag delivery. These findings demonstrate that Ag-specific B cells provide different, yet compensatory, signals for Tfh cell differentiation, while reconciling conflicting data indicating a requirement for ICOS-L expression on cognate B cells for Tfh cell development with those demonstrating that the latter requirement could be bypassed in lieu of that tendered by noncognate B cells. Our findings clarify the separable roles of delivery of Ag and ICOS-L by cognate B cells for Tfh cell maturation and function, and have implications for using therapeutic ICOS blockade in settings of abundantly available Ag, such as in systemic autoimmunity.

Topics: Animals; Antigens; Antigens, CD19; B-Lymphocytes; Cell Proliferation; DNA-Binding Proteins; Flow Cytometry; Inducible T-Cell Co-Stimulator Ligand; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Nitrophenols; Ovalbumin; Phenylacetates; Proto-Oncogene Proteins c-bcl-6; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; T-Lymphocytes, Helper-Inducer

Whereas NO is known to regulate T cell responses, its role in regulating B cell responses remains unclear. Previous studies suggested that inducible NO synthase 2 (NOS2/iNOS) is required for normal IgA Ab responses but inhibits antiviral IgG2a Ab responses. In this study we used NOS2(-/-) mice to determine the role of NO in T cell-dependent and T cell-independent (TI)-2 Ab responses. Whereas T cell-dependent Ab responses were only modestly increased in NOS2(-/-) mice, IgM and IgG3 Ab responses as well as marginal zone B cell plasma cell numbers and peritoneal B1b B cells were significantly elevated after immunization with the TI-2 Ag 4-hydroxy-3-nitrophenyl acetyl (NP)-Ficoll. The elevated TI-2 responses in NOS2(-/-) mice were accompanied by significant increases in serum levels of BAFF/BLyS and by increases in BAFF-producing Ly6C(hi) inflammatory monocytes and monocyte-derived dendritic cells (DCs), suggesting that NO normally inhibits BAFF expression. Indeed, we found that NOS2(-/-) DCs produced more BAFF than did wild-type DCs, and addition of a NO donor to NOS2(-/-) DCs reduced BAFF production. Bone marrow chimeric mice that lack NOS2 in either nonhematopoietic or hematopoietic cells had intermediate IgM and IgG3 Ab responses after NP-Ficoll immunization, suggesting that NOS2 from both hematopoietic and nonhematopoietic sources regulates TI-2 Ab responses. Similar to NOS2(-/-) mice, depletion of Ly6C(hi) inflammatory monocytes and monocyte-derived DCs enhanced NP-specific IgM and IgG3 responses to NP-Ficoll. Thus, NO produced by inflammatory monocytes and their derivative DC subsets plays an important role in regulating BAFF production and TI-2 Ab responses.

Topics: Animals; Antibody Formation; B-Cell Activating Factor; B-Lymphocyte Subsets; Ficoll; Haptens; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrophenols; Phenylacetates; T-Lymphocyte Subsets

The ability to recognize small organic molecules and chemical modifications of host molecules is an essential capability of the adaptive immune system, which until now was thought to be mediated mainly by B cell antigen receptors. Here we report that small molecules, such as cyanine 3 (Cy3), a synthetic fluorescent molecule, and 4-hydroxy-3-nitrophenylacetyl (NP), one of the most noted haptens, are γδ T cell antigens, recognized directly by specific γδ TCRs. Immunization with Cy3 conjugates induces a rapid Cy3-specific γδ T cell IL-17 response. These results expand the role of small molecules and chemical modifications in immunity and underscore the role of γδ T cells as unique adaptive immune cells that couple B cell-like antigen recognition capability with T cell effector function.

Topics: Animals; Carbocyanines; Cell Membrane; Haptens; Immunity; Immunization; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell, gamma-delta

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Topics: Adoptive Transfer; Animals; Autoantibodies; B-Lymphocyte Subsets; Cell Movement; Cellular Microenvironment; Coculture Techniques; Crosses, Genetic; Germinal Center; Haptens; Immunoglobulin G; Interleukin-17; Interleukins; Kidney; Lymphocyte Cooperation; Lymphopoiesis; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Receptors, Interleukin-17; T-Lymphocytes, Helper-Inducer

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP-specific or anti-Pn14-specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A-specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.

Topics: Animals; Antibodies, Bacterial; Antibody Affinity; Antibody Diversity; Antibody Formation; B-Lymphocytes; Bacterial Capsules; Cell Lineage; Cytidine Deaminase; Enzyme Activation; Gene Knock-In Techniques; Germinal Center; Haptens; Housing, Animal; Immunization; Immunoglobulin Class Switching; Immunoglobulin G; Mice; Mice, Knockout; Nitrophenols; Phenylacetates; Signal Transduction; Somatic Hypermutation, Immunoglobulin; Spleen; Streptococcal Infections; Streptococcus pneumoniae; TOR Serine-Threonine Kinases

Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7(+) B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.

Topics: Administration, Oral; Adoptive Transfer; Animals; Antibody Affinity; Antigens; B-Lymphocytes; Cholera Toxin; Gastrointestinal Tract; Gene Order; Germinal Center; Immunization; Immunoglobulin A; Immunoglobulin Heavy Chains; Mice; Models, Immunological; Nitrophenols; Peyer's Patches; Phenylacetates; Plasma Cells; Receptors, Antigen, B-Cell

The mechanistic basis for efficient combating of the infinite range of foreign Ags by the limited repertoire of naive Abs expressed on primary B cell surfaces during their first encounter was addressed through elegantly designed crystallographic analyses. Resolution of the discrepancy arising from the limited number of possible germline Ab receptors on primary B cells for recognizing the unlimited pool of possible Ags has been attempted by invoking the degenerate recognition potential of the germline Abs. Structural analyses of germline mAb BBE6.12H3 in an Ag-free state, as well as bound to four different peptide Ags, established the correlation of its degenerate specificity with conformational versatility of the paratope. Six distinct paratope topologies observed for a single germline mAb provided a quantitative description of the primary Ag recognition repertoire at the tertiary structural level. Each of the four different peptide Ags was bound specifically to a distinct conformation of the paratope, which was also different from that of the Ag-free states of the same germline mAb. A minimal conserved motif in the pristine Ag-combining site essential for multispecificity and Ag binding-mediated change in the elbow angle of Fab was also discernible. It is proposed that the generation of a primary Ab repertoire involves large, yet finite, germline Ab clones, each capable of adopting discrete conformations, which in turn exhibit diverse binding modes.

Topics: Animals; Antibodies, Monoclonal; Antigens; B-Lymphocytes; Binding Sites, Antibody; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred C57BL; Models, Molecular; Nitrophenols; Phenylacetates; Protein Conformation; Protein Structure, Tertiary; Receptors, Antigen, B-Cell

Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.

Topics: Animals; Antigens, Surface; Apoptosis Regulatory Proteins; B-Lymphocyte Subsets; Cell Differentiation; Chickens; Dendritic Cells, Follicular; gamma-Globulins; Germinal Center; Haptens; Immunization, Secondary; Immunoglobulin G; Immunoglobulin M; Immunologic Memory; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Programmed Cell Death 1 Receptor; Receptors, CXCR5; Receptors, Interleukin-21; T-Lymphocytes, Helper-Inducer

We searched for memory B cells responsible for high-affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody production by C57BL/6 mice immunized with NP-chicken γ-globulin (CGG), using flow cytometry. We first prepared transfectants expressing B-cell antigen receptor (BCR) of known affinity as a memory B-cell model as well as NP-allophycocyanin (APC) of different NP valences, NP(lo), NP(med) and NP(hi). We then used the latter as probes capable of distinguishing BCR affinities: NP(lo)-APC bound to BCRs with an affinity higher than 3.4 × 10(6) M(-1), while NP(med)-APC bound to those with a higher than germline affinity. B cells capable of binding to NP(lo)-APC appeared in spleens on day 14 post-immunization, and harbored Tyr95 (Tyr95 type) as well as a mutation from Trp33 to Leu. B cells with BCRs harboring Gly95 (Gly95 type) appeared only in the NP(med)-APC-binding fraction on day 56 and in the NP(lo)-APC-binding fraction on day 77, indicating that this long duration was necessary for Gly95 type B cells to acquire high affinity and to become a member of the group of memory B cells with high affinity. Administration of NP-CGG on day 77 caused little change in the proportion of the Gly95 type in NP(lo)-APC-binding B cells in the following 2 weeks but brought about an increase in the number of high-affinity antibody-secreting cells (ASC), suggesting that the memory B-cell compartment established was maintained at a later stage and supplied high-affinity ASCs. The relationship between these Gly95 type memory B cells and ASCs is discussed.

Topics: Animals; Antibodies; Antibody Affinity; B-Lymphocytes; Cell Communication; Cell Separation; Flow Cytometry; gamma-Globins; Immunization; Immunologic Memory; Mice; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Nitrophenols; Phenylacetates; Phycocyanin; Protein Engineering; Receptors, Antigen, B-Cell; Transgenes

Rapid production of neutralizing antibody can be critical for limiting the spread of infection. Such early antibody results when B-cell blasts mature directly to plasmablasts without forming germinal centers. These extrafollicular responses can involve Ig class switch recombination (CSR), producing antibody that can readily disseminate through infected tissues. The present study identifies the differentiation stage where CSR occurs in an extrafollicular response induced by 4-hydroxy-3-nitrophenyl acetyl (NP) conjugated to Ficoll (NP-Ficoll). To do this, we took advantage of the antigen dose dependency of CSR in this response. Thus, while both 30 and 1 μg NP-Ficoll induce plasmablasts, only the higher antigen dose induces CSR. Activation-induce cytidine deaminase (AID) is critical for CSR and in keeping with this a proportion of NP-specific B-cell blasts induced by 30 μg NP-Ficoll express AID. None of the B blasts responding to the non-CSR-inducing 1 μg dose of NP-Ficoll express AID. We confirmed that CSR occurs in B blasts by demonstrating the presence of rearranged heavy-chain transcripts in B blasts in the 30 μg response. CSR in this extrafollicular response is confined to B blasts, because NP-specific plasmablasts, identified by expressing CD138 and Blimp-1, no longer express AID and cannot undergo CSR.

Topics: Animals; B-Lymphocytes; Cytidine Deaminase; Ficoll; Immunoconjugates; Immunoglobulin Class Switching; Immunoglobulin Heavy Chains; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Plasma Cells; Positive Regulatory Domain I-Binding Factor 1; Syndecan-1; Transcription Factors

CD248 is a cell surface receptor that specifically identifies fibroblasts and pericytes during development and in association with cancer and inflammation. However, its function is poorly defined and its role in lymphoid organs not studied. Here, we used (4-hydroxy-3-nitrophenyl)acetyl chicken gamma-globulin immunisation and mice lacking CD248 to study whether CD248 modulates popliteal LN (pLN) expansion and subsequent immune responses. We have found that CD248 is required for complete pLN expansion but not for co-ordination of B and T cell compartmentalisation or antibody production following (4-hydroxy-3-nitrophenyl)acetyl chicken gamma-globulin immunisation. In vitro, we show that CD248 expression in human MG63 stromal cells and mouse embryonic fibroblasts leads to a pro-proliferative and pro-migratory phenotype. This correlates with a proliferating CD248(+) population observed in vivo during pLN expansion. Taken together, these data highlight a role for CD248 in secondary lymphoid organ remodelling during adaptive immune responses.

Topics: Animals; Antibody Formation; Antigens, CD; Antigens, Neoplasm; B-Lymphocytes; Biomarkers; Cell Communication; Cell Line; Cell Movement; Cell Proliferation; Chickens; Fibroblasts; gamma-Globulins; Haptens; Humans; Immunization; Lymph Nodes; Mice; Mice, Knockout; Neoplasm Proteins; Nitrophenols; Pericytes; Phenylacetates; Stromal Cells; T-Lymphocytes

Although CD40 signaling is required for activation and differentiation of B cells, including germinal center (GC) formation and generation of memory B cells, in vivo generation of CD40 signaling augments plasma cell differentiation but disrupts GCs. Thus, CD40 signaling is thought to direct B cells to extrafollicular plasma cell fate rather than GC formation. In this study, we analyzed CD40L transgenic (CD40LTg) mice that constitutively express CD40L on B cells. After immunization, activation of B cells, but not dendritic cells, was augmented, although dendritic cells can be activated by CD40 ligation. Bone marrow chimera carrying CD40LTg and nontransgenic B cells showed increased Ab production from transgenic, but not from coexisting nontransgenic, B cells, suggesting that CD40L on a B cell preferentially stimulates the same B cell through an autocrine pathway, thereby augmenting Ab production. Although GCs rapidly regressed after day 5 of immunization and failed to generate late-appearing high-affinity Ab, CD40LTg mice showed normal GC formation up to day 5, as well as normal generation of long-lived plasma cells and memory B cell responses. This observation suggests that CD40 signaling does not block GC formation or differentiation of GC B cells, but it inhibits sustained expansion of GC B cells and augments B cell differentiation.

Topics: Adjuvants, Immunologic; Animals; B-Lymphocyte Subsets; CD40 Ligand; Cell Differentiation; Cells, Cultured; Female; Germinal Center; Growth Inhibitors; Haptens; Immunoglobulin G; Immunoglobulin M; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Mice, Transgenic; Molecular Sequence Data; Nitrophenols; Phenylacetates; Up-Regulation

Mechanisms by which cell surface levels of the BCR are regulated remain largely unknown. We found that B cells lacking the lysosomal-associated protein transmembrane 5 (LAPTM5) expressed higher levels of cell surface BCR than did wild-type (WT) B cells after Ag stimulation in vitro and in vivo. In addition, LAPTM5-deficient mice contained an increased frequency of Ag-specific B cells and produced greater amounts of Abs than did WT mice after immunization with a T-dependent Ag. Adoptive transfer of LAPTM5-deficient B cells with WT T cells into RAG1-deficient mice revealed that the increased surface BCR levels and the enhanced B cell activation and Ab production were due to a B cell intrinsic defect. As they aged, the LAPTM5-deficient mice had increased titers of serum IgM and autoantibodies and immune complex deposition in the kidney. Immunofluorescent and biochemical analysis revealed that LAPTM5 physically interacted with the BCR complex and promoted its degradation in the lysosomal compartment in mouse B cells. These results demonstrate a role for LAPTM5 in the negative regulation of cell surface BCR levels and B cell activation.

Topics: Animals; Antibody Affinity; B-Lymphocyte Subsets; Cell Line, Tumor; Cells, Cultured; Chickens; Down-Regulation; Epitopes, B-Lymphocyte; gamma-Globulins; Haptens; Immediate-Early Proteins; Immunoglobulin M; Lymphocyte Activation; Lysosomes; Membrane Proteins; Mice; Mice, Knockout; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Up-Regulation

It has long been presumed that after leaving the germinal centers (GCs), memory B cells colonize the marginal zone or join the recirculating pool. Here we demonstrate the preferential localization of nitrophenol-chicken gamma-globulin-induced CD38(+)IgG1(+) memory B cells adjacent to contracted GCs in the spleen. The memory B cells in this region proliferated after secondary immunization, a response that was abolished by depletion of CD4(+) T cells. We also found that these IgG1(+) memory B cells could present antigen on their surface, and that this activity was required for their activation. These results implicate this peri-GC region as an important site for survival and reactivation of memory B cells.

Topics: ADP-ribosyl Cyclase 1; Animals; B-Lymphocytes; CD4-Positive T-Lymphocytes; Chickens; Flow Cytometry; gamma-Globulins; Germinal Center; Immunization; Immunoglobulin G; Immunohistochemistry; Immunologic Memory; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nitrophenols; Phenylacetates; Spleen

Apobec3/Rfv3 is an innate immune factor that promotes the neutralizing Ab response against Friend retrovirus (FV) in infected mice. Based on its evolutionary relationship to activation-induced deaminase, Apobec3 might directly influence Ab class switching and affinity maturation independently of viral infection. Alternatively, the antiviral activity of Apobec3 may indirectly influence neutralizing Ab responses by reducing early FV-induced pathology in critical immune compartments. To distinguish between these possibilities, we immunized wild-type and Apobec3-deficient C57BL/6 (B6) mice with (4-hydroxy-3-nitrophenyl) acetyl (NP) hapten and evaluated the binding affinity of the resultant NP-specific Abs. These studies revealed similar affinity maturation of NP-specific IgG1 Abs between wild-type and Apobec3-deficient mice in the absence of FV infection. In contrast, hapten-specific Ab affinity maturation was significantly compromised in Apobec3-deficient mice infected with FV. In highly susceptible (B6 x A.BY)F(1) mice, the B6 Apobec3 gene protected multiple cell types in the bone marrow and spleen from acute FV infection, including erythroid, B, T, and myeloid cells. In addition, B6 Apobec3 deficiency was associated with elevated Ig levels, but decreased induction of splenic germinal center B cells and plasmablasts during acute FV infection. These data suggest that Apobec3 indirectly influences FV-specific neutralizing Ab responses by reducing virus-induced immune dysfunction. These findings raise the possibility that enabling Apobec3 activity during acute infection with human pathogenic retroviruses, such as HIV-1, may similarly facilitate stronger virus-specific neutralizing Ab responses.

Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Antibody Affinity; Bone Marrow Cells; Cytidine Deaminase; Female; Flow Cytometry; Friend murine leukemia virus; Humans; Immunization; Leukemia, Experimental; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Retroviridae Infections; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Tumor Virus Infections

Upon antigen (Ag) encounter, B cells require T-cell help to enter the germinal center (GC). They obtain this help by presenting Ag-derived peptides on MHC class II (MHCII) for recognition by the T-cell receptor (TCR) of CD4(+) T cells. Peptides are loaded onto MHCII in endosomal compartments in a process catalyzed by the MHCII-like protein H2-M (HLA-DM in humans). This process is modulated by another MHCII-like protein, H2-O (HLA-DO in humans). H2-O is a biochemical inhibitor of peptide loading onto MHCII; however, on the cellular level, it has been shown to have varying effects on Ag presentation. Thus, the function of H2-O in the adaptive immune response remains unclear. Here, we examine the effect of H2-O expression on the ability of Ag-specific B cells to enter the GC. We show that when Ag specific WT and H2-O(-/-) B cells are placed in direct competition, H2-O(-/-) B cells preferentially populate the GC. This advantage is confined to Ag-specific B cells and is due to their superior ability to obtain Ag-specific T-cell help when T-cell help is limiting. Overall, our work shows that H2-O expression reduces the ability of B cells to gain T-cell help and participate in the GC reaction.

Topics: Adoptive Transfer; Animals; Antigen Presentation; B-Lymphocytes; Germinal Center; Histocompatibility Antigens Class II; Humans; Mice; Mice, Congenic; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Recombinant Proteins; T-Lymphocytes, Helper-Inducer

Fc receptor-like A (FCRLA) and FCRLB have homology to the transmembrane FCRL family members (FCRL 1-6) and to the conventional receptors for the Fc portion of immunoglobulin, but uniquely are cytosolic proteins expressed in B cells. Here we describe the phenotype of Fcrlb-gene targeted mice. B cell development and in vitro responses are normal; however, antibody responses to a T-dependent antigen are elevated. The gene encoding the inhibitory FcγRIIb is located nearby Fcrlb. Although Fcrlb-gene targeting had no effect on the function or basal expression of FcγRIIb, its expression was reduced following activation. This abnormal regulation was due to co-inheritance of Fcgr2b and the mutant Fcrlb allele from the 129 ES cells. A promoter polymorphism in the 129/Sv Fcgr2b allele results in diminished upregulation of FcγRIIb following B cell activation. Thus, we speculate that the enhanced antibody response seen in the FCRLB-deficient mice may be due to the Fcgr2b promoter.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Antinuclear; Antibody Formation; B-Lymphocytes; Cell Count; Cell Proliferation; Gene Expression; Immunoglobulin Fab Fragments; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Polymorphism, Genetic; Promoter Regions, Genetic; Receptors, Fc; Receptors, IgG; Vaccination

Resting antigen-experienced memory B cells are thought to be responsible for the more rapid and robust antibody responses after antigen reencounter, which are the hallmark of memory humoral responses. The molecular basis for the development and survival of memory B cells remains largely unknown. We report that phospholipase C (PLC) gamma2 is required for efficient formation of germinal center (GC) and memory B cells. Moreover, memory B cell homeostasis is severely hampered by inducible loss of PLC-gamma2. Accordingly, mice with a conditional deletion of PLC-gamma2 in post-GC B cells had an almost complete abrogation of the secondary antibody response. Collectively, our data suggest that PLC-gamma2 conveys a survival signal to GC and memory B cells and that this signal is required for a productive secondary immune response.

Topics: Animals; B-Lymphocytes; Cell Cycle; Cell Survival; Gene Deletion; Germinal Center; Immunization; Immunoglobulin G; Immunologic Memory; Mice; Mice, Knockout; Nitrophenols; Phenylacetates; Phospholipase C gamma

The effect of antibody affinity on molecular forms of immune complexes was investigated by measuring antigen-antibody interactions using surface plasmon resonance (SPR), electrospray ionization time-of-flight mass spectrometry under non-denaturing conditions (MS), analytical ultracentrifugation (AUC), and transmission electron microscopy (TEM). (4-Hydroxy-3-nitrophenyl)acetic acid (NP) of different valences was conjugated to bovine serum albumin (BSA) and these conjugates were used as antigens. In the interaction between N1G9, a low affinity antibody, and NP(7)-BSA, a 1:1 immune complex was detected as the major product and higher molecular weight complexes were not obtained by any of the methods employed. These results suggested that N1G9 predominantly formed an intramolecular divalent complex with NP(7)-BSA using the two Fab arms of an antibody. Although complexes of various sizes were detected by MS, AUC, and TEM in the interaction between C6, a high affinity antibody, and NP(7)-BSA, only 1:1 immune complexes were observed by SPR. These results showed that two NP(7)-BSA molecules cannot simultaneously bind to an antibody, irrespective of antibody affinity strength, when the Fc region is immobilized to a flexible dextran matrix on sensor chip but are able to do so with high affinity antibodies free in solution. The results also showed that the stoichiometry of the antigen-antibody interaction is altered by restricting the movement of the Fc region. Since immunoglobulins exist as antibodies in solution or as B cell receptors on the cell surface, it is suggested that interactions of B cell receptors with polyvalent antigens such as NP-BSA might be different from those of antibodies free in solution.

Topics: Animals; Antibody Affinity; Antigen-Antibody Complex; Antigens; Cattle; Immobilized Proteins; Mass Spectrometry; Models, Immunological; Nitrophenols; Phenylacetates; Serum Albumin, Bovine

DAP-related apoptotic kinase-2 (DRAK2), a death-associated protein kinase family member, is highly expressed in B and T lymphocytes in the human and the mouse. To determine whether DRAK2 plays a role in B-cell activation and differentiation, we analyzed germinal centers (GCs) and the specific antibody response to NP in drak2-/- mice immunized with the thymus-dependent (TD) conjugated hapten NP16-CGG. In drak2-/- mice, spleen GCs were normal in size and morphology, but their number was reduced by as much as 5-fold, as compared to their wild-type littermates. This was not due to a defect in B-cell proliferation, as the BrdU uptake was comparable in DRAK2-deficient and wild-type B cells. Rather, the proportion of apoptotic GC B and T cells in drak2-/- mice was significantly higher than that in wild-type control mice, as shown by 7-AAD and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining. In drak2-/- mice, the generation high affinity IgG antibodies was impaired in spite of the seemingly normal somatic hypermutation and class switch DNA recombination machineries in drak2-/- B cells. In NP16-CGG-immunized drak2-/- mice, T-cell-intrinsic Bcl-xL transgene expression increased the number of GCs and rescued the high affinity IgG response to NP. These findings suggest a novel role for DRAK2 in regulating the GC reaction and the response to TD antigens, perhaps through increased survival of T cells and enhanced B-cell positive selection. They also suggest that DRAK2-deficiency is not involved in regulating intrinsic B-cell apoptosis.

Topics: Animals; Antibody Formation; Antibody Specificity; Apoptosis; B-Lymphocytes; bcl-X Protein; Cell Proliferation; Cells, Cultured; Germinal Center; Immunoglobulin Class Switching; Immunoglobulin G; Mice; Mice, Knockout; Microscopy, Fluorescence; Nitrophenols; Phenylacetates; Protein Serine-Threonine Kinases; Somatic Hypermutation, Immunoglobulin; Spleen; T-Lymphocytes

Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to the promoters of the tynA and feaB genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR caused small increases in the activities of the tynA and feaB promoters in cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and caused a marked repression of the tynA and feaB promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the tynA and feaB genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the tynA and feaB promoters. Mutation of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by tynA and feaB, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.

Topics: DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Nitric Oxide; Nitrophenols; Oxidation-Reduction; Phenethylamines; Phenylacetates; Promoter Regions, Genetic; Transcription Factors; Tyramine

Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when preexisting clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1-8i(+/-) mice, which feature a high frequency of B cells with nitrophenyl (NP)-binding specificity, respond to NP-haptenated proteins with the production of NP-specific Abs, but affinity maturation is impaired due to insufficient generation of high-affinity Ab-producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naive, wild-type recipients. Remarkably, when 10(4) B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 10(6) B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation.

Topics: Animals; Antibody-Producing Cells; Cell Adhesion; Cell Differentiation; Cell Proliferation; Clone Cells; Germinal Center; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nitrophenols; Phenylacetates; Phycocyanin; Receptors, Antigen, B-Cell; Signal Transduction; Somatic Hypermutation, Immunoglobulin

Newborn infants are exposed to various sources of oxidative and/or nitrative stress, which refers to either oxidation and/or nitration of endogenous proteins including loss of their original function. Nitrative stress is predominantly caused following synthesis of peroxynitrite. Particularly preterm infants with immature defense mechanisms against free radical injury appear at risk.. To test the feasibility of quantifying the degradation products of the peroxynitrite marker nitrotyrosine [3-nitro-4-hydroxyphenylacetic acid (NHPA) and para-hydroxyphenylacetic acid (PHPA)] in neonatal urine samples.. NHPA and PHPA were determined by gas chromatography/mass spectroscopy in urinary samples of preterm and term infants (mean gestational age = 28.4 and 39.6 weeks, respectively).. The urinary NHPA levels were lower in preterm infants in comparison with term infants. When the NHPA levels were adjusted to the urinary PHPA levels, no differences were found between the two groups.. Nitrotyrosine can be quantified in urinary samples of even the most immature infants. Nitration of endogenous PHPA in the gastrointestinal tract of term infants may have masked potentially higher levels of NHPA in preterm infants.

Topics: Free Radicals; Gastrointestinal Tract; Gestational Age; Humans; Infant, Newborn; Infant, Premature; Nitrophenols; Oxidative Stress; Oxygen; Phenylacetates; Tyrosine

Mature B cells co-express both IgM and IgD types of antigen receptors before activation. Our earlier work has shown that the co-expression of IgD and IgM plays an important role in regulating the composition of antibody repertoire during a primary immune response. However, the roles of these two B cell receptors in the development of B cell memory responses remain unclear. The present study shows that during the secondary immune response to (4-hydroxy-3-nitrophenyl)acetyl (NP), IgM-/- mice secreted significant amount of NP-specific IgD antibodies. The kinetics of antigen-specific IgD antibodies produced in IgM-/- mice was similar to that of IgM antibodies in wild type mice during the secondary response. However, the production of antigen-specific class-switched antibodies in IgM-/- mice was significantly reduced compared to that in wild type mice, particularly at the early phase of the memory response. In addition, germinal center (GC) reaction was significantly diminished in IgM-/- mice after secondary challenge with soluble antigen. Nevertheless, affinity maturation of antibodies appears largely intact in IgM-/- mice during memory response. Thus, our studies demonstrate that IgM-mediated signaling plays an important role in the development of efficient B cell memory responses.

Topics: Animals; Antibody Formation; Antibody Specificity; B-Lymphocytes; Germinal Center; Immunoglobulin D; Immunoglobulin M; Immunologic Memory; Mice; Mice, Knockout; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Signal Transduction

Germinal center (GC) responses to T-dependent Ags require effective collaboration between Th cells, activated B cells, and follicular dendritic cells within a highly organized microenvironment. Studies using gene-targeted mice have highlighted nonredundant molecules that are key for initiating and maintaining the GC niche, including the molecules of the ICOS, CD40, and lymphotoxin (LT) pathways. Signaling through ICOS has multiple consequences, including cytokine production, expression of CD40L on Th cells, and differentiation into CXCR5(+) follicular Th cells, all of which are important in the GC reaction. We have therefore taken advantage of ICOS(-/-) mice to dissect which downstream elements are required to initiate the formation of GC. In the context of a T-dependent immune response, we found that GC B cells from ICOS(-/-) mice express lower levels of LTalphabeta compared with wild-type GC B cells in vivo, and stimulation of ICOS on T cells induces LTalphabeta on B cells in vitro. Administration of agonistic anti-LTbeta receptor Ab was unable to restore the GC response in ICOS(-/-) mice, suggesting that additional input from another pathway is required for optimal GC generation. In contrast, treatment with agonistic anti-CD40 Ab in vivo recovered GC networks and restored LTalphabeta expression on GC B cells in ICOS(-/-) mice, and this effect was dependent on LTbeta receptor signaling. Collectively, these data demonstrate that ICOS activation is a prerequisite for the up-regulation of LTalphabeta on GC B cells in vivo and provide a model for cooperation between ICOS, CD40, and LT pathways in the context of the GC response.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; B-Lymphocytes; CD40 Antigens; Chickens; gamma-Globulins; Germinal Center; Inducible T-Cell Co-Stimulator Protein; Lymphotoxin beta Receptor; Lymphotoxin-alpha; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Signal Transduction

In order to quantitate the contribution of amino acid replacements to an increase in affinity during affinity maturation, we measured thermodynamic parameters of the antigen-antibody interaction for a group of anti-(4-hydroxy-3-nitrophenyl) acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. We prepared a common ancestor and hypothetical intermediate clones that might occur on the affinity maturation pathway, by employing site-directed mutagenesis. Isothermal calorimetric titration of the antigen-antibody reaction revealed that antibody evolution proceeds in two steps. The first step is driven by a decrease in enthalpy, in which two amino acid replacements in the VL region play an essential role. Further accumulation of amino acid replacements in VH and VL regions during the second step induce a progressive increase in affinity, which is driven by an increase in entropy, which has a cooperative mutational effect.

Topics: Amino Acid Sequence; Amino Acid Substitution; Aminocaproic Acid; Antibodies, Monoclonal; Antibody Affinity; Antibody Formation; Calorimetry; Circular Dichroism; Clone Cells; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Models, Molecular; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Protein Conformation; Thermodynamics

The quasimonoclonal (QM) mouse provides a model to analyze B cell selection because major B cell antigen receptors (BCR) are composed of the knockin V(H)DJ(H) 17.2.25 (V(H)T) encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for (4-hydoxy-3-nitrophenyl)acetyl (NP). We have reported that during a T-dependent antibody (Ab) response for a low-affinity NP analog p-nitrophenylacetyl (pNP), although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. The initial affinity of V(H)T/lambda2 B cells for pNP was approximately 50-100-fold higher than that of V(H)T/lambda1 B cells, suggesting a role of BCR affinity in recruiting B cells to affinity maturation processes. Here, we investigated whether the intensity of BCR signals could contribute to the selection of V(H)T/lambda2 B cells for affinity maturation. V(H)T/lambda2 B cells were more responsive to pNP than V(H)T/lambda1 B cells in vitro. When CFSE-labeled QM B cells were transferred into the wild type mice where T cells had been primed with chicken gamma-globulin (CGG), QM B cells challenged by pNP-conjugated CGG could be observed to get activated and migrate to GCs in the early phase of the T-dependent response to pNP-CGG. Adoptive transfer of sorted populations revealed that the V(H)T/lambda2 B cell population was more potent in migration into GCs than the V(H)T/lambda1 counterpart. Thus, it is suggested that the higher BCR affinity of V(H)T/lambda2 B cells may be an initial cue for their recruitment to GCs during a T-dependent Ab response.

Topics: Animals; B-Lymphocytes; Cell Movement; Germinal Center; Immunoglobulin Heavy Chains; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; T-Lymphocytes

MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.

Topics: Animals; B-Lymphocytes; Cell Differentiation; Cells, Cultured; Cytokines; Germinal Center; Immunoglobulin G; Lymphocyte Activation; Lymphotoxin-alpha; Lymphotoxin-beta; Mice; Mice, Knockout; Mice, Transgenic; MicroRNAs; Nitrophenols; Peyer's Patches; Phenylacetates; Somatic Hypermutation, Immunoglobulin; Spleen; T-Lymphocytes; Th1 Cells; Th2 Cells; Tumor Necrosis Factor-alpha

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Topics: Animals; Antigens, CD; Antigens, Viral; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cell Count; Cytochromes c; Gene Expression; Immunization, Secondary; Immunoglobulin Heavy Chains; Immunologic Memory; Interleukin-4; Lymph Nodes; Mammary Tumor Virus, Mouse; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell, alpha-beta; Receptors, CCR7; Receptors, Chemokine; Vaccination

In aging, both primary and secondary antibody responses are impaired. One of the most notable changes in age-associated immune deficiency is the diminished germinal center (GC) reaction. This impaired GC response reduces antibody affinity maturation, decreases memory B cell development, and prevents the establishment of long-term antibody-forming cells in the bone marrow. It is of great importance to explore novel strategy in improving GC response in the elderly. In this study, the efficacy of immunization with immune complexes in overcoming age-associated deficiency in GC response was investigated. We show that the depressed GC response in aged mice can be significantly elevated by immunization with immune complexes. Importantly, there is a significant improvement of B cell memory response and long-lived plasma cells. Our results demonstrate that immune complex immunization may represent a novel strategy to elicit functional GC response in aging, and possibly, to overcome age-related immune deficiency in general.

Topics: Aging; Animals; Antibody Formation; Antigen-Antibody Complex; Bone Marrow; Female; Germinal Center; Immunization; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Plasma Cells; T-Lymphocytes

Deficiencies in early complement components are associated with the development of systemic lupus erythematosus (SLE) and therefore early complement components have been proposed to influence B lymphocyte activation and tolerance induction. A defect in apoptosis is a potential mechanism for breaking of peripheral B cell tolerance, and we hypothesized that the lack of the early complement component C4 could initiate autoimmunity through a defect in peripheral B lymphocyte apoptosis. Previous studies have shown that injection of a high dose of soluble antigen, during an established primary immune response, induces massive apoptotic death in germinal centre B cells. Here, we tested if the antigen-induced apoptosis within germinal centres is influenced by early complement components by comparing complement C4-deficient mice with C57BL/6 wild-type mice. We demonstrate that after the application of a high dose of soluble antigen in wild-type mice, antibody levels declined temporarily but were restored almost completely after a week. However, after antigen-induced apoptosis, B cell memory was severely limited. Interestingly, no difference was observed between wild-type and complement C4-deficient animals in the number of apoptotic cells, restoration of antibody levels and memory response.

Topics: Animals; Antigens; Apoptosis; B-Lymphocytes; Complement C4; Enzyme-Linked Immunosorbent Assay; Female; Germinal Center; Immune Tolerance; Immunologic Memory; Lupus Erythematosus, Systemic; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

We previously found that there are two distinct antibody (Ab) maturation pathways for the immune response of C57BL/6 mice to 4-hydroxy-3-nitrophenylacetyl (NP), one involving Abs with high evolvability (group-H) and the other involving Abs with low evolvability (group-L). Commitment to whichever pathway is followed pre-determined in B cells at an early developmental stage. Candidates for the group-L or -H pathway are thus expected to pre-exist in the initial repertoire of the immune response. In the present study, we examined the initial Ab repertoire from the viewpoint of the latent potential of these Abs for effective affinity maturation. At first, we prepared anti-NP B cell hybridomas at 1 week postimmunization. Although the diversity of the obtained repertoire was maintained mainly by the third complementarity determining region of the heavy chain (CDR-H3), their changes in the near UV circular dichroism resulting from NP-binding allowed for classification into three groups according to the same rules applied in the pathway classification of the maturated Abs. This suggested that the innate structural properties of CDR-H3 were conserved throughout maturation. In other words, in exploring the structure of CDR-H3, it is possible to distinguish the latent potentials of Abs in effective affinity maturation even those making up the initial Ab repertoire. We then examined an artificially designed group-H Ab prototype and found its NP-binding ability sufficient for engagement in the initial repertoire. The question arose here as to why the majority of the actual initial repertoire consisted of the group-L ancestors regardless of their middling NP-binding affinity, which called for further discussion from the viewpoint of the dynamics possibly shaping the repertoire.

Topics: Amino Acid Sequence; Animals; Antibodies; Antibody Affinity; Antibody Formation; Antibody Specificity; Antigen-Antibody Complex; B-Lymphocytes; Circular Dichroism; Complementarity Determining Regions; Haptens; Immunoglobulin Fab Fragments; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Models, Molecular; Molecular Sequence Data; Nitrophenols; Peptide Library; Phenylacetates

The cascade of reactive nitrogen species generated from nitric oxide causes modification of proteins, lipids, and nucleic acids in a wide range of organisms. 3-Nitrotyrosine is one of the most common products of the action of reactive nitrogen species on proteins. Although a great deal is known about the formation of 3-nitrotyrosine, the subsequent metabolism of this compound is a mystery. Variovorax paradoxus JS171 and Burkholderia sp. strain JS165 were isolated from soil slurries when 3-nitrotyrosine was provided as the sole carbon, nitrogen, and energy source. During growth on 3-nitrotyrosine stoichiometric amounts of nitrite were released along with approximately one-half of the theoretically available ammonia. The catabolic pathway involving oxidative denitration is distinct from the pathway for tyrosine metabolism. The facile isolation and the specific, regulated pathway for 3-nitrotyrosine degradation in natural ecosystems suggest that there is a significant flux of 3-nitrotyrosine in such environments.

Topics: 3,4-Dihydroxyphenylacetic Acid; Biodegradation, Environmental; Burkholderia; Comamonadaceae; Models, Biological; Molecular Sequence Data; Nitrophenols; Phenylacetates; RNA, Bacterial; RNA, Ribosomal, 16S; Soil Microbiology; Tyrosine

Intrastriatal injection of 3-nitrotyrosine, which is a biomarker for nitrating oxidants, provokes dopaminergic neuronal death in rats by unknown mechanisms. Herein, we show that extracellular 3-nitrotyrosine is transported via the l-aromatic amino acid transporter in nondopaminergic NT2 cells, whereas in dopaminergic PC12 cells, it is transported by both the l-aromatic amino acid and the dopamine transporters. In both cell lines, 3-nitrotyrosine is a substrate for tyrosine tubulin ligase, resulting in its incorporation into the C terminus of alpha-tubulin. In NT2 cells, incorporation of 3-nitrotyrosine into alpha-tubulin induces a progressive, reversible reorganization of the microtubule architecture. In PC12 cells, 3-nitrotyrosine decreases intracellular dopamine levels and is metabolized by the concerted action of the aromatic amino acid decarboxylase and monoamine oxidase. Intracellular levels of 133 micromol of 3-nitrotyrosine per mole of tyrosine did not alter NT2 viability but induced PC12 apoptosis. The cell death was reversed by caspases and aromatic amino acid decarboxylase and monoamine oxidase inhibitors. 3-Nitrotyrosine induced loss of tyrosine hydroxylase-positive primary rat neurons, which was also prevented by an aromatic amino acid decarboxylase inhibitor. These findings provide a novel mechanism by which products generated by reactive nitrogen species induce dopaminergic neuron death and thus may contribute to the selective neurodegeneration in Parkinson's disease.

Topics: Amino Acid Transport Systems; Animals; Apoptosis; Aromatic-L-Amino-Acid Decarboxylases; Cell Death; Cell Line, Tumor; Cell Physiological Phenomena; Cell Survival; Cells; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Mesencephalon; Microtubules; Monoamine Oxidase; Neurons; Nitrophenols; PC12 Cells; Phenylacetates; Rats; Tubulin; Tyrosine

Naturally occurring IgM and IgA levels are remarkably stable between different individuals. In mice lacking joining chain (J-chain), the steady-state levels of IgM are reduced, while IgA levels are elevated. We have here analysed the IgM and IgA responses as well as the regulation of naturally occurring antibodies in mice that delete all J-chain expressing B cells (JDTA mice) and have been back-crossed to C57BL/6 mice. The IgM response to a T-cell-dependent antigen was reduced in JDTA mice but still easily detectable. In contrast, a very pronounced primary IgA response could be detected in JDTA mice while wild type controls showed no detectable primary IgA response. With regard to naturally occurring antibodies, bone marrow chimeras between JDTA and control C57BL/6 mice had a donor cell phenotype with regard to serum IgM and IgA. Mixed bone marrow chimeras had an intermediate phenotype, indicating that the naturally occurring antibody IgM and IgA levels are B-cell autonomous and not subjected to feed-back control. This was confirmed by transfer of the dominant naturally occurring IgM/IgA phenotype to the recipient by peritoneal exudate cells.

Topics: Animals; Bone Marrow Transplantation; Crosses, Genetic; Epitopes; Feedback, Physiological; gamma-Globulins; Gene Deletion; Gene Targeting; Humans; Immunoglobulin A; Immunoglobulin J-Chains; Immunoglobulin M; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitrophenols; Phenylacetates; Radiation Chimera

The expression of FcgammaR by human skin-derived mast cells of the MC(TC) type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of beta-hexosaminidase, PGD(2), leukotriene C(4) (LTC(4)), IL-5, IL-6, IL-13, GM-CSF, and TNF-alpha. FcgammaRIIa was consistently detected along with FcepsilonRI at the mRNA and protein levels; FcgammaRIIc was sometimes detected only by RT-PCR; but FcgammaRIIb, FcgammaRI, and FcgammaRIII mRNA and protein were not detected. FcgammaRIIa-specific mAb caused skin MC(TC) cells to degranulate and secrete PGD(2), LTC(4), GM-CSF, IL-5, IL-6, IL-13, and TNF-alpha in a dose-dependent fashion. FcepsilonRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC(4), which was not released by this agonist. Simultaneous but independent cross-linking of FcepsilonRI and FcgammaRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MC(TC) cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.

Topics: Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, CD; Cell Degranulation; Cells, Cultured; Cross-Linking Reagents; Gene Expression Regulation; Humans; Immunoglobulin E; Immunoglobulin G; Lung; Mast Cells; Nitrophenols; Phenylacetates; Receptor Aggregation; Receptors, IgG; RNA, Messenger; Serum Albumin, Bovine; Skin

Mathematical models have been used to study different aspects of the germinal centre reaction, in particular, affinity maturation of antibodies and the hypothesis of recycling. So far, interpretation of several theoretical and experimental results has pointed to the existence of recycling. However, theoretical models have seldom been compared with experimental data from specific immune responses and the potential relevance of recycling in the germinal centre is still an open problem. In this article, we propose a model without recycling that takes into account selection mechanisms that were previously uncovered experimentally. We apply the model to several experimental systems that use different Ag and compare the results with experimental data of affinity maturation whenever available. The results obtained for a primary immune response to the hapten (4-hydroxy-3-nitrophenyl)-acetyl show that recycling is not a necessary mechanism to achieve the level of affinity maturation observed in germinal centre reactions. Similar levels of affinity maturation are obtained for other responses, although for antibodies involving several affinity-enhancing mutations the affinity maturation obtained with the model is much lower. Interpretation of these results and consequences towards the concept of recycling are discussed.

Topics: Animals; Antibody Affinity; B-Lymphocytes; Germinal Center; Histocompatibility Antigens Class II; Models, Immunological; Nitrophenols; Phenylacetates

Previous studies examining the primary germinal center (GC) response to SRBC in mice demonstrated a steady ratio of IgM(+) to isotype-switched GC B cells and a persistent population of GC B cells with a founder phenotype. These characteristics held true at the inductive, plateau, and dissociative phases of the GC response, suggesting a steady-state environment. To test whether these characteristics apply to the primary response of other T cell-dependent Ags, the present study examined the GC response after challenge with (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Multiparameter flow cytometric analysis was used to assess the phenotype of splenic NP-reactive cells at multiple time points after immunization. Results of these studies demonstrated the characteristics of the SRBC-induced GC reaction to be fully maintained in the NP response. In particular, there was a steady ratio of nonswitched to switched B cells, with the majority of NP-reactive GC B cells displaying IgM. In addition, a substantial frequency of B220(-) NP-binding cells was observed in the spleen at later time points after NP challenge. Although these cells were IgE(+), they were found to express both kappa and lambda L chains and display the high-affinity IgE Fc (FcepsilonRI) receptor, suggesting that this population is not of B cell origin. Adoptive transfer studies further demonstrated the B220(-) NP-binding subset to be derived from the myeloid lineage.

Topics: Animals; B-Lymphocyte Subsets; Epitopes, B-Lymphocyte; Female; Germinal Center; Haptens; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Protein Binding

Germinal centers (GCs) have been identified as site at which the somatic mutation of immunoglobulins occurs.However, somatic mutations in immunoglobulins have also been observed in animals that normally do not harbor germinal centers. This clearly indicates that somatic mutations can occur in the absence of germinal centers. We therefore attempted to determine whether or not GCs exist in TNFR1-deficient mice, and are essential for the somatic mutation of immunoglobulins, using (4-hydroxy-3-nitropheny)acetyl-ovalbumin (NP-OVA). Both wild-type and TNFR1-deficient mice were immunized with NPOVA, and then examined with regard to the existence of GCs. No typical B-cell follicles were detected in the TNFR1-deficient mice. Cell proliferation was detected throughout all splenic tissue types, and no in vivo immunecomplex retention was observed in the TNFR1-deficient mice. All of these data strongly suggest that no GCs were formed in the TNFR1-deficient mice. Although TNFR1-deficient mice are unable to form GCs, serological analyses indicated that affinity maturation had been achieved in both the wild-type and TNFR1-deficient mice. We therefore isolated and sequenced several DNA clones from wild-type and the TNFR1-deficient mice. Eight out of 12 wild-type clones, and 11 out of 14 clones of the TNFR-1-deficient mice contained mutations at the CDR1 site. Thus, the wild-type and TNFR1-deficient mice were not extremely different with regard to types and rates of somatic mutation. Also, high-affinity antibodies were detected in both types of mice. Collectively, our data appear to show that affinity maturation may occur in TNFR1-deficient mice, which completely lack GCs.

Topics: Animals; Antibody Affinity; B-Lymphocytes; Base Sequence; Complementarity Determining Regions; Enzyme-Linked Immunosorbent Assay; Germinal Center; Immunoglobulin Class Switching; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Ovalbumin; Phenylacetates; Receptors, Tumor Necrosis Factor, Type I; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Somatic Hypermutation, Immunoglobulin; Spleen; T-Lymphocytes

Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.

Topics: Animals; Antibodies, Monoclonal; B-Lymphocytes; Cell Line; Chickens; Cytidine Deaminase; Nitrophenols; Phenylacetates; Serum Albumin, Bovine; Somatic Hypermutation, Immunoglobulin

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

Topics: Animals; Autoantibodies; B-Lymphocytes; Bone Marrow Cells; Cell Differentiation; Cell Movement; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunophenotyping; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred MRL lpr; Mice, Knockout; Mice, Mutant Strains; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Signal Transduction; Spleen

Immunization with a T cell-dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated V(H) genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1-expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220(+) memory B cells, and this is sufficient to account for the process of affinity maturation.

Topics: Adoptive Transfer; Animals; Antigens, CD19; B-Lymphocyte Subsets; Bone Marrow Cells; Chemokine CXCL13; Chemokines, CXC; Flow Cytometry; Germinal Center; Haptens; Hemocyanins; Immunization; Immunoglobulin G; Immunoglobulin Variable Region; Immunologic Memory; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Nitrophenols; Phenylacetates; Plasma Cells; Positive Regulatory Domain I-Binding Factor 1; Repressor Proteins; Spleen; Transcription Factors

The IGF-1 receptor (IGF-1R) is expressed on T and B lymphocytes, and the expression of the insulin- and IGF-1-signaling machinery undergoes defined changes throughout lineage differentiation, offering a putative role for IGF-1 in the regulation of immune responses. To study the role of the IGF-1R in lymphocyte differentiation and function in vivo, we have reconstituted immunodeficient RAG2-deficient mice with IGF-1R(-/-) fetal liver cells. Despite the absence of IGF-1Rs, the development and ex vivo activation of B and T lymphocytes were unaltered in these chimeric mice. By contrast, the humoral immune response to the T cell-independent type 2 Ag 4-hydroxy-3-nitrophenyl acetyl-Ficoll was significantly reduced in mice reconstituted with IGF-1R-deficient fetal liver cells, whereas responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenyl acetyl-chicken globulin were normal. Moreover, in an in vitro model of T cell-independent type 2 responses, IGF-1 promoted Ig production potently upon polyvalent membrane-IgD cross-linking. These data indicate that functional IGF-1R signaling is required for T cell-independent B cell responses in vivo, defining a novel regulatory mechanism for the immune response against bacterial polysaccharides.

Topics: Animals; Antigens, T-Independent; B-Lymphocyte Subsets; Cell Differentiation; Down-Regulation; Fetal Tissue Transplantation; Ficoll; Immunoglobulins; Insulin-Like Growth Factor I; Liver Transplantation; Lymphocyte Activation; Lymphopoiesis; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Phosphorylation; Phosphotyrosine; Radiation Chimera; Receptor, IGF Type 1; Signal Transduction; T-Lymphocytes; Up-Regulation

During humoral immune responses, naive B cells differentiate into Ab-secreting plasma cells within secondary lymphoid organs. Differentiating plasma cells egress from their sites of generation and redistribute to other tissues, predominantly the bone marrow and mucosal tissues. In this study, we demonstrate that within peripheral lymph nodes newly generated plasma cells localize to medullary cords which express the beta(2) integrin ligand ICAM-1. In beta(2) integrin-deficient mice plasma cells accumulate inside the lymph nodes, resulting in severely reduced plasma cell numbers in the bone marrow. Since plasma cells isolated from beta(2) integrin-deficient animals migrate efficiently into the bone marrow when transferred i.v., our findings provide profound evidence that beta(2) integrins are required for the egress of plasma cells from peripheral lymph nodes.

Topics: Adoptive Transfer; Animals; Bone Marrow Cells; CD18 Antigens; Cell Differentiation; Cell Movement; Cholera Toxin; Haptens; Intercellular Adhesion Molecule-1; Lymph Nodes; Lymphocyte Count; Mesentery; Mice; Mice, Inbred C57BL; Mice, Knockout; Neck; Nitrophenols; Phenylacetates; Plasma Cells

We prepared IgG and IgM with identical combining sites to a hapten, (4-hydroxy-3-nitrophenyl)acetic acid (NP), and used surface plasmon resonance to evaluate the association constants (Ka) in interactions of these antibodies (Abs) with antigens (Ags) which differed in the size of carriers and NP valence as well as in the stoichiometry of Ag to Ab in the immune complexes. It was found that IgM was unable to form an Ag1Ab1 complex with the highly haptenated Ag, NP(18.6)-bovine serum albumin (BSA), such that one NP(18.6)-BSA molecule was held by multiple contacts with Fab arms from five subunits, although IgM was capable of forming an Ag4Ab1 complex in which each subunit was bound to one NP(18.6)-BSA molecule. IgM was superior to IgG in interactions with large Ags of low hapten density. The Ka values of IgM to these Ags were estimated to be approximately 1x10(9) M(-1), about 20-fold higher than those of IgG. Reduction of inter-subunit and inter-chain disulfide bonds resulted in a decrease in Ka values to large Ags but no change in those to small Ags.

Topics: Animals; Antigens; Chromatography, Gel; Chromatography, High Pressure Liquid; Circular Dichroism; Haptens; Immunoglobulin M; Mice; Nitrophenols; Phenylacetates; Protein Binding

The deficiency in generating high-affinity antibodies due to impaired somatic hypermutation of Ig genes in the germinal center (GC) is considered the major mechanism responsible for the compromised humoral responses in aging. Since the intrinsic capability of aged B lymphocytes to respond to initial antigenic stimuli is largely intact and the expression of activation-induced cytidine deaminase, a key component required for Ig somatic hypermutation, is comparable between B cells from aged and young mice, it is possible to restore the age-related deficiency in the humoral response by circumventing the requirement for signals from other immune components. Here, we show that GC B cells from aged mice during a memory response carried mutated Ig genes with mutational frequencies comparable to that of GC B cells from young mice. Additionally, characterization of mutations in VDJ segments, and analysis of antibody-forming cells and antibodies demonstrated that the processes of antigen-driven clonal selection and affinity maturation are largely intact in aged animals. Thus, we conclude that the diminished antibody responses in aged animals may be significantly improved by repeated immunizations. These findings may have important implications in designing vaccines and immunization protocols for the elderly population and patients with certain immune deficiencies such as AIDS.

Topics: Age Factors; Aging; Animals; Antibody Affinity; Antibody-Producing Cells; B-Lymphocytes; Base Sequence; Bone Marrow; Bone Marrow Cells; Cell Count; Complementarity Determining Regions; Cytidine Deaminase; DNA; Gene Expression; Gene Rearrangement, B-Lymphocyte; Genes, Immunoglobulin; Immunogenetics; Immunoglobulin G; Immunoglobulin Variable Region; Immunologic Memory; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Phenylacetates; Sequence Analysis, DNA; Spleen; Vaccination

We prepared a series of hapten-BSA conjugates with varying ratios of biotin to measure ligand-receptor interactions on B cells by flow cytometry using avidin for detection. Surface plasmon resonance measurements of the interaction with a monoclonal anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody suggested that NP(5)-BSA or NP(7)-BSA harboring 29 or 23 biotin molecules (NP(5)-BSA-bio(29) or NP(7)-BSA-bio(23)) would be suitably sensitive for flow cytometric analysis. By using NP-BSA-bio, we analyzed NP-binding cells in immunized mice. Unexpectedly, 30-40% of spleen cells expressing IgM could bind to NP(5)-BSA or NP(7)-BSA after immunization of mice with NP(40)-chicken gamma-globulin. The proteins binding to NP(7)-BSA-bio(23) on the cell surface were analyzed by immunoprecipitation and western blotting. Surprisingly, most of the proteins binding NP-BSA-bio on the cell surface were not the membrane form of IgM monomer, but a secreted IgM pentamer. It is likely that the IgM pentamer bound through Fc receptors for polymeric IgA or IgM and contributed to antigen binding. Comparison of the binding ratio of NP(0.9)-BSA:NP(5)-BSA between B cells of primary and secondary immunization suggested that the affinity of IgM matured during immunization.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Avidin; B-Lymphocytes; Biotin; Cell Line; Flow Cytometry; gamma-Globulins; Gene Expression; Immunization; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunoglobulin M; Mice; Mice, Inbred A; Nitrophenols; Phenylacetates; Protein Binding; Receptors, Fc; Serum Albumin, Bovine; Spleen

This chapter describes the analysis of long- and short-lived plasma cells on tissue sections. Mice are immunized with 4-hydroxy-3-nitrophenyl acetyl (NP) coupled to a T-cell-dependent carrier. Antigen-specific germinal center cells and extrafollicular plasma cells are identified by immunohistology on tissue sections. Plasma cells labeled with 5-bromo-2'-deoxyuridine (BrdU) pulses given during different phases of B-cell response are identified on spleen sections. To identify mutated cells originating from cells in germinal centers, antigen-specific cells from spleen sections are isolated by microdissection. From these NP-specific recombined VDJ genes are amplified with family-specific primers by polymerase chain reaction (PCR) for deoxyribonucleic acid (DNA) sequencing. The methods described can be used to characterize origins and life-span of plasma cells in vivo.

Topics: Animals; B-Lymphocytes; Bromodeoxyuridine; Cell Differentiation; Chickens; Germinal Center; Immunization; Mice; Microdissection; Mutation; Nitrophenols; Phenylacetates; Plasma Cells; Polymerase Chain Reaction; Receptors, Antigen, B-Cell; Sheep; Spleen; T-Lymphocytes, Helper-Inducer

With increasing age, the ability to produce protective antibodies in response to immunization declines, leading to a reduced efficacy of vaccination in the elderly. To examine the effect of age on the cognate function of CD4 T cells, we have used a novel adoptive transfer model that allows us to compare identical numbers of antigen-specific naive T cells from young and aged TCR transgenic (Tg) donors. Upon transfer of aged donor CD4 T cells to young hosts, there was significantly reduced expansion and germinal center (GC) differentiation of the antigen-specific B cell population after immunization. This reduced cognate helper function was seen at all time points and over a wide range of donor cell numbers. In hosts receiving aged CD4 cells, there were also dramatically lower levels of antigen-specific IgG. These age-related defects were not due to defects in migration of the aged CD4 T cells, but may be attributable to reduced CD154 (CD40L) expression. Furthermore, we found that there was no difference in B cell expansion and differentiation or in IgG production when young CD4 T cells were transferred to young or aged hosts. Our results show that, in this model, age-related reductions in the cognate helper function of CD4 T cells contribute significantly to defects in humoral responses observed in aged individuals.

Topics: Aging; Animals; Antibody Formation; Antigens, CD; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Movement; Gene Expression Regulation; Germinal Center; Immunoglobulin G; Mice; Mice, Knockout; Mice, Transgenic; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell

We determined thermodynamic and kinetic parameters for the antigen-antibody interaction using a group of anti-(4-hydroxy-3-nitrophenyl)acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. These monoclonal antibodies were considered to have originated from a common ancestor clone and to represent progression along the affinity maturation pathway. The kinetic measurements showed that both association and dissociation rate constants of the antigen-antibody interaction decreased during maturation. Thermodynamic measurements revealed that an increase in affinity was obtained by an increase in entropy without any significant change in enthalpy. These results suggested that the mechanism for the antigen-antibody interaction shifted from a "zipper" type to a "lock-and-key" type during antibody evolution.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Circular Dichroism; Evolution, Molecular; Haptens; Kinetics; Mice; Models, Immunological; Molecular Sequence Data; Nitrophenols; Phenylacetates; Sequence Homology, Amino Acid; Somatic Hypermutation, Immunoglobulin; Surface Plasmon Resonance; Thermodynamics

We have developed a simple method using flow cytometry to estimate the relative affinity of B cell receptor (BCR) possessing the hapten-binding activity. Bovine serum albumin (BSA) was conjugated with a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP) and biotin (NP-BSA-bio). The interaction between NP-BSA-bio and anti-NP monoclonal antibodies (mAb) was studied as a model of the BCR reaction by surface plasmon resonance (SPR) using a biosensor chip immobilized with mAbs through anti-Fc antibody (Ab). The relative affinity of these mAbs was estimated on the basis of resonance units for the binding of NP(0.5)-BSA-bio(21) relative to that of NP(7.4)-BSA-bio(21) expressed as a ratio (NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21)). In combination with streptavidin (SA)-R-phycoerythrin (PE), we measured the binding of NP-BSA-bio to BCR by flow cytometry and found that a high number of biotin molecules was necessary to improve the sensitivity of detection of the bound NP-BSA-bio without steric hindrance in the NP-BCR interaction. We demonstrated that the ratio of the mean fluorescence intensity (MFI) of NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21) at a concentration of 10(-8) M could be used as a practical measure of the affinity. This method is expected to be useful for the study of affinity maturation on the cellular level.

Topics: Antibodies, Monoclonal; Antibody Affinity; Biotin; Cell Membrane; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Haptens; Hybridomas; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Surface Plasmon Resonance; Tumor Cells, Cultured

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

Topics: Animals; Deuterium; Gas Chromatography-Mass Spectrometry; Humans; Injections, Intravenous; Lipopolysaccharides; Male; Models, Chemical; Nitrates; Nitrophenols; Nitrosation; Phenylacetates; Rats; Rats, Sprague-Dawley; Reference Standards; Tyrosine

A small number of key somatic mutations lead to high-affinity binding in the anti-hapten immune responses to 2-phenyl-5-oxazolone (phOx) and (4-hydroxy-3-nitrophenyl)acetyl (NP). Affinity maturation models of the germinal center hold that B cells carrying these key mutations are preferentially selected for expansion within the germinal centers. However, additional factors are required to account for some quantitative aspects of affinity maturation in vivo. Radmacher et al. have shown that key mutants are observed in vivo significantly less frequently than expected by these models. To account for this finding, they propose that selection is a stochastic process where key mutants may be overlooked by positive selection or recruited out of the germinal center. While acknowledging that a minimal amount of stochastic selection is probably unavoidable in the germinal center, we instead propose a structural explanation for this key mutant discrepancy. This model is based on the existence of a large number of blocking mutations whose presence can prevent the ability of key mutations to confer high-affinity binding. Using mathematical modeling and computer simulation, we show that in addition to reconciling the key mutant discrepancy, the blocking model accounts for other aspects of experimental data that are not predicted by the stochastic selection model. In particular, the blocking model is consistent with the observation that key mutants generally exhibit a higher number of mutations per sequence in the phOx response, but a lower number in the NP response.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; Base Sequence; Clone Cells; Computer Simulation; Germinal Center; Haptens; Models, Genetic; Models, Immunological; Molecular Sequence Data; Mutation; Nitrophenols; Oxazolone; Phenylacetates; Selection, Genetic; Stochastic Processes

Germinal centers (GCs) form in B cell follicles and require specific signals for development and maintenance. B cell-activating factor belonging to the TNF family (BAFF) is a fundamental B cell survival factor and therefore may influence GC reactions and subsequent Ab responses. To test this possibility, the effect of BAFF neutralization in immunized mice was assessed. Using B cell maturation Ag-Fc, we demonstrate that BAFF blockade does not inhibit GC formation or somatic hypermutation. However, GCs in B cell maturation Ag-Fc-treated mice dissipated more rapidly than those of control mice and did not form a mature follicular dendritic cell reticulum. Examination of immunized BAFF-null mice validated the BAFF-independent nature of GC formation. Furthermore, Ab responses, including high-affinity responses, were attenuated. This is the first evidence that BAFF is required for maintenance, but not initiation, of the GC reaction, and it further hints that somatic hypermutation within the GC and selection of Ag-specific high-affinity Ab could be uncoupled.

Topics: Animals; B-Cell Activating Factor; B-Cell Maturation Antigen; B-Lymphocyte Subsets; Cell Differentiation; Dendritic Cells, Follicular; Down-Regulation; Female; Gene Frequency; Germinal Center; Humans; Immunoglobulin G; Injections, Intraperitoneal; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Multigene Family; Nitrophenols; Phenylacetates; Receptors, Tumor Necrosis Factor; Somatic Hypermutation, Immunoglobulin; T-Lymphocytes; Tumor Necrosis Factor-alpha

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig micro H chain Ab and anti-Ig delta H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.

Topics: Animals; Antibodies, Anti-Idiotypic; Antigens; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; CD40 Antigens; Cell Adhesion Molecules; Cell Cycle; Cell Division; Cell Survival; Cells, Cultured; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Lectins; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Phosphorylation; Receptors, Antigen, B-Cell; Sialic Acid Binding Ig-like Lectin 2; Signal Transduction; Spleen

The paradigm of T helper-1 (Th-1) and Th-2 cells developing from non-committed naive precursors is firmly established. Th1 cells are characterized by IFNgamma production and, in mice, the selective switching to IgG2a. Conversely IL-4 production and selective switching to IgG1 and IgE characterize Th2 cells. Analysis of Th2 induction in vitro indicates that this polarization develops gradually in T cells activated by anti-CD3 in the presence of IL-4; conversely anti-CD3 and IFNgamma induce Th1 cells. In this report, we explore evidence that indicates that the T helper cell polarization in vivo cannot solely be explained by the cytokine environment. This is provided by studying the early acquisition of Th1 and Th2 activities during responses to a mixture of Th1 and Th2-inducing antigens. It is shown that these divergent forms of T cell help can rapidly develop in cells within a single lymph node. It is argued that early polarization to show Th-1 or Th-2 behavior can be induced by signals delivered during cognate interaction between virgin T cells and dendritic cells, in the absence of type 1 or type 2 cytokines. This contrasts with the critical role of the cytokines in reinforcing the Th-phenotype and selectively expanding T helper clones.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Bromodeoxyuridine; CD3 Complex; gamma-Globulins; Immunization; Immunoglobulin D; Immunoglobulin G; Interferon-gamma; Interleukin-4; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Nitrophenols; Pertussis Vaccine; Phenylacetates; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Th1 Cells; Th2 Cells

It has not been resolved whether gammadelta T cells can collaborate with germinal center B cells and support Ig hypermutation during an Ab response to a truly defined T-dependent Ag. In this study, we show that in the absence of alphabeta T cells, immunization with the well-defined T-dependent Ag, (4-hydroxy-3-nitrophenyl) acetyl (NP) conjugate, was able to induce Ig hypermutation. However, the clonotypes of B cells responding to NP were dramatically altered in TCR beta(-/-) mice. Unlike B cells in wild-type mice that use canonical VDJ rearrangements, most NP-responding B cells in mutant mice use analog genes of the J558 gene family. In addition, the majority of anti-NP Abs produced in mutant mice use kappaL chain instead of lambda1L chain, which dominates in mice of Igh(b) background. Thus, the B cell population that collaborates with gammadelta T cells is distinct from B cells interacting with conventional alphabeta Th cells.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; B-Lymphocyte Subsets; Cell Differentiation; Chickens; Clone Cells; Female; gamma-Globulins; Germinal Center; Haptens; Immunophenotyping; Lymphocyte Cooperation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Somatic Hypermutation, Immunoglobulin; T-Lymphocyte Subsets

Ligation of CD27 on B cells has been shown to inhibit terminal differentiation of activated murine B cells into plasma cells. We show in this study that this inhibition is accompanied by an enhanced movement of activated B cells toward differentiation into memory cells. Treatment of mice with anti-CD27 during immunization leads to the generation of greater numbers of Ag-binding B cells in draining lymph nodes that persist for longer periods of time, and they contain a greater proportion of cells of a postgerminal center phenotype. Limiting dilution analyses reveal that they contain a higher frequency of cells that can be stimulated to secrete specific IgG, and adoptive transfer experiments confirm that they can generate higher secondary responses in carrier-primed recipients. Remarkably, significant secondary responses are also seen following primary immunization with a T-independent Ag in the presence of anti-CD27, confirming that ligation of CD27 on B cells during priming induces differentiation into the memory lineage. Treatment with anti-CD27 during priming also increases the average affinity of the secondary response, suggesting that high affinity clones generated early in a primary response may normally differentiate preferentially into plasma cells and are rescued from this fate by CD27 ligation. Anti-CD40 treatment shows similar effects in vivo. However, unlike CD27, CD40 coligation also enhances proliferation, survival, and isotype switching of LPS-stimulated B cells, suggesting that the two receptors may enhance commitment to B cell memory by different mechanisms, or that a common mechanism is used through both receptors that does not involve cell cycle control or survival.

Topics: Adoptive Transfer; Animals; B-Lymphocyte Subsets; CD40 Antigens; Epitopes, B-Lymphocyte; gamma-Globulins; Immune Sera; Immunization, Secondary; Immunologic Memory; Lymphocyte Activation; Lymphocyte Count; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Plasma Cells; Protein Binding; Signal Transduction; Tumor Necrosis Factor Receptor Superfamily, Member 7; Up-Regulation; Vaccination

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-gamma as they begin to proliferate. Parallel respective induction of B cells to produce gamma1 or gamma2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and gamma1 and epsilon switch transcript production were underway 48-72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The gamma1 and epsilon switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.

Topics: Animals; B-Lymphocytes; Cell Differentiation; Chickens; Epitopes, T-Lymphocyte; gamma-Globulins; Haptens; Immunization; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulins; Injections, Subcutaneous; Interleukin-13; Interleukin-4; Lymphocyte Activation; Lymphocyte Cooperation; Lymphopenia; Mice; Mice, Inbred BALB C; Mice, Knockout; Nitrophenols; Phenylacetates; Plasma Cells; RNA, Messenger; Th1 Cells; Th2 Cells; Up-Regulation

The primary burst of Ab and germinal center (GC) formation in response to T-dependent Ag is compromised in aging mice. Here we examine the effects of aging on the post-GC phase of memory B cell differentiation and the late Ab repertoire maturation in bone marrow (BM) in mice immunized with a hapten nitrophenyl coupled to chicken gamma-globulin. Specific Ab-forming cells (AFC) with mutated V(H) genes accumulated preferentially in the BM of aged mice, although the AFC numbers and average number of mutations per V(H) were lower, and the D gene usage was less restricted compared with those in the young animals. However, the repertoire of AFC after an Ag boost demonstrated the hallmarks of Ag selection, including the recurrent mutations and canonical VD rearrangements, similar to the late primary response in young animals. It is postulated that the Ab repertoire maturation in aged mice is delayed and may be notably improved by repeated immunizations.

Topics: Aging; Animals; B-Lymphocytes; Bone Marrow Cells; Chickens; gamma-Globulins; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Germ-Line Mutation; Germinal Center; Haptens; Hemolytic Plaque Technique; Immunization, Secondary; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunologic Memory; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Sequence Analysis, DNA; Spleen

Mice with a disrupted C4 locus (C4(-/-)) have an impaired immune response to thymus-dependent Ags. To test the role of bone marrow-derived C4 in humoral immunity, we reconstituted deficient animals with wild-type bone marrow or an enriched fraction of bone marrow-derived macrophages. C4 chimeras were immunized with 4-hydroxy-3-nitrophenyl(5) conjugated to keyhole limpet hemocyanin (NP(5)- KLH) or infected with HSV-1, and the Ab response was evaluated. Wild-type bone marrow rescued the humoral immune response to both Ags, i.e., the soluble Ag and HSV-1, demonstrating that local C4 production is sufficient for humoral responses. Although the C4 chimeric animals lacked detectable C4 in their sera, C4 mRNA was identified in splenic sections by in situ hybridization, and C4 protein deposits were identified in the germinal center areas of splenic follicles by immunofluorescence staining. Macrophages derived from bone marrow produced sufficient C4 protein to restore the humoral response to NP(5)-KLH in C4-deficient animals when administered along with Ag. Cell-sorting experiments, followed by C4-specific RT-PCR, identified splenic macrophages (CD11b(+), CD11c(-)) as a cellular source for C4 synthesis within the spleen.

Topics: Adoptive Transfer; Animals; Antibody Formation; Bone Marrow Transplantation; Cells, Cultured; Complement C4; Haptens; Hemocyanins; Immunoglobulin G; Immunologic Deficiency Syndromes; Injections, Intravenous; Macrophages; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitrophenols; Phenylacetates; Radiation Chimera; Spleen

Apoptosis constitutes the primary mechanism by which noncycling plasma cells are eliminated after the secretion of Ag-specific Abs in a humoral immune response. The underlying mechanism is not known. Here, we demonstrate that the expression of both TRAIL/Apo-2 ligand and the death receptors (DR) DR5 and DR4, but not Fas, are sustained in IL-6-differentiated Ig-secreting human plasma cells as well as primary mouse plasma cells generated in a T-dependent immune response. Plasma cell apoptosis is induced by both endogenous and exogenous TRAIL ex vivo, suggesting that TRAIL-mediated killing may, in part, be plasma cell autonomous. By contrast, resting and activated B cells are resistant to TRAIL killing despite comparable expression of TRAIL and DRs. The preferential killing of plasma cells by TRAIL correlates with decreased expression of CD40 and inactivation of NF-kappaB. These results provide the first evidence that primary plasma cells synthesize TRAIL and are direct targets of TRAIL-mediated apoptosis, which may relate to the inactivation of the NF-kappaB survival pathway.

Topics: 3T3 Cells; Animals; Antigens, Differentiation, T-Lymphocyte; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocyte Subsets; Caspases; CD40 Antigens; Cell Differentiation; Cell Line, Transformed; Cells, Cultured; Coculture Techniques; Down-Regulation; fas Receptor; gamma-Globulins; Haptens; Humans; Interleukin-6; Jurkat Cells; Ligands; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; NF-kappa B; Nitrophenols; Phenylacetates; Plasma Cells; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Due to characteristic clinical signs, immunoglobulins of isotype E (IgE) are believed to be involved in several allergic diseases of the horse. To date, closer investigations have been hampered by the fact that neither purified equine IgE nor anti-equine IgE monoclonal antibodies were available for IgE isotype determination. As an approach to solve this problem, we constructed a stable cell line (EqE6) that expresses recombinant equi-murine IgE specific for 4-(hydroxy-3-nitro-phenyl) acetyl (NP). Biochemical analysis of the purified protein revealed a highly glycosilated IgE monomer of approximately 230,000 Da. The biological ability of the NP-IgE to mediate histamine release after crosslinking with antigen was demonstrated in vitro using equine blood leucocytes. In vivo, the intradermal application of NP-IgE followed by antigen crosslinking induced a type I hypersensitivity skin reaction in horses. Both results indicate that the recombinant NP-IgE contains an intact and functional Fc(epsilon) RI binding site and mediates effector functions in equine basophils and cutaneous mast cells. This equi-murine IgE can be used for the production of IgE-specific polyclonal and monoclonal antibodies. In addition, the NP specificity allows the antigen-specific activation of equine Fc(epsilon)-receptor-expressing cells, such as mast cells and basophils. This property could be used to investigate IgE-mediated mechanisms for a better understanding of equine type I allergic diseases.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Basophils; Blotting, Western; Cell Line; Electrophoresis, Polyacrylamide Gel; Female; Histamine Release; Horse Diseases; Horses; Hypersensitivity, Immediate; Immunoglobulin E; Male; Mast Cells; Molecular Sequence Data; Nitrophenols; Phenylacetates; Polymerase Chain Reaction; Receptors, IgE; Sequence Homology, Amino Acid; Species Specificity

The quasi-monoclonal mouse has limited B cell diversity, whose major (approximately 80%) B cell Ag receptors are comprised of the knockin V(H) 17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of V(H)T (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated V(H)T-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of V(H)T/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that V(H)T/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than V(H)T/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody Diversity; Antibody Specificity; B-Lymphocyte Subsets; Base Sequence; Cell Differentiation; Cells, Cultured; Clone Cells; Epitopes, B-Lymphocyte; Haptens; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Mice; Mice, Knockout; Molecular Sequence Data; Nitrophenols; Phenylacetates; Point Mutation; Receptors, Antigen, B-Cell

Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed. The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the lambda1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by lambda1 positive antibodies but fail to produce high affinity NP-binding IgG1 antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.

Topics: Animals; Antibodies; Antibody Affinity; B-Lymphocytes; Germinal Center; Haptens; Immunization; Immunoglobulin G; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Radiation Chimera; Spleen; T-Lymphocytes

Exceptionally germinal center formation can be induced without T cell help by polysaccharide-based antigens, but these germinal centers involute by massive B cell apoptosis at the time centrocyte selection starts. This study investigates whether B cells in germinal centers induced by the T cell-independent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to Ficoll undergo hypermutation in their immunoglobulin V region genes. Positive controls are provided by comparing germinal centers at the same stage of development in carrier-primed mice immunized with a T cell-dependent antigen: NP protein conjugate. False positive results from background germinal centers and false negatives from non-B cells in germinal centers were avoided by transferring B cells with a transgenic B cell receptor into congenic controls not carrying the transgene. By 4 d after immunization, hypermutation was well advanced in the T cell-dependent germinal centers. By contrast, the mutation rate for T cell-independent germinal centers was low, but significantly higher than in NP-specific B cells from nonimmunized transgenic mice. Interestingly, a similar rate of mutation was seen in extrafollicular plasma cells at this stage. It is concluded that efficient activation of hypermutation depends on interaction with T cells, but some hypermutation may be induced without such signals, even outside germinal centers.

Topics: Adoptive Transfer; Animals; Animals, Congenic; Antigens, T-Independent; B-Lymphocytes; Base Sequence; DNA; Germinal Center; Immunization; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Signal Transduction; T-Lymphocytes

To examine how B cell receptor affinity affects clonal selection in thymus-independent type 2 (TI-2) immune responses, we produced mice with antibodies that showed a 40-fold difference in affinity for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). The difference in the responses of high- and low-affinity B cells to NP-Ficoll was only twofold. However, in competition experiments only the high-affinity B cells responded to antigen. CD19 deficiency increased the affinity threshold of TI-2 responses, whereas Lyn deficiency enhanced clonal expansion but abrogated B cell terminal differentiation. Thus, in TI-2 immune responses, large differences in affinity produce only small differences in the intrinsic ability of B cells to respond to antigen, and selection for high-affinity clones is due to clonal competition during the earliest stages of the response.

Topics: Animals; Antigens, CD; Antigens, CD19; Antigens, Differentiation, B-Lymphocyte; Apoptosis; B-Lymphocytes; Binding, Competitive; Cell Adhesion Molecules; Cell Division; Gene Targeting; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Lectins; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Sialic Acid Binding Ig-like Lectin 2; src-Family Kinases; T-Lymphocytes

Human saliva contained 4-hydroxyphenylacetic acid (HPA) (2-10 microM) and nitrite (60-300 microM). HPA was nitrated to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA) when HPA and sodium nitrite were mixed at pH 1.0. NO2HPA was also formed when saliva was incubated under acidic conditions. These results suggest that salivary HPA is nitrated to NO2HPA when saliva is swallowed into the stomach.

Topics: Gastric Mucosa; Humans; Hydrogen-Ion Concentration; Nitrites; Nitrophenols; Phenylacetates; Saliva; Sodium Nitrite

In chickens, a single set of unique functional segments of both Ig H and L chain genes is rearranged during early embryogenesis to generate a pool of B cell progenitors that will be diversified in the bursa by gene conversion, forming the preimmune repertoire. After hatching, bursal cells are exposed to environmental Ags in the bursal lumen. We prepared B cells from each single bursal follicle and used PCR-directed Ig L chain gene analysis to study the differentiation of B cells and the effect of antigenic stimulation from the bursal lumen on the neonatal chicken B cell repertoire formation. Selective amplification of B cell clones with a productive V-J joint was observed during the late embryonic stage, possibly by the interaction with ligands expressed on the bursal stroma and further accelerated in the neonatal chicken. Administration of the artificial Ags into the bursal lumen before the isolation of bursa by bursal duct ligation in the embryo caused a significant increase in lymphocytes with a productive V-J joint in the neonatal chicken bursa compared with the isolated bursa. Intra- and interclonal diversity of a complementarity-determining region measured by an evolutionary distance increased during bursal development. Clonal diversification did not require stimulation by artificial Ags from the bursal lumen. Thus, the preimmune repertoire in the bursa is generated by gene conversion during Ag-independent B cell proliferation, and antigenic stimulation from the bursal epithelium to bursal B cells plays roles in the selection of clones with a productive V-J joint.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Antibody Diversity; B-Lymphocyte Subsets; Base Sequence; Bursa of Fabricius; Cell Differentiation; Chickens; Clone Cells; Cloning, Molecular; Complementarity Determining Regions; Evolution, Molecular; Gene Rearrangement, B-Lymphocyte, Light Chain; Germ-Line Mutation; Immunoglobulin Joining Region; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Molecular Sequence Data; Nitrophenols; Phenylacetates

Exposure to airborne pollutants such as tobacco smoke is associated with increased activation of inflammatory-immune processes and is thought to contribute to the incidence of respiratory tract disease. We hypothezised that cigarette smoke (CS) could synergize with activated inflammatory/immune cells to cause oxidative injury or result in the formation of unique reactive oxidants. Isolated human neutrophils were exposed to gas-phase CS, and the production of nitrating and chlorinating oxidants following neutrophil stimulation was monitored using the substrate 4-hydroxyphenylacetate (HPA). Stimulation of neutrophils in the presence of CS resulted in a reduced oxidation and chlorination of HPA, suggesting inhibition of NADPH oxidase or myeloperoxidase (MPO), the two major enzymes involved in inflammatory oxidant formation. Peroxidase assays demonstrated that neutrophil MPO activity was not significantly affected after CS-exposure, leaving the NADPH oxidase as a likely target. The inhibition of neutrophil oxidant formation was found to coincide with depletion of cellular GSH, and a similar modification of critical cysteine residues, such as those in NADPH oxidase components, might be involved in reduced respiratory burst activity. As alpha,beta-unsaturated aldehydes such as acrolein have been implicated in thiol modifications by CS, we exposed neutrophils to acrolein prior to stimulation, and observed inhibition of NADPH oxidase activation in relation to GSH depletion. Additionally, translocation of the cytosolic components of NADPH oxidase to the membrane, a necessary requirement for enzyme activation, was inhibited. Protein adducts of acrolein (or related aldehydes) could be detected in several neutrophil proteins, including NADPH oxidase components, following neutrophil exposure to either CS or acrolein. Alterations in neutrophil function by exposure to (environmental) tobacco smoke may affect inflammatory/infectious conditions and thereby contribute to tobacco-related disease.

Topics: Acrolein; Cells, Cultured; Chromatography, High Pressure Liquid; Drug Interactions; Glutathione; Humans; NADPH Oxidases; Neutrophils; Nitrophenols; Oxidation-Reduction; Peroxidase; Phenylacetates; Respiratory Burst; Smoke

The germinal center reaction (GCR) of vertebrate immunity provides a remarkable example of evolutionary succession, in which an advantageous phenotype arises as a spontaneous mutation from the parental type and eventually displaces the parental type altogether. In the case of the immune response to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), as with several other designed immunogens, the process is dominated by a single key mutation, which greatly simplifies the modeling of and analysis of data. We developed a two-stage model of this process in which the primary stage represents the appearance and establishment of the mutant population as a stochastic process while the second stage represents the growth and dominance of the clone as a deterministic process, conditional on its time of establishment from stage one. We applied this model to the analysis of population samples from several germinal center (GC) reactions and used maximum-likelihood methods to estimate the waiting times to arrival and to dominance of the mutant clone. We determined the sampling properties of the maximum-likelihood estimates using Monte Carlo methods and compared them to their asymptotic distributions. The methods we present here are well-suited for use in the analysis of other systems, such as tumor growth and the experimental evolution of bacteria.

Topics: Animals; Antibody Formation; B-Lymphocytes; Computer Simulation; Genes, Dominant; Germinal Center; Likelihood Functions; Models, Genetic; Monte Carlo Method; Mutation; Nitrophenols; Phenylacetates; Selection, Genetic; Stochastic Processes; Vertebrates

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens; Basophils; beta-N-Acetylhexosaminidases; Binding Sites, Antibody; Binding, Competitive; Calcium; Cell Membrane; CHO Cells; Cricetinae; Epitopes; Histamine Antagonists; Histamine Release; Humans; Immunoglobulin E; Immunoglobulin Fab Fragments; Intracellular Fluid; Kinetics; Leukocytes, Mononuclear; Leukotriene Antagonists; Leukotrienes; Mice; Molecular Sequence Data; Nitrophenols; Peptide Fragments; Phenylacetates; Rats; Receptors, IgE; Recombinant Proteins; Transfection; Tumor Cells, Cultured

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.

Topics: Amino Acid Sequence; Animals; B-Lymphocyte Subsets; Base Sequence; Bone Marrow Cells; Cell Differentiation; Cell Survival; Epitopes, B-Lymphocyte; Female; Germinal Center; Haptens; Immunoglobulin E; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunologic Memory; Immunophenotyping; Leukocyte Common Antigens; Macrophage-1 Antigen; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Spleen

H2-DM (DM, previously H2-M) facilitates the exchange of peptides bound to MHC class II molecules. In this study, we have used H2-DM-deficient (DM(-/-)) mice to analyze the influence of DM in the priming of B cell responses in vivo and for Ag presentation by B cells in vitro. After immunization, IgG Abs could be raised to a T-dependent Ag, 4-hydroxy-5-nitrophenylacetyl-OVA, in DM(-/-) mice, but closer analysis revealed the IgG response to be slower, diminished in titer, and composed of low-affinity Abs. The Ab response correlated with a vast reduction in the number of germinal centers in the spleen. The presentation of multiple epitopes by H2-A(b) from distinct Ags was found to be almost exclusively DM-dependent whether B cells internalized Ags via fluid phase uptake or using membrane Ig receptors. The poor B cell response in vivo could be largely, but not completely restored by expression of a H2-Ea(d) transgene, despite the fact that Ag presentation by H2-E(d/b) molecules was found to be highly DM dependent. Hence, while substantial Ab responses can be raised in the absence of DM, this molecule is a crucial factor both for Ag processing and for the normal maturation of T-dependent humoral immune responses in vivo.

Topics: Animals; Antibody Affinity; Antibody Formation; Antigen Presentation; B-Lymphocyte Subsets; Germinal Center; H-2 Antigens; Haptens; Hemocyanins; Histocompatibility Antigens Class II; HLA-D Antigens; Hybridomas; Immunoglobulin G; Injections, Intraperitoneal; Mice; Mice, Knockout; Mice, Transgenic; Muramidase; Nitrophenols; Ovalbumin; Peptide Fragments; Phenylacetates; Receptors, Fc; T-Lymphocytes; Transgenes

Mucosal immunoglobulin (Ig)A dominance has been proposed to be associated with preferential class switch recombination (CSR) to the IgA heavy chain constant region, Calpha. Here, we report that B cell activation in nasal-associated lymphoid tissue (NALT) upon stimulation with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gamma globulin caused an anti-NP memory response dominated by high affinity IgA antibodies. In the response, however, NP-specific IgG(+) B cells expanded and sustained their number as a major population in germinal centers (GCs), supporting the view that CSR to IgG heavy chain constant region, Cgamma, operated efficiently in NALT. Both IgG(+) and IgA(+) GC B cells accumulated somatic mutations, indicative of affinity maturation to a similar extent, suggesting that both types of cell were equally selected by antigen. Despite the selection in GCs, high affinity NP-specific B cells were barely detected in the IgG memory compartment, whereas such cells dominated the IgA memory compartment. Taken together with the analysis of the V(H) gene clonotype in GC and memory B cells, we propose that NALT is equipped with a unique machinery providing IgA-specific enrichment of high affinity cells into the memory compartment, facilitating immunity with high affinity and noninflammatory secretory antibodies.

Topics: Administration, Intranasal; Animals; Antibody Affinity; Antigens; B-Lymphocytes; Cells, Cultured; Chemotaxis, Leukocyte; Germinal Center; Haptens; Immunoglobulin A; Immunoglobulin G; Immunoglobulin Isotypes; Immunologic Memory; Injections, Intraperitoneal; Lymph Nodes; Lymphoid Tissue; Mice; Mice, Inbred C57BL; Mice, Knockout; Nasal Cavity; Nitrophenols; Phenylacetates

Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.

Topics: Animals; Antigens, CD; B-Lymphocytes; B7-1 Antigen; B7-2 Antigen; CD40 Antigens; Ficoll; Germinal Center; Haptens; Membrane Glycoproteins; Mice; Mice, Nude; Mice, Transgenic; Nitrophenols; Peanut Agglutinin; Phenylacetates; Signal Transduction; Spleen; T-Lymphocytes

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; Antibody-Producing Cells; Base Sequence; Binding Sites, Antibody; Bone Marrow Cells; Chickens; Dose-Response Relationship, Immunologic; Female; gamma-Globulins; Germinal Center; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Variable Region; Lymphocyte Count; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Receptors, Complement 3b; Receptors, Complement 3d

Cr2-/- mice have an impairment in humoral immunity, as shown by the decrease in the Ab titers against T cell-dependent Ags and abnormalities in germinal center formation. Germinal centers are present, but they are decreased in size and number, indicating problems in their development. In this study, we investigated whether this abnormality in germinal center development is associated with problems in the establishment of optimal affinity maturation and the generation of memory B cells, processes closely related to the germinal center reaction. We immunized the Cr2-/- animals with different Ags with or without adjuvants. We showed that, when immunized without adjuvants, complement receptors are absolutely required for optimal affinity maturation. Although limited affinity maturation is elicited in the Cr2-/- Ab response, it is decreased as compared with normal animals. Memory B cell generation is also impaired. In the presence of adjuvants, germinal center development in the Cr2-/- mice is still abnormal, as demonstrated by their decreased size and number. Surprisingly, adjuvants establish optimal affinity maturation and partially restore the amount of Ab produced during the primary response and memory B cell generation. However, adjuvants cannot improve the ability of follicular dendritic cells to retain Ags in the form of immune complexes. These observations indicate that immunization with inflammatory Ags offset some of the immunological abnormalities found in the Cr2-/- mice and show that optimal affinity maturation in the Cr2-/- mice can be achieved in the absence of normal germinal centers.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Affinity; Antigens; B-Lymphocyte Subsets; Base Sequence; Cell Differentiation; Dendritic Cells, Follicular; Germinal Center; Haptens; Hemocyanins; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunologic Deficiency Syndromes; Immunologic Memory; Injections, Intraperitoneal; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Receptors, Complement 3d; Spleen

C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.

Topics: Animals; Apoptosis; B-Lymphocytes; Base Sequence; DNA, Complementary; fas Receptor; Germinal Center; Haptens; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Molecular Sequence Data; Nitrophenols; Phenylacetates

Serum antibody (Ab) can play several roles during B cell immune responses. Among these is to promote the deposition of immune complexes (ICs) on follicular dendritic cells (FDCs). ICs on FDCs are generally thought to be critical for normal germinal center (GC) formation and the development and selection of memory B cells. However, it has been very difficult to test these ideas. To determine directly whether FDC-bound complexes do indeed function in these roles, we have developed a transgenic (Tg) mouse in which all B lymphocytes produce only the membrane-bound form of immunoglobulin M. Immune Tg mice have 10,000-fold less specific Ab than wild-type mice and lack detectable ICs on FDCs. Nonetheless, primary immune responses and the GC reaction in these mice are robust, suggesting that ICs on FDCs do not play critical roles in immune response initiation and GC formation. Moreover, as indicated by the presence and pattern of somatic mutations, memory cell formation and selection appear normal in these IC-deficient GCs.

Topics: Amino Acid Sequence; Animals; Antibodies; Antigen-Antibody Complex; B-Lymphocytes; Dendritic Cells, Follicular; Female; Genes, Immunoglobulin; Germinal Center; Haptens; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Male; Mice; Mice, Inbred BALB C; Mice, Transgenic; Molecular Sequence Data; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell

B lymphocyte stimulator (BLyS) is a newly identified monocyte-specific TNF family cytokine. It has been implicated in the development of autoimmunity, and functions as a potent costimulator with antiimmunoglobulin M in B cell proliferation in vitro. Here we demonstrate that BLyS prominently enhances the humoral responses to both T cell-independent and T cell-dependent antigens, primarily by attenuation of apoptosis as evidenced by the prolonged survival of antigen-activated B cells in vivo and in vitro. BLyS acts on primary splenic B cells autonomously, and directly cooperates with CD40 ligand (CD40L) in B cell activation in vitro by protecting replicating B cells from apoptosis. Moreover, although BLyS alone cannot activate the cell cycle, it is sufficient to prolong the survival of naive resting B cells in vitro. Attenuation of apoptosis by BLyS correlates with changes in the ratios between Bcl-2 family proteins in favor of cell survival, predominantly by reducing the proapoptotic Bak and increasing its prosurvival partners, Bcl-2 and Bcl-xL. In either resting or CD40L-activated B cells, the NF-kappaB transcription factors RelB and p50 are specifically activated, suggesting that they may mediate BLyS signals for B cell survival. Together, these results provide direct evidence for BLyS enhancement of both T cell-independent and T cell-dependent humoral immune responses, and imply a role for BLyS in the conservation of the B cell repertoire. The ability of BLyS to increase B cell survival indiscriminately, at either a resting or activated state, and to cooperate with CD40L, further suggests that attenuation of apoptosis underlies BLyS enhancement of polyclonal autoimmunity as well as the physiologic humoral immune response.

Topics: 2,4-Dinitrophenol; Animals; Antibodies, Bacterial; Antibody Formation; Apoptosis; B-Cell Activating Factor; B-Lymphocytes; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; CD40 Ligand; Cells, Cultured; Female; gamma-Globulins; Humans; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; NF-kappa B; NF-kappa B p50 Subunit; Nitrophenols; Phenylacetates; Pneumococcal Vaccines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Serum Albumin, Bovine; T-Lymphocytes; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha

Topics: Animals; Antibodies; B-Lymphocytes; Cell Survival; Haptens; Immunologic Memory; Integrases; Mice; Nitrophenols; Phenylacetates; Phycoerythrin; Viral Proteins

Immunological memory in the antibody system is generated in T-cell-dependent responses and carried by long-lived memory B cells that recognize antigen by high-affinity antibodies. But it remains controversial whether these B cells represent true 'memory' cells (that is, their maintenance is independent of the immunizing antigen), or whether they are a product of a chronic immune response driven by the immunizing antigen, which can be retained in the organism for extended time periods on the surface of specialized antigen-presenting cells (follicular dendritic cells). Cell transfer experiments provided evidence in favour of a role of the immunizing antigen; however, analysis of memory cells in intact animals, which showed that these cells are mostly resting and can persist in the absence of detectable T-cell help or follicular dendritic cells, argued against it. Here we show, by using a genetic switch mediated by Cre recombinase, that memory B cells switching their antibody specificity away from the immunizing antigen are indeed maintained in the animal over long periods of time, similar to cells retaining their original antigen-binding specificity.

Topics: Alleles; Animals; Antibody Specificity; Antigens; B-Lymphocytes; Cloning, Molecular; Genes, Switch; Genotype; Haptens; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunologic Memory; Integrases; Interferon Type I; Molecular Sequence Data; Nitrophenols; Phenylacetates; Phycoerythrin; Recombination, Genetic; Viral Proteins

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor that is sufficient to trigger terminal differentiation in the B cell lymphoma BCL-1. In this study, we have determined the expression pattern of Blimp-1 in vivo in primary and secondary lymphoid organs of humans and immunized mice. Blimp-1 is expressed in plasma cells derived from either a T-independent or T-dependent response in plasma cells that have undergone isotype switching and those resulting from secondary immunization. Blimp-1 is also present in long-lived plasma cells residing in the bone marrow. However, Blimp-1 was not detected in memory B cells. This expression pattern provides further evidence of a critical role for Blimp-1 in plasma cell development, supporting earlier studies in cultured lines. Significantly, Blimp-1 was also found in a fraction (4-15%) of germinal center B cells in murine spleen and human tonsils. Blimp-1 expression in the germinal center is associated with an interesting subset of cells with a phenotype intermediate between germinal center B cells and plasma cells. In the mouse, Blimp-1(+) germinal center B cells peak at day 12 postimmunization and disappear soon thereafter. They are not apoptotic, some are proliferating, they express germinal center markers peanut agglutinin or CD10 but not Bcl-6, and most express CD138 (syndecan-1), IRF4, and cytoplasmic Ig. Together, these data support a model in which B cell fate decisions occur within the germinal center and Blimp-1 expression is critical for commitment to a plasma cell, rather than a memory cell, fate.

Topics: Animals; Antigens, T-Independent; B-Lymphocyte Subsets; Bone Marrow Cells; Cell Differentiation; Cell Lineage; Ficoll; Germinal Center; Haptens; Hemocyanins; Humans; Immunization, Secondary; Immunologic Memory; Immunophenotyping; Injections, Intraperitoneal; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Plasma Cells; Positive Regulatory Domain I-Binding Factor 1; Repressor Proteins; Transcription Factors

To determine the role of the pleiotropic cytokine TGF-beta in B cells, we generated mice lacking the TGF-beta receptor (TbetaR) type II selectively in this cell type through conditional mutagenesis (Cre/loxP). The absence of TbetaRII in B cells leads to a reduced life span of conventional B cells, expansion of peritoneal B-1 cells, B cell hyperplasia in Peyer's patches, elevated serum immunoglobulin, and substantial IgG3 responses to a normally weak immunogen. This B cell hyperresponsiveness is associated with a virtually complete serum IgA deficiency. The data reveal differential roles of TbetaR in homeostasis and antigen responsiveness of B cell subpopulations and establish a critical function of the TGF-beta receptor ligand pair in the induction of IgA responses in vivo.

Topics: Animals; Antigens; B-Lymphocyte Subsets; B-Lymphocytes; Cell Division; Chickens; Crosses, Genetic; Female; Ficoll; gamma-Globulins; IgA Deficiency; Immunoglobulin A; Immunoglobulin Isotypes; Intestinal Mucosa; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Knockout; Nitrophenols; Peyer's Patches; Phenylacetates; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Deletion

In order to obtain further information on the interaction between antigens (Ags) and B cell Ag receptors (BCR) for a better understanding of the relationship between signals resulting from Ag binding and B cell activation, effects of Ag valence and size on the apparent association constant, i.e. the avidity as well as the molecular stoichiometry of immune complexes in Ag-antibody (Ab) interactions were studied. Hapten conjugates using proteins of various molecular weights, such as hen egg lysozyme (HEL), ovalbumin (OVA), bovine serum albumin (BSA), and chicken gammaglobulin (CGG), were prepared for this purpose. Different ratios of the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP), to the protein were used for conjugation, and interactions between anti-NP monoclonal Abs (mAbs) and the NP conjugates were evaluated by surface plasmon resonance. It was founded that the two binding sites of an Ab were able to simultaneously accommodate two NP(1)-HEL, resulting in a tri-molecular complex, Ag(2)Ab(1). However, NP conjugates of the higher-molecular-weight proteins, OVA and BSA, formed only Ag(1)Ab(1), irrespective of hapten valence. This was thought to be due to steric hindrance caused by the binding of the first Ag. These results suggested that the stoichiometry depended largely on the size of the Ag involved and that mAbs with a low affinity are more efficient at raising the binding strength through divalent interaction since the avidity of two mAbs in interactions with highly haptenated BSA was not significantly different in spite of a 10-fold difference in affinity to the monovalent NP(1)-HEL.

Topics: Antibody Affinity; Antigen-Antibody Reactions; Antigens; gamma-Globulins; Haptens; Models, Immunological; Muramidase; Nitrophenols; Ovalbumin; Phenylacetates; Serum Albumin, Bovine; Surface Plasmon Resonance; Thermodynamics

The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav-/- B cells. In vivo, vav-/- mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav-/- B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav-/- mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag.

Topics: Animals; Antibodies, Viral; Antigens; Antigens, Viral; B-Lymphocytes; Cell Cycle Proteins; Guanine Nucleotides; Haptens; Immunoglobulin M; Injections, Intravenous; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Ovalbumin; Phenylacetates; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Receptors, Antigen, B-Cell; Vesicular stomatitis Indiana virus

We developed a universal recombinant bispecific molecule (BiMol) that is capable of redirecting cytotoxic T cells to tumor cells via tagged anti-tumor ligands such as antibody fragments or cytokines. A recombinant bispecific diabody with binding specificities for the CD3 molecule on T cells as well as for the hapten nitrophenyl (NIP) was produced. This bispecific molecule is capable of redirecting cytotoxic T cells to kill a series of malignant cells, including B cell lymphoma, Hodgkin's lymphoma, and colon carcinoma via NIP-conjugated ligands to tumor-associated antigens. Cytotoxic activity of the diabody was found to be comparable to tetradoma-derived bispecific antibodies with similar specificities. Our findings demonstrate that universal CD3xanti-NIP diabodies could be used for T cell based cellular immunotherapy in a variety of human malignancies. Additionally, these bispecific molecules allow fast and economic testing of tumor-associated antigens on malignant cells for their potential use as immunotherapeutic target structures if corresponding hapten-conjugated antibodies or ligands are available.

Topics: Antibodies, Bispecific; Antigens, Neoplasm; Calcium; CD3 Complex; Cell Division; Haptens; Humans; Hybridomas; Jurkat Cells; Neoplasms; Nitrophenols; Phenylacetates; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

We found that two distinct antibody maturation pathways exist in the immune response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl and that the junctional amino acid introduced by a process far preceding somatic hypermutation determined the pathway of affinity maturation. Antibodies belonging to each pathway clearly separated into two separate branches of a phylogenic tree. We also constructed a three-dimensional fitness landscape for antibody evolution by introducing the association constants of the antibodies into the phylogenic tree as the third axis, allowing us to comprehend the significance of junctional diversity in the "evolvability" of antibodies. Thermodynamic analyses of the antigen-antibody interactions suggested that a high conformational versatility in the antigen-combining site allows for the enhanced evolvability of antibodies.

Topics: Amino Acid Sequence; Amino Acids; Animals; Antibody Formation; B-Lymphocytes; Base Sequence; Calorimetry; Haptens; Hybridomas; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Phenylacetates; Sequence Homology, Amino Acid

Immunization with T-dependent antigens induces a rapid differentiation of B cells to plasmacytes that produce the primary immunoglobulin M (IgM) and IgG antibodies with low affinities for the immunogen. It is proposed that the IgG antibody forms immune complexes with the residual antigen which provide an important stimulus for the formation of germinal centres (GC) and the activation of somatic mutation. This hypothesis was tested by passive administration of hapten-specific antibody into mice shortly after the immunization with nitrophenyl (NP) coupled to chicken gamma globulin (NP-CGG) in an environment of limited T-cell help. Athymic mice that received normal T helper cells at 72 hr after the administration of antigen produced low levels of anti-NP antibody and the splenic GC formation was delayed until day 12 after the antigen administration. The analysis of VDJ segments from NP-reactive GC B cells showed very few mutations in the VH genes. Passive injection of anti-NP IgG1 monoclonal antibody - but, not IgM - stimulated the GC formation up to normal levels and the somatic mutation activity in the GC B cells was fully restored. In addition, GC B cells in the recipients of IgG1 antibody demonstrated a change in the usage of germline-encoded VH genes which was not apparent among the primary antibody-forming cells. These results suggest the existence of a specific feedback mechanism whereby the IgG antibody regulates the GC formation, clonotypic repertoire and somatic mutation in GC B cells.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibody-Producing Cells; B-Lymphocytes; CD4-Positive T-Lymphocytes; Feedback; Germinal Center; Immunization, Passive; Immunoglobulin G; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Mutation; Nitrophenols; Phenylacetates

Protection against infection with encapsulated bacteria is mediated by IgG antibodies against the capsular polysaccharides. Production of such antibodies is impaired during infancy, when susceptibility to bacterial meningitis is greatest. Recent studies have proposed the use of anti-CD40 antibody to increase responsivenesses to polysaccharide antigens. We show here that the IgG response to a model polysaccharide antigen is greatly increased, but retains thymus-independent characteristics--switching continues to be mainly to IgG3 and neither germinal centers nor memory B cells are formed. Furthermore, anti-CD40 causes striking splenomegaly in mice, which is accompanied by dramatic cellular redistribution and proliferation of dendritic cells, macrophages, T cells and endothelium, as well as B cells. These findings raise the possibility that the anti-CD40 effect is due mainly to increased activity of accessory cells that affect plasmablast growth and differentiation rather than mimicry of T cell help.

Topics: Animals; Antibodies; Antigens, T-Independent; CD40 Antigens; Ficoll; Germinal Center; Immunity, Mucosal; Immunoglobulin Class Switching; Immunoglobulin G; Immunologic Memory; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Nitrophenols; Phenylacetates; T-Lymphocytes, Helper-Inducer; Time Factors

Antigen-specific-effected immunoregulation by T lymphocytes is mediated by extracellular proteins produced by T lymphocytes. These immunoproteins bind specifically to nonprocessed antigen and either induce antigen-specific immunoregulatory T cells (tsfi) or effect regulation (tsfe). T cell proteins that bind specifically to nonprocessed antigen have ben termed "T cell antigen-binding molecules" (TABM), and by definition, tsfe and tsfi are, in part, TABM. To characterize tsfi, tsfe, and TABM and understand the relationships and function of these immunoproteins, we have combined the efforts of two laboratories to compare tsfi, tsfe, and TABM isolated by each laboratory. Data obtained in one laboratory were reproduced by the other, and all reagents prepared by each laboratory were exchanged. TABM, tsfi, and tsfe were found to express TCRCalpha epitopes but not TCRCbeta epitopes. The amino acid sequence of a tryptic peptide of a T cell hybridoma TABM specific for nitrophenylhydroxy acetate (NP) is similar to a TCRalpha chain and TCR pre-alpha chain amino acid sequence. ELISA and immunoblotting demonstrated that Mr 77,000 T cell hybrid-derived tsfi, tsfe, and TABM are noncovalently associated with Mr 15,000-16,000 interleukin-10 (IL-10). ELISA also demonstrated that tsfi and tsfe are associated with I-J. The ability of tsfi and tsfe to suppress a mixed lymphocyte reaction was prevented by anti-IL-10 or anti-I-J antibodies, suggesting that antigen-specific immunoregulatory T cell proteins function by an antigen-specific focusing of immunoregulatory cytokines.

Topics: Amino Acid Sequence; Animals; Antibodies; Antigens; Blood Proteins; Cytokines; Epitopes; Immunity, Cellular; Immunoblotting; Interleukin-10; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Nitrophenols; Peptide Fragments; Phenylacetates; Protein Binding; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; Sequence Homology, Amino Acid

To examine the role of germinal centers (GCs) in the generation and selection of high affinity antibody-forming cells (AFCs), we have analyzed the average affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific AFCs and serum antibodies both during and after the GC phase of the immune response. In addition, the genetics of NP-binding AFCs were followed to monitor the generation and selection of high affinity AFCs at the clonal level. NP-binding AFCs gradually accumulate in bone marrow (BM) after immunization and BM becomes the predominant locale of specific AFCs in the late primary response. Although the average affinity of NP-specific BM AFCs rapidly increased while GCs were present (GC phase), the affinity of both BM AFCs and serum antibodies continued to increase even after GCs waned (post-GC phase). Affinity maturation in the post-GC phase was also reflected in a shift in the distribution of somatic mutations as well as in the CDR3 sequences of BM AFC antibody heavy chain genes. Disruption of GCs by injection of antibody specific for CD154 (CD40 ligand) decreased the average affinity of subsequent BM AFCs, suggesting that GCs generate the precursors of high affinity BM AFCs; inhibition of CD154-dependent cellular interactions after the GC reaction was complete had no effect on high affinity BM AFCs. Interestingly, limited affinity maturation in the BM AFC compartment still occurs during the late primary response even after treatment with anti-CD154 antibody. Thus, GCs are necessary for the generation of high affinity AFC precursors but are not the only sites for the affinity-driven clonal selection responsible for the maturation of humoral immune responses.

Topics: Animals; Antibodies; Antibody Affinity; Antibody-Producing Cells; Bone Marrow Cells; Germinal Center; Immunization; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

The behavior of p-azophenylarsonate (Ars)-specific B cell clones during the primary T cell-dependent splenic response of A/J mice was investigated using an immunohistochemical approach. The earliest Ars-specific B cells were observed as isolated cells in the red pulp by day 3 after immunization with Ars-keyhole limpet hemocyanin, (KLH) and at day 6, large clusters of Ars-specific B cells were first detected in germinal centers, which continued to be observed for an additional 8 to 15 days. Surprisingly, no Ars-specific B cell foci were observed in or near the CD4 T cell-rich periarteriolar lymphoid sheath (PALS) during the entire primary response. Nevertheless, A/J mice immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma globulin (NP-CGG) or Ars-CGG mounted robust splenic (4-hydroxy-3-nitrophenyl)acetyl or CGG-specific PALS-associated focus reactions, respectively. In contrast, no Ars-specific PALS B cell foci were detected in A/J mice immunized with Ars-CGG. These data add to a growing body of evidence indicating that B cell proliferation and differentiation in CD4 T cell-rich microenvironments are not prerequisites for the GC reaction. Taken together with previous results obtained using other model Ags, the data suggest that the specificity of the B cell Ag receptor may strongly influence the lymphoid microenvironment in which a B cell clone first undergoes Ag-driven clonal expansion and differentiation.

Topics: Animals; Arterioles; B-Lymphocytes; Cell Differentiation; Chickens; Epitopes; gamma-Globulins; Germinal Center; Lymphoid Tissue; Mice; Mice, Inbred A; Nitrophenols; p-Azobenzenearsonate; Phenylacetates

Imbalances of Th1- and Th2-type responses have been postulated to be a predisposing factor for both humoral and cellular mediated autoimmune diseases. To further define their roles in systemic autoimmunity, IL-4 and IFN-gamma gene knockout mice were studied for susceptibility to the prototypic Th2-mediated mercury-induced autoimmunity. A predominant Th2-type response following HgCl2 treatment of wild-type B10.S mice was confirmed by the findings of a significant increase in splenic IL-4 and hypergammaglobulinemia primarily of the IgG1 isotype, without an increase in IFN-gamma levels. Paradoxically, IL-4-deficient mice developed the characteristic anti-nucleolar autoantibodies and tissue deposition of immune complexes, while IFN-gamma-deficient mice had very low autoantibody levels and essentially normal immunohistology. Studies to define defects in Ab responses of IFN-gamma-deficient mice, using the T-dependent Ag (4-hydroxy-3-nitrophenyl)acetyl, revealed an attenuated IgG response to low and to a lesser extent high doses of (4-hydroxy-3-nitrophenyl)acetyl-hemocyanin, but maintenance of affinity maturation. These results indicate that Th1/Th2 imbalance does not directly play a role in susceptibility to mercury-induced autoimmunity, and suggest that the dependence on Th1-type responses in certain autoimmune diseases is due to the requirement for IFN-gamma for Ab production to weakly antigenic self molecules.

Topics: Animals; Autoantibodies; Autoimmune Diseases; Disease Susceptibility; Dose-Response Relationship, Immunologic; Haptens; Interferon-gamma; Interleukin-4; Mercuric Chloride; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenylacetates; Th1 Cells; Th2 Cells

In the first week of the primary immune response to the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten, plasmacytic foci and germinal centers (GCs) in C57BL/6 mice are comprised of polyclonal populations of B lymphocytes bearing the lambda1 L-chain (lambda1+). The Ig H-chains of these early populations of B cells are encoded by a variety of VH and D exons undiversified by hypermutation while later, oligoclonal populations are dominated by mutated rearrangements of the VH186.2 and DFL16.1 gene segments. To assess directly Ab affinities within these defined splenic microenvironments, representative VDJ rearrangements were recovered from B cells participating in the early immune response to NP, inserted into Ig H-chain expression cassettes, and transfected into J558L (H-; lambda1+) myeloma cells. These transfectoma Abs expressed a remarkably wide range of measured affinities (Ka = 5 x 10(4)-1.3 x 10(6) M(-1)) for NP. VDJs recovered from both foci and early GCs generated comparable affinities, suggesting that initial differentiation into these compartments occurs stochastically. We conclude that Ag normally activates B cells bearing an unexpectedly wide spectrum of Ab affinities and that this initial, promiscuous clonal activation is followed by affinity-driven competition to determine survival and clonal expansion within GCs and entry into the memory and bone marrow plasmacyte compartments.

Topics: Animals; Antibody Affinity; Antibody-Producing Cells; Arginine; B-Lymphocytes; Chickens; gamma-Globulins; Germinal Center; Glycine; Haptens; Immunoglobulin M; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Plasma Cells; Point Mutation; Spleen

Several mechanisms that diversify the adult immune repertoire, such as terminal deoxynucleotidyl transferase-dependent N region addition, are not available to the neonatal mouse. One important process that contributes to protective immunity in the adult is somatic mutation, which plays a major role in the generation of high affinity memory B cells. It is not clear whether B cells in the neonatal mouse can activate the somatic mutation machinery. To investigate this, we immunized neonates with poly(L-Tyr,L-Glu)-poly-D,L-Ala-poly-L-Lys complexed with methylated BSA, or (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin. Eight to fourteen days after priming, V(D)J rearrangements of known V(H) genes (V(H)SM7 family) were screened for mutations using a temperature-melt hybridization assay and oligonucleotide probes specific for complementarity-determining regions I and II; possible mutations were confirmed by sequence analysis. More mutations per sequence were found in heavy chains from neonates immunized with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin than in those from neonates immunized with poly(L-Tyr,L-Glu)-poly-D,L-Ala-poly-L-Lys complexed with methylated BSA. Mutations were found in heavy chains lacking N regions, suggesting that B cells of the putative fetal lineage can somatically mutate and diversify an initially limited repertoire. Since neonates immunized as early as 1 or 2 days after birth had mutations, the somatic mutation machinery can be activated soon after birth, suggesting that early vaccination should result in affinity maturation and protective immunity in the neonate.

Topics: Animals; Animals, Newborn; Base Sequence; Chickens; Female; gamma-Globulins; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Immunoglobulin; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Multigene Family; Mutation; Nitrophenols; Peptides; Phenylacetates; Serum Albumin, Bovine

Immunization with protein-containing antigens results in two types of antigen-specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re-exposure to antigen. The issue of the inter-relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen-specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub-population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity-enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B-cell population contains few, if any, cells from the early response and is less stringently selected.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; B-Lymphocyte Subsets; Base Sequence; Bone Marrow; Bone Marrow Cells; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunologic Memory; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Sequence Homology, Nucleic Acid; Spleen; Vaccination

Previous work on the cis-acting elements that control heavy chain variable region (VH) gene somatic hypermutation has indicated the presence of an as yet unidentified element(s) 3' of the intron enhancer that is necessary for high rate mutation. Examination of cis-acting elements involved in kappa light chain V gene hypermutation has demonstrated a requirement for both the intronic and 3' kappa enhancers in this process. To examine whether the 3'alpha heavy chain enhancer [3'alpha E(hs1,2)] is required for somatic hypermutation of VH genes, we generated two types of transgenic mice. One type was generated using a construct containing a VH promoter, a rearranged VDJ, the heavy chain intronic enhancer, and the murine heavy chain 3'alpha E(hs1,2). The transgenes in the second lines were similar to the transgenes in the first with the addition of a second complete matrix attachment region (MAR) 3' of the heavy chain intronic enhancer, and splice acceptor and polyadenylation sites between the two enhancers. Analysis of both transgenes revealed levels of mutation at least 10-fold lower than endogenous VH genes. These data suggest that the 3'alpha E(hs1,2) does not play a role analogous to the 3' kappa enhancer in the regulation of the hypermutation process. Moreover, in one of the transgenes, the presence of the 3'alpha E(hs1,2) resulted in a lack of transcription in vivo, suggesting a negative regulatory role for this enhancer in certain contexts.

Topics: Animals; B-Lymphocytes; Enhancer Elements, Genetic; Genes, Immunoglobulin; Haptens; Hybridomas; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nitrophenols; p-Azobenzenearsonate; Peyer's Patches; Phenylacetates; Transcription, Genetic; Transgenes

We have designed and constructed a bacterial expression vector to produce a fusion protein of hapten-specific single-chain Fv (ScFv) and alkaline phosphatase (PhoA) in Escherichia coli. The ScFv gene was assembled using genes encoding the heavy and light chain variable domains of anti-NP (4-hydroxy-3-nitrophenyl acetyl) mouse monoclonal antibody. The ScFv gene was then fused to the 5' terminus of the E. coli PhoA coding region. The expressed fusion protein ScFv(NP)-PhoA was purified using an NP affinity column, and gel-filtration. Characterization of the fusion protein was then performed. The estimated molecular weight by gel filtration was approximately 151 kDa, suggesting the dimerization of the protein. Kinetic constants of ScFv(NP)-PhoA were calculated and compared with those of wild-type PhoA. The k(cat) values of ScFv(NP)-PhoA and wild-type PhoA were 103 (s(-1)) and 96.1 (s(-1)), respectively, showing that PhoA activity was somewhat increased by tethering the molecules. The equilibrium binding constant of ScFv(NP)-PhoA was determined using two different haptens, NP-capronate and NIP(3-iodo-4-hydroxy-5-nitrophenyl acetyl) by means of fluorescence quenching measurements. The obtained binding constants were 2.2 x 10(5) (M-1) for NP-capronate and 1.O x 10(6) (M(-1)) for NIP, respectively. No apparent difference in binding constants was seen between ScFv(NP) and ScFv(NP)-PhoA, showing that sufficient specificity and binding affinity were retained when ScFv(NP) was tethered to alkaline phosphatase. ScFv(NP)-PhoA can be used to detect nanogram concentrations of NP-BSA in ELISA without the use of chemically conjugated secondary antibodies.

Topics: Alkaline Phosphatase; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Affinity; Base Sequence; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Gene Expression; Genetic Vectors; Haptens; Immunoglobulin Fragments; Kinetics; Mice; Molecular Sequence Data; Molecular Weight; Nitrophenols; Peptides; Phenylacetates; Recombinant Fusion Proteins

The pathogenesis of neuronal degeneration in both sporadic and familial amyotrophic lateral sclerosis (ALS) associated with mutations in superoxide dismutase may involve oxidative stress. A leading candidate as a mediator of oxidative stress is peroxynitrite, which is formed by the reaction of superoxide with nitric oxide. 3-Nitrotyrosine is a relatively specific marker for oxidative damage mediated by peroxynitrite. In the present study, biochemical measurements showed increased concentrations of 3-nitrotyrosine and 3-nitro-4-hydroxyphenylacetic acid in the lumbar and thoracic spinal cord of ALS patients. Increased 3-nitrotyrosine immunoreactivity was observed in motor neurons of both sporadic and familial ALS patients. Neurologic control patients with cerebral ischemia also showed increased 3-nitrotyrosine immunoreactivity. These findings suggest that peroxynitrite-mediated oxidative damage may play a role in the pathogenesis of both sporadic and familial ALS.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Antibodies, Monoclonal; Family Health; Female; Humans; Male; Middle Aged; Motor Neurons; Mutation; Nissl Bodies; Nitrophenols; Oxidative Stress; Phenylacetates; Spinal Cord; Superoxide Dismutase; Tyrosine

As a model for studying the generation of antibody diversity, a gene-targeted mouse was produced that is hemizygous for a rearranged V(D)J segment at the immunoglobulin (Ig) heavy chain locus, the other allele being nonfunctional. The mouse also has no functional kappa light chain allele. The heavy chain, when paired with any lambda light chain, is specific for the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). The primary repertoire of this quasi-monoclonal mouse is monospecific, but somatic hypermutation and secondary rearrangements change the specificity of 20 percent of the antigen receptors on B cells. The serum concentrations of the Ig isotypes are similar to those in nontransgenic littermates, but less than half of the serum IgM binds to NP, and none of the other isotypes do. Thus, neither network interactions nor random activation of a small fraction of the B cell population can account for serum Ig concentrations.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; B-Lymphocytes; Base Sequence; Cell Line; Cloning, Molecular; DNA; Flow Cytometry; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Isotypes; Immunoglobulin J-Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Leukocyte Common Antigens; Leukosialin; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Nitrophenols; Phenylacetates; Recombinant Proteins; Sialoglycoproteins

Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; Antibody Formation; Antigen-Antibody Reactions; Antigens; B-Lymphocytes; Base Sequence; Dendritic Cells; DNA; Dose-Response Relationship, Immunologic; Germinal Center; Immunization; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunologic Memory; Lymphotoxin-alpha; Mice; Molecular Sequence Data; Nitrophenols; Ovalbumin; Phenylacetates; Spleen; T-Lymphocytes

We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA). Human PMN from healthy volunteers was stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml) at 37 degrees C in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-buffered Hank's balanced salt solution (pH 7.4) with HPA (1 mM). NO2HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO2HPA was eliminated by N-monomethyl-L-arginine (100 microM). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO2HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO2HPA.

Topics: Animals; Arginine; Enzyme Inhibitors; Humans; Luminescent Measurements; Macrophages, Alveolar; Neutrophils; Nitrates; Nitrites; Nitrophenols; omega-N-Methylarginine; Peroxidase; Phenylacetates; Rats; Superoxides; Tetradecanoylphorbol Acetate

In this study, we examine the relationship between primary and secondary T cell-dependent immune responses using the response of xid mice to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) as an experimental model. The reduced serologic primary immune response of xid mice was demonstrated to be caused by a substantially decreased Ab-forming cell (AFC) generation. Furthermore, the germinal center reaction in the primary xid immune response was diminished and the frequency of NP-specific memory B cells prior to secondary immunization was reduced 10-fold. Despite the poor primary response of xid mice, secondary exposure to Ag generated a response that was qualitatively and quantitatively equal to that of wt mice. The number of IgG1 AFCs in spleen and bone marrow increased equally in both groups, as did the proportion of AFCs secreting high affinity Ab in both locations. The extent and distribution of somatic mutations in the V(H) genes of xid secondary response B cells was also found to be normal, indicating that the xid gene product is not critical for the processes that result in affinity maturation. Thus, although xid mice generate memory B cells of normal phenotype but at a substantially lower frequency, this does not limit the magnitude of the secondary response. Therefore, our results imply that the reduced memory B cell frequency in xid mice is still above some threshold value necessary for a normal secondary immune response.

Topics: Animals; Antibody-Producing Cells; Antigens, CD; B-Lymphocytes; B7-2 Antigen; CD40 Ligand; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genetic Linkage; Haptens; Hematopoietic Stem Cells; Hemocyanins; Immunization; Immunization, Secondary; Immunoglobulin G; Immunologic Deficiency Syndromes; Immunologic Memory; Lymphocyte Activation; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Mutant Strains; Mutation; Nitrophenols; Phenylacetates; Transfection; X Chromosome

Monoclonal antibodies (mAbs) specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) were prepared at various times after immunization and the amino acid sequences of VH and V lambda 1 in these mAbs were deduced from cDNA nucleotide sequences. Replacements due to somatic mutation were not found in day 7 mAbs but were found in those of days 14, 84 and 294. The affinity of day 7 mAbs to NP-glycine(NP-Gly) was in the order of 10(4) M-1 and it increased about 8000-fold with time after immunization. The extrinsic circular dichroism (CD) spectrum of the NP-epsilon-aminocaproic acid (NP-Cap)/Ab complex was unique for each mAb, although the spectra were grouped into two types, which tended to shift from one type to another with time, suggesting a variation in the micro-environments around NP-Cap in the combining sites. All these data indicate that the structure of the combining site was altered by somatic mutation; however, the fine-specificity measured by cross-reactivity with hapten analogues did not change significantly with time. We examined the amino acid residues in CDRs responsible for recognition of NP-haptens by comparing the amino acid sequences of anti-NP mAbs. Analyses revealed the presence of several conserved amino acid residues in CDRs of VH and V lambda 1, such as Tyr-32H, and Tyr-60H, in addition to a core segment involving Arg-50H.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Circular Dichroism; Conserved Sequence; Haptens; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Phenylacetates; Sequence Analysis

The three-dimensional structures of the Fab fragment, in its unliganded and liganded crystals, of mouse anti-(4-hydroxy-3-nitrophenyl)acetate (NP) antibody N1G9 have been determined by the molecular replacement method. The unliganded and NP-liganded structures were refined at 2.4 A resolution to crystallographic R-factors of 0.194 and 0.196, respectively. Antibody N1G9 bears lambda light chains, and is one of the primary immune response antibodies. Fab N1G9 exhibits an elbow angle of 197 degrees in both structures. This large angle is ascribed to the VL-CL interface formed by lambda-chain residues. A hydrophobic pocket surrounded by the complementarity-determining regions except L2 is identified as a hapten-binding site. Between the liganded and unliganded structures, root-mean-square (r.m.s.) positional deviations are 0.42 A for the main-chain atoms, and 0.74 A for all the protein atoms. The major structural differences between these structures are localized in the hapten-binding site, and yield an r.m.s. deviation of 1.03 A for the side-chain atoms. The soaked NP ligand is in van der Waals contact with the aromatic side-chains of Tyr32L and Trp91L of the light chain, and Trp33H and Tyr97H of the heavy chain, and is hydrogen-bonded to the side-chains of Trp96L, His35H, Arg50H, Tyr95H, and Ser100aH. The side-chain of Lys58H is salt-bridged to the NP hydroxyl group. The side-chains of Arg50H, Trp33H, and Tyr97H are shifted toward the NP carboxyl group. The side-chain of Trp33H, whose replacement to Leu increases affinity by tenfold, is sandwiched between the Arg50H and Tyr97H side-chains, and is in cramped contact both with the ligand and with these side-chains. Affinity increases in the maturation of the anti-NP antibodies are ascribable to conformational relief of these cramped contacts through the replacement of Trp33H or through suitable structural alterations in the H3 region.

Topics: Animals; Antigen-Antibody Complex; Binding Sites, Antibody; Computer Graphics; Crystallography, X-Ray; Fourier Analysis; Haptens; Immunoglobulin Fab Fragments; Mice; Models, Molecular; Nitrophenols; Phenylacetates; Protein Conformation

Germinal centers (GCs) are the sites of antigen-driven V(D)J gene hypermutation and selection necessary for the generation of high affinity memory B lymphocytes. Despite the antigen dependence of this reaction, injection of soluble antigen during an established primary immune response induces massive apoptotic death in GC B cells, but not in clonally related populations of nonfollicular B lymphoblasts and plasmacytes. Cell death in GCs occurs predominantly among light zone centrocytes, is antigen specific, and peaks within 4-8 h after injection. Antigen-induced programmed death does not involve cellular interactions mediated by CD40 ligand (CD40L) or Fas; disruption of GCs by antibody specific for CD40L was not driven by apoptosis and C57BL/6.lpr mice, though unable to express the Fas death trigger, remained fully susceptible to soluble antigen. Single injections of antigen did not significantly decrease GC numbers or average size, but repeated injections during an 18-h period resulted in fewer and substantially smaller GCs. As cell loss appeared most extensive in the light zone, decreased GC cellularity after prolonged exposure to soluble antigen implies that the Ig- centroblasts of the dark zone may require replenishment from light zone cells that have survived antigenic selection. GC cell death is avidity-dependent; oligovalent antigen induced relatively little apoptosis and GC B cells that survived long exposures to multivalent antigen expressed atypical VDJ rearrangements unlikely to encode high affinity antibody. Antigen-induced apoptotic death in GCs may represent a mechanism for the peripheral deletion of autoreactive B cell mutants much as the combinatorial repertoire of immature B lymphocytes is censored in the bone marrow.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; Apoptosis; B-Lymphocytes; Base Sequence; CD40 Ligand; Cells, Cultured; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Immunoglobulin; Germinal Center; Immune Tolerance; Immunohistochemistry; Immunologic Memory; Lymphocyte Cooperation; Membrane Glycoproteins; Mice; Molecular Sequence Data; Nitrophenols; Phenylacetates; Spleen

The development of memory B cells takes place in germinal centers (GC) of lymphoid follicles where antigen-driven lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells. However, the mechanisms that drive this complex response are not well understood. We explored the relationship between GC formation and the onset of hypermutation in response to the hapten phosphorylcholine (PC) coupled to antigenic proteins in mice bearing different frequencies of CD4+ T cells. PC-reactive GC were identified by staining frozen splenic sections with peanut agglutinin (PNA) and with monoclonal Abs against AB1-2, a dominant idiotope of T15+ anti-PC antibody. The nucleotide sequences of rearranged T15 VH1 genes were determined from polymerase chain reaction amplifications of genomic DNA from microdissected GC B cells. T15+ GC became fully developed by day 6-7 after primary immunization of euthymic mice with either PC-keyhole limpet hemocyanin (KLH) or PC-chicken gamma globulin (CGG). Yet the VH1 gene segments recovered from the primary GC as late as day 10-14 had low numbers of mutations, in contrast to responses to the haptens nitrophenyl or oxazolone that sustain high levels of hypermutation after GC formation. PC-reactive B cells proliferate in histologically typical GC for considerable periods with no or little somatic hypermutation; the signals for GC formation are independent of those for the activation of hypermutation. We then examined GC 7 d after secondary immunization with PC-KLH in euthymic mice, in nu/nu mice reconstituted with limited numbers of normal CD4+ cells before priming (CD4(+)-nu/nu) and in nu/nu mice. All of these animals develop T15+ GC after antigen priming, however, the patterns of V gene mutations in the secondary GC reflected the levels of CD4+ cells present during the primary response. VDJ sequences from secondary GC of euthymic mice were heavily mutated, but most of these mutations were shared among all related (identical VDJ joints) sequences suggesting the proliferation of mutated, memory B cells, with little de novo somatic hypermutation. In contrast, the patterns of V gene diversity in secondary GC from CD4(+)-nu/nu mice suggested that there was ongoing mutation and clonal diversification during the first week after rechallenge. The secondary GC from T cell-deficient, nu/nu mice showed little evidence for mutational and/or recombinational diversity of T15+ B cells. We conclude that

Topics: Amino Acid Sequence; Animals; Antibody Diversity; B-Lymphocytes; Base Sequence; Chickens; gamma-Globulins; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Immunoglobulin; Haptens; Hemocyanins; Immunization; Immunization, Secondary; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunologic Memory; Immunotherapy, Adoptive; Lymphoid Tissue; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Models, Immunological; Molecular Sequence Data; Nitrophenols; Oxazolone; Phenylacetates; Phosphorylcholine; Sequence Alignment; Sequence Homology, Amino Acid; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer

The primary immune response to T cell-dependent Ags develops in two pathways. These comprise the extrafollicular pathway, in which foci of Ab-secreting cells develop, and the intrafollicular pathway that gives rise to germinal centers and affinity maturation. We have previously shown that de-aggregated (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to the protein carrier human serum albumin (HSA), (NP-HSA), injected 6 days after challenge immunization with aggregated NP-HSA, resulted in impaired development of NP-specific, higher-affinity cells. Studies presented here describe the cellular basis underlying this impairment of affinity maturation. Using multiparameter flow cytometry, we show that mice injected with soluble NP-HSA ("tolerant" mice) develop significantly fewer NP-binding IgG1+ B220+ cells of germinal center origin than do the control ("immune") mice. In addition, using immunohistology, we noted that the spleens of tolerant mice had a marked reduction in the number of germinal centers that contained lambda-bearing cells, these being characteristic of the NP response in C57BL/6 mice. Curiously, germinal centers in the spleens of tolerant mice had more than twice the volumes of those in the immune spleens. In contrast to its effect on the germinal center pathway, soluble Ag enhanced the extrafollicular pathway, reflected by the increased numbers of B cells secreting IgM and IgG1 Abs specific for NP, HSA, and undefined Ags. Thus, soluble NP-HSA given after challenge immunization can impede affinity maturation of NP-specific cells, but enhance the extrafollicular pathway. These results are discussed in the context of the known capacity of some persisting Ags, e.g., malaria parasites, to frustrate affinity maturation and memory cell generation.

Topics: Animals; Antibody Formation; Antigens, Surface; Cell Differentiation; Cell Division; Flow Cytometry; Humans; Immune Tolerance; Immunization; Immunoglobulin G; Leukocyte Common Antigens; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Serum Albumin; Solubility; Specific Pathogen-Free Organisms; Spleen

After injection with immunogenic conjugates of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), two distinct B cell populations can be identified in the spleen during the primary response. One of these populations is specialized for Ab production; the other, the germinal centers (GCs), has been identified as the site of Ig somatic hypermutation. Ag-driven selection of GC B cells bearing mutated receptors with higher affinity leads to the affinity maturation of serum Ab and increased protective humoral immunity. Microdissection of GC B cell populations specific for NP and sequencing of the recovered Ig heavy chain variable region genes revealed that the somatic hypermutation process is absent in the GCs of aged C57BL/6 mice. However, selection for Ag appears to occur in the absence of hypermutation in the form of competition between unmutated clones of Ag-activated B lymphocytes. Thus, affinity maturation in these animals is limited to the affinities of Ab encoded by the germline.

Topics: Age Factors; Animals; Antigen Presentation; B-Lymphocytes; Base Sequence; Clone Cells; Female; Immunity; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Spleen

Sera were analysed for levels of IgA- and IgG-class and IgA-subclass antibodies against tetanus toxoid and synthesized, tetanus-toxoid, beta-chain peptides. Forty-five peptides, deduced from the amino-acid sequence of the tetanus-toxoid beta-chain, were synthesized, in order to analyse epitope-binding of antibodies. Monoclonal, human, anti-tetanus antibodies were used to evaluate the synthesized peptides. Five synthesized peptides, bound by high, medium or low levels of IgA antibodies, were selected for the quantification of IgA1 and IgA2 antibodies. A set of chimeric, IgA-subclass antibodies with NP-specificity were used to quantify the antigen-specific IgA-subclass antibodies in a novel assay. Antibodies against the synthesized peptides were predominantly of IgA1 subclass.

Topics: Adult; Amino Acid Sequence; Antibodies, Monoclonal; Binding Sites, Antibody; Haptens; Humans; Immunoglobulin A; Immunoglobulin G; Molecular Sequence Data; Nitrophenols; Peptide Fragments; Phenylacetates; Tetanus Toxoid

To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 degrees C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Binding Sites, Antibody; Calorimetry; Haptens; Immunoglobulin Fab Fragments; In Vitro Techniques; Mice; Nitrophenols; Osmolar Concentration; Phenylacetates; Temperature; Thermodynamics

Germinal centres are dynamic microenvironments of B-lymphocyte differentiation, which develop in secondary lymphoid tissues during immune responses. Within germinal centres, activated B lymphocytes proliferate and point mutations are rapidly introduced into the genes encoding their immunoglobulin receptors. As a result, new specificities of B cells are created, including those with a heightened capacity to bind the immunizing antigen. Immunoglobulin gene mutation can also lead to reactivity to self antigens. It has been suggested that any newly formed self-reactive B cells are eliminated within the germinal centre in order to avoid autoimmunity. Here we present evidence that antigen-specific, high-affinity, germinal-centre B cells are rapidly killed by apoptosis in situ when they encounter soluble antigen. The effect seems to act directly on the B cells, rather than through helper T cells. Furthermore, the apoptosis is unique to germinal-centre cells, and is only incompletely impeded by constitutive expression of the proto-oncogene bcl-2. This phenomenon may reflect clonal deletion of self-reactive B cells within germinal centres.

Topics: Animals; Antigens; Apoptosis; Autoimmunity; B-Lymphocytes; Cell Differentiation; Complement System Proteins; Haptens; Humans; Immunologic Memory; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitrophenols; Phenylacetates; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Serum Albumin; Solubility; Spleen; T-Lymphocytes, Helper-Inducer

Previous studies utilizing NP (4-hydroxy, 3-nitrophenyl acetyl hapten)-specific, T suppressor hybridomas have indicated that expression of TCR-alpha, but not TCR-beta, mRNA is required for expression of Ag-specific suppressor factor bioactivity. Suppressor-effector factor has been shown to be Ag specific and I-J restricted. Although the expression of TCR-alpha mRNA was necessary for suppressor activity, the role of TCR-alpha, as it pertained to the functional properties of T cell suppressor factor (TsF), was not established. To determine which properties of TsF could be accounted for by TCR-alpha expression, TCR-alpha cDNA, derived from NP-specific, suppressor T cell (Ts) hybridomas, was transfected into recipient Ts hybridomas of a second Ag specificity. The resulting heterologous transfectants displayed NP-specific, genetically restricted TsF activity. The Ag specificity corresponded to that of the TCR-alpha donor; however, the genetic restriction was influenced by the recipient cell, implying that TCR-alpha did not control genetic restriction of the TsF. Examination of TCR-beta expression in one of the MHC-restricted transfectants indicated that the genetic restriction of TsF could not be accounted for by TCR-beta gene products. The data support the conclusion that TCR-alpha expression is not only obligate for TsF bioactivity, but that the Ag specificity of the TCR-alpha dictates the Ag specificity of the resulting suppressor factor.

Topics: Animals; Antigens; Base Sequence; DNA Primers; Haptens; Hemolytic Plaque Technique; Hybridomas; Hypersensitivity, Delayed; Mice; Molecular Sequence Data; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell, alpha-beta; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory; Transfection

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; Antibody Formation; Antibody-Producing Cells; B-Lymphocytes; Base Sequence; fas Receptor; Germinal Center; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Immunologic Memory; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Selection, Genetic; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid

Cultivation of an ice nucleation-active strain of Xanthomonas campestris in the presence (1 ppm) of 4-hydroxy-3-nitrophenylacetic acid resulted in enhancement of its ice-nucleation activity. Both the ice-nucleation-active protein, InaX, and its mRNA were effectively expressed in the bacterial cells cultured in the presence of this compound. This indicates that this compound stimulated the biosynthesis of the ice-nucleation-active protein.

Topics: Bacterial Proteins; Freezing; Genes, Bacterial; Nitrophenols; Phenylacetates; Transcription, Genetic; Xanthomonas campestris

Topics: Animals; B-Lymphocytes; Immunoglobulin kappa-Chains; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates

On the basis of multinuclear NMR data, the structures of the antibody combining sites of anti-4-hydroxy-3-nitrophenylacetyl (NP) antibodies were compared for N1G9, which is one of the primary response antibodies with low affinity for NP, and 3B62, which is one of the secondary response antibodies with high affinity for NP. It has been concluded, on the basis of the results of antibody engineering, that in most secondary response antibodies a Trp-->Leu exchange at position 33 of the heavy chain is primarily responsible for the increased affinity for NP [Allen, D., Simon, T., Sablitzky, F., Rajewsky, K., & Cumano, A. (1988) EMBO J. 7, 1995-2001]. Although 3B62 exhibits one of the highest affinities for NP, it lacks the Trp-->Leu exchange at position 33 of the heavy chain. A variety of stable isotope-labeled Fab analogues of N1G9 and 3B62 have been prepared. Chain-specific resonance assignments were made by recombination of the heavy chains and light chains of the Fab fragments. Binding experiments of a spin-labeled hapten and NOESY experiments have demonstrated that, compared with the environment of the antibody combining site of N1G9, the combination of mutations (including one codon deletion) and the particular D-JH rearrangement in the heavy chain of 3B62 affords a more hydrophobic environment, which is formed by one Tyr residue originating from the light chain and two Tyr residues originating from the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Affinity; Binding Sites, Antibody; Cell Line; Immunoglobulin Fab Fragments; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Sequence Homology, Amino Acid

The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.

Topics: Amino Acid Sequence; Animals; Antibody-Producing Cells; Antigens; B-Lymphocytes; Base Sequence; Cell Differentiation; Cells, Cultured; Female; Genes, Immunoglobulin; Immunization; Immunoglobulin Variable Region; Immunologic Memory; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates

In the murine spleen, germinal centers are the anatomic sites for antigen-driven hypermutation and selection of immunoglobulin (Ig) genes. To detail the kinetics of Ig mutation and selection, 178 VDJ sequences from 16 antigen-induced germinal centers were analyzed. Although germinal centers appeared by day 4, mutation was not observed in germinal center B cells until day 8 postimmunization; thereafter, point mutations favoring asymmetrical transversions accumulated until day 14. During this period, strong phenotypic selection on the mutant B lymphocytes was inferred from progressively biased distributions of mutations within the Ig variable region, the loss of crippling mutations, decreased relative clonal diversity, and increasingly restricted use of canonical gene segments. The period of most intense selection on germinal center B cell populations preceded significant levels of mutation and may represent a physiologically determined restriction on B cells permitted to enter the memory pathway. Noncanonical Ig genes recovered from germinal centers were mostly unmutated although they probably came from antigen-reactive cells. Together, these observations demonstrate that the germinal center microenvironment is rich and temporally complex but may not be constitutive for somatic hypermutation.

Topics: Animals; B-Lymphocytes; Base Sequence; DNA, Single-Stranded; Female; Genes, Immunoglobulin; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Kinetics; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenotype; Phenylacetates; Polymerase Chain Reaction; Spleen

Somatic mutations and cell lineage relationships were examined in a large panel of hybridomas derived from a single mouse 21 days after a primary immunization with NP-CGG. Among 21 lambda-bearing anti-NP hybridomas 18 distinct cell lineages were observed. Ten of the hybridomas used the V186-2 gene which is the most frequently utilized VH gene in the anti-NP response. Analysis of DNA sequence of the entire VH region of these antibodies revealed extensive somatic mutations. The selection for certain codon changes and the level of mutation observed is comparable to that observed in an early secondary anti-NP response. An unexpected observation was that one-third of the hybridomas produced IgM antibodies. Two IgM antibodies expressing the V186-2 gene contained extensive mutations in the VH region. These results indicate that once the somatic mutation process is initiated, it progresses rapidly and continues for at least two weeks during the development of the response. A highly mutated repertoire of memory B cells is formed by three weeks post-immunization that can be rapidly utilized to generate the secondary immune response.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Southern; DNA, Single-Stranded; Escherichia coli; Female; Gene Rearrangement; Genes, Immunoglobulin; Haptens; Immunoglobulin delta-Chains; Immunoglobulin Heavy Chains; Immunoglobulin J-Chains; Immunoglobulin kappa-Chains; Immunoglobulin M; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Nucleic Acid; Transfection

CD5+ B cells have been shown to be disproportionately associated with autoimmune diseases and transformation. In many cases, their apparent ability to bypass self-tolerance is manifested by the production of autoantibodies. These observations, plus the hypothesis that CD5+ B cells represent a distinct B cell lineage, encourage studies into the conditions and factors that influence their development. In the present study, we employed a well-established assay for murine CD5+ B cell function, i.e., their ability to augment the responses of IgHb-linked idiotypic determinants on anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) antibody (Nbb) idiotype-bearing CD5- B cells to a T-independent antigen, together with multiple methods of cell surface phenotyping, to evaluate the potential for interleukin (IL) 4 to effect maturation of CD5+ B cells, CD5+, IgM+, Thy-1-, and NPb idiotype-specific cells panned on antibody-coated petri dishes or sorted by flow cytometry from spleen were capable of augmenting NPb idiotypic responses of NP-KLH-primed responder cells to NP-Ficoll. Splenic B cell populations depleted of CD5+ B cells failed to affect idiotype expression even after 2 days in culture, a time when a small percentage of CD5+ B cells appeared to be regenerated. However, idiotype-specific regulatory activity could be restored in CD5- splenic B cell populations by culture for 2 days with recombinant IL-4. Cells responsible for idiotype-specific regulatory activity after culture with IL-4 were in fact CD5+, IgM+, and Thy-1.2- B cells, demonstrating that IL-4 can drive the functional, if not the phenotypic, maturation of splenic B cells associated with the CD5+ B cell lineage. The results illustrate one possible mechanism by which T cells could control the maturation of cells belonging to the CD5+ B cell lineage.

Topics: Animals; Antigens, CD; B-Lymphocytes; CD5 Antigens; Cells, Cultured; Epitopes; Flow Cytometry; Immunoglobulin Idiotypes; Immunophenotyping; Interleukin-4; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Spleen

We have studied the effects of a four residue insertion into the FR3 loop of the heavy chain variable region from the anti-NP antibody B1-8. The insertion mutant is obtained as secreted antibody without major defects in biosynthesis, indicating that antibody variable domains can accommodate length variation not only in complementarity determining regions (CDRs), but also in framework region (FR) loops. The B1-8 antigen binding site is not affected by the change in a neighbouring loop. FR3 insertions represent a new method of antibody engineering with a potential to obtain strong antigen binding by designing additional antigen contacting residues.

Topics: Amino Acid Sequence; Base Sequence; Binding Sites, Antibody; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Molecular Sequence Data; Mutagenesis, Insertional; Nitrophenols; Phenylacetates; Protein Conformation; Protein Engineering

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM- antigen-specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipopolysaccharide, interleukin (IL)-2, IL-4 and IL-5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti-NP antibody-secreting cells and non-secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody-secreting, IgG1+ cells were uniquely identified by co-expression of the matrix receptor, syndecan. The NP-specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ lambda+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.

Topics: Animals; B-Lymphocytes; Base Sequence; Cells, Cultured; Female; Haptens; Hemocyanins; Immunization; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Variable Region; Immunologic Memory; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Phenylacetates; Polymerase Chain Reaction

In the genetically restricted response that follows immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to protein carriers, two distinct populations of B cells are observed in the spleens of C57BL/6 mice. By 48 h postimmunization, foci of antigen-binding B cells appear along the periphery of the periarteriolar lymphoid sheaths. These foci expand to contain large numbers of antibody-forming cells that neither bind the lectin, peanut agglutinin, nor mutate the rearranged immunoglobulin variable region loci. Germinal centers containing peanut agglutinin-positive B cells can be observed by 96-120 h after immunization. Although specific for the immunizing hapten, these B cells do not produce substantial amounts of antibody, but are the population that undergoes somatic hypermutation and affinity-driven selection. Both focus and germinal center populations are pauciclonal, founded, on average, by three or fewer B lymphocytes. Despite the highly specialized roles of the focus (early antibody production) and germinal center (higher affinity memory cells) B cell populations, analysis of VH to D to JH joins in neighboring foci and germinal centers demonstrate that these B cell populations have a common clonal origin.

Topics: Animals; B-Lymphocytes; Base Sequence; Clone Cells; DNA; Female; Haptens; Immunoenzyme Techniques; Immunoglobulin Heavy Chains; Lymphoid Tissue; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Phenylacetates; Sequence Homology, Nucleic Acid

The accumulation of somatic mutants in splenic B lymphocytes early after primary immunization with the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) coupled to chicken gamma globulin (CG) was determined. Rearranged V186.2 heavy chain genes were amplified by the polymerase chain reaction from genomic DNA and subjected to nucleotide sequence analysis. Somatic antibody mutants become detectable on day 6 after immunization, and most of the somatic mutations accumulating in the memory compartment are introduced until day 14. At this time strong selection for mutants expressing high binding affinity for NP is apparent. Extrapolation from the mutation frequency increases between day 6 and day 14 to the previously determined mutation frequency at week 6 (Weiss. U. and Rajewsky, K., J. Exp. Med. 1990, 172: 1681) leads to the prediction that the process of mutant generation ceases to operate around day 22 after primary immunization.

Topics: Amino Acid Sequence; Animals; B-Lymphocytes; Base Sequence; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Immunoglobulin; Immunization; Immunologic Memory; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Polymerase Chain Reaction; Time Factors

Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electrophoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and lambda 1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.

Topics: Animals; Bacterial Outer Membrane Proteins; Binding Sites, Antibody; Cell Membrane; Escherichia coli; Gene Expression; Genes, Immunoglobulin; Haptens; Immunoglobulin Fragments; Mice; Nitrophenols; Phenylacetates; Protein Engineering; Protein Sorting Signals

After primary immunization with an immunogenic conjugate of (4-hydroxy-3-nitrophenyl)acetyl, two anatomically and phenotypically distinct populations of antibody-forming cells arise in the spleen. As early as 2 d after immunization, foci of antigen-binding B cells are observed along the periphery of the periarteriolar lymphoid sheaths. These foci expand, occupying as much as 1% of the splenic volume by day 8 of the response. Later, foci grow smaller and are virtually absent from the spleen by day 14. A second responding population, germinal center B cells, appear on day 8-10 and persist at least until day 16 post-immunization. Individual foci and germinal centers represent discrete pauciclonal populations that apparently undergo somatic evolution in the course of the primary response. We suggest that foci may represent regions of predominantly interclonal competition for antigen among unmutated B cells, while germinal centers are sites of intraclonal clonal competition between mutated sister lymphocytes.

Topics: Animals; Antibodies; Antibody Formation; Antigens; B-Lymphocytes; Base Sequence; Cell Division; DNA; Immunoglobulin Isotypes; Immunoglobulin lambda-Chains; Immunoglobulin Switch Region; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Nucleic Acid Hybridization; Phenotype; Phenylacetates; Spleen

The generation and selection of somatic antibody mutants are key elements of acquired immunity, essential for the affinity maturation of antibody responses dependent on T cells. The mutants are generated through a mechanism that introduces point mutations at high rate into rearranged variable (V) region genes in the course of cell proliferation. Their appearance coincides with the generation of germinal centres, which are characterized by oligoclonal B-cell proliferation and have been suggested to be the microenvironment in which antibody mutants are generated. We report here direct evidence for this hypothesis. Rearranged V-region genes were amplified from the genomic DNA of cells picked from individual germinal centres. The sequence analysis of these genes revealed that most represent cells of distinct B-cell clones which expanded locally, generating somatic antibody mutants at high rate. By contrast, antigen-induced proliferation of B cells at another site, periarteriolar lymphocyte sheath-associated foci, was not associated with somatic hypermutation.

Topics: Amino Acid Sequence; Animals; Antibody Affinity; B-Lymphocytes; Base Sequence; Clone Cells; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Immunoglobulin; Immunoglobulin Variable Region; Lymphoid Tissue; Mice; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates

The expression of lambda L and microH chain cDNA was examined in Chinese hamster ovary cells. Each cDNA was linked to a different, amplifiable, selectable drug marker gene, and expression was monitored in the presence of increasing concentrations of the selective drugs. Cells were obtained that produced greater than 60 micrograms/10(6) cells/48 h of assembled antibody. This Chinese hamster ovary cell-synthesized IgM was polymeric, and exhibited specific hapten binding and C fixation. The expression strategy employed here may prove useful for the future production of genetically engineered antibodies and other multi-subunit proteins.

Topics: Animals; Antibodies; Antibody Specificity; Base Sequence; Carrier Proteins; Cell Line; Cloning, Molecular; Complement Fixation Tests; Cricetinae; Cricetulus; DNA; Endoplasmic Reticulum Chaperone BiP; Genetic Engineering; Heat-Shock Proteins; Immunoglobulin lambda-Chains; Immunoglobulin mu-Chains; Immunoglobulins; Molecular Chaperones; Molecular Sequence Data; Nitrophenols; Phenylacetates; Recombinant Proteins; Transfection

The anti-(4-hydroxy-3-nitro-phenyl)acetyl (NP) response is dominated by lambda 1 chain-bearing antibodies expressing the VH gene V186.2 in combination with the D element DFL16.1. lambda 1-positive B cells were isolated from the spleens of mice immunized with NP-chicken gamma globulin 6 wk earlier. Rearranged V186.2 genes were amplified from the genomic DNA of these cells and sequenced. In cases where the rearrangement was typical for secondary anti-NP antibodies, the VHDJH sequences were generally heavily mutated. The frequency and the nature of the nucleotide exchanges mirrored those of secondary response antibodies. V186.2 genes with other rearrangements and V186.2-related genes isolated concomitantly were essentially unmutated. These results demonstrate: (a) that somatic antibody mutants are largely restricted to a small compartment of peripheral B cells, namely, that of memory cells; (b) that the memory compartment is strongly selected for high affinity precursors and largely purged from antigen-binding loss mutants; and (c) that the repertoire of binding specificities expressed in the secondary response is established in its final form before secondary immunization.

Topics: Amino Acid Sequence; Animals; B-Lymphocytes; Base Sequence; Female; Gene Rearrangement; Genes, Immunoglobulin; Haptens; Immunization; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Immunologic Memory; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; Spleen

A T cell hybridoma producing a T suppressor factor (TsF) with specificity for the hapten nitrophenyl was converted to long term growth in serum-free medium and its product tested by serology, bioactivity, and Western blot analysis. Results indicated that Ag-specific suppressive activity was present in serum-free medium and this TsF could exhibit the characteristics ascribed to it by various groups: it could bind nominal Ag with specificity, it was bound by anti-TsF mAb, and it could mediate Ag-specific suppression both in vivo and in vitro. Western blot and SDS-PAGE analysis of this purified TsF revealed a 43-kDa single chain protein.

Topics: Animals; Blotting, Western; Culture Media; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Haptens; Hybridomas; Hypersensitivity, Delayed; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Nitrophenols; Phenylacetates; Suppressor Factors, Immunologic

We have previously demonstrated that IgG antibody binding to microfilariae of Dirofilaria immitis increased in the presence of purified C1q. The present study was designed to examine the mechanism of the C1q effect using a system with an antihapten monoclonal antibody (MoAb) and a hapten as an antigen. Microtiter plates were coated with 4-hydroxy-3-nitrophenyl-acetyl (NP)-bovine serum albumin (BSA), and mouse anti-NP MoAb (IgG) was added in the presence of C1q. The amount of IgG which bound to NP-BSA increased with the addition of C1q (p less than 0.01) when the antibody had both specificity to the antigen and ability to fix C1q. The C1q effect, examined using two anti-NP MoAbs with different affinities, was more apparent with the low-affinity antibody (LAMoAb) than with the high-affinity (HAMoAb; percent enhancement of IgG binding was 19 vs. 12%). The C1q effect on LAMoAb binding was doubled when a small amount of HAMoAb was incubated with LAMoAb. The C1q effect on IgG binding might be operative in the early phase of infection, where a small amount of high-affinity antibody and a relatively large amount of low-affinity antibody are produced in the host.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Binding Sites, Antibody; Complement C1q; Dose-Response Relationship, Immunologic; Haptens; Immunoglobulin G; Mice; Nitrophenols; Phenylacetates; Serum Albumin, Bovine

Fusion between the thioguanine resistant myeloma cell line MOPC-315 [which produces alpha, lambda-2 antibodies specific to the 2,4-dinitrophenyl (DNP) hapten] and a long term in vivo maintained hybridoma 6100.15 [which produces mu, lambda-1 antibodies specific to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten] resulted in the generation of 12 hybridomas. These hybridomas secrete a mixed family of immunoglobulins (Ig) that bind both DNP and NP and express both IgM and IgA serological determinants. Affinity purified molecules from NP, DNP, anti-mu, or anti-alpha immunosorbents react with both anti-mu and anti-alpha antisera, suggesting that these Ig represent IgM-IgA hybrid molecules. This conclusion was supported by idiotypic analyses. To determine the roles of individual immunoglobulin chains in determining antibody specificity this IgM-IgA hybridoma was used for immunoselection. Following lysis with specific anti-mu and anti-idiotype antibodies, an alpha+, mu- variant clone (A12) was identified, which secreted Ig that binds DNP but not NP. The DNP binding proteins were shown to express alpha, lambda-1 and lambda-2 chains. In contrast, the Ig which lack DNP binding activity only expressed alpha and lambda-1 determinants. The combined results demonstrate that the lambda-1 chain from 6100.15 hybridoma cannot replace lambda-2 of MOPC-315 for DNP binding activity. These data imply that these closely related lambda chains carry sites critical for antigen binding activity. An IgM-IgA hybridoma variant (MA2) which secretes Ig that binds to NP and DNP and expresses mu, alpha and lambda-2 chains was also characterized. This molecule lacked a lambda-1 chain. To determine if the Ig prepared with heterologous mu and lambda-2 chains had NP binding activity required immunoselection of a fourth clone (M2). M2 secretes homogeneous Ig bearing only mu and lambda-2 chains. In contrast to either parental Ig, the M2 antibody molecules express dual binding activity to both NP and DNP. Thus, critical amino acid substitutions in the MOPC-315 lambda-2 sequence are required for DNA binding specificity.

Topics: Animals; Antibody Specificity; Binding Sites; Cell Line; Dinitrobenzenes; Hybridomas; Immunoglobulin A; Immunoglobulin lambda-Chains; Immunoglobulin M; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Tumor Cells, Cultured

The effects of aging on cellular and molecular components of the 4-hydroxy-3-nitrophenyl acetyl-specific suppressor T (Ts) cell circuit were analyzed in vitro using inducer (Ts1), transducer (Ts2), and effector (Ts3) cells and activating factors (TsF1 and TsF2) derived from young or old mice. The activation of Ts2 cells by TsF1 and of Ts3 cells by TsF2 was found age-restricted, suggesting a loss of Ts2 and Ts3 cell subsets in old mice. However, the activation of Ts3 cells by small amounts of TsF2 is more efficient when both are derived from old rather than from young mice while the same level of maximum suppression is attained. Higher affinity of the interactions involved in Ts cell activation may compensate for loss of Ts cell subsets in old mice. No age restriction was found for antigen presentation to Ts1 cells and for the interaction between Ts3 cells and target B cells. Thus, the effects of aging on immunosuppression result from changes within the Ts cell circuit.

Topics: Aging; Animals; Cells, Cultured; Immunoglobulin Idiotypes; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Nitrophenols; Phenylacetates; T-Lymphocytes, Regulatory

The Fab fragment of the anti-4-hydroxy-3-nitrophenylacetic acid monoclonal antibody, 88C6/12 has been crystallized in the presence of the eliciting hapten, 4-hydroxy-3-nitrophenacetyl-epsilon-aminocaproic acid (NP-aminocap) and the heteroclitic iodinated analog, 4-hydroxy-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-aminocap). Crystals obtained by precipitation with 32% (w/v) polyethylene glycol 3400 in the presence of 40 to 400 microM of either NP-aminocap or NIP-aminocap, belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 81.2 A, b = 86.9 A, c = 131.1 A. The cell volume suggests the presence of two molecules of the complex per asymmetric unit. Analysis of the Patterson function indicates that these two molecules are related by a local 2-fold axis parallel to the crystallographic b axis located at x = 0.218 and z = 0.25.

Topics: Antibodies, Monoclonal; Crystallization; Haptens; Macromolecular Substances; Nitrophenols; Phenylacetates; X-Ray Diffraction

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.

Topics: Animals; Antigen-Presenting Cells; Clone Cells; Epitopes; Hybridomas; Hypersensitivity, Delayed; Lymphocyte Activation; Macrophages; Mice; Nitrophenols; Phenylacetates; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Trinitrobenzenes

In a number of different strains of inbred mice, immunization with a hapten coupled to a protein carrier results in production of homogeneous serum antibodies. At the genetic level this corresponds to the use of a very limited set of variable region genes in the actively secreting B-cells. In contrast, immunization with the same hapten coupled to a T-cell independent (TI) carrier produces a heterogeneous antibody response. Here we show that successive immunizations of C57BL/6 mice, first with the hapten NP coupled to ficoll, a TI carrier, and then one month later with a subliminal dose of the same hapten coupled to a protein carrier, generate a novel set of hybridomas. These hybridomas produce antibodies which are of the IgM isotope and which lack somatic mutation. Some of these antibodies have a much higher affinity for NP than do antibodies which use the prototypical gene combination (VH186.2-lamda 1) of the strain specific response in C57BL/6 mice.

Topics: Animals; Antibody Affinity; Antibody-Producing Cells; Antigens, T-Independent; Base Sequence; DNA; Ficoll; Genes, Immunoglobulin; Hemolytic Plaque Technique; Hybridomas; Immunoglobulin M; Immunologic Memory; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Phenylacetates; Polysaccharides

Two anti-phenyloxazolone (phOx3) and one anti-GAT MAbs from C57BL mice are shown to be coded by VH gene 186.2. This gene has been found earlier to code for several anti-NP (NNP) antibodies (Bothwell et al., 1981) and anti-GT antibodies (Rocca-Serra et al., 1983; Carmack and Pincus, 1986). The L chain partner of the VH 186.2 gene is different in anti-NP and anti-GAT antibodies (Bothwell et al., 1981; Rocca-Serra et al., 1983; Carmack and Pincus, 1986); in anti-phOx antibodies two new unrelated kappa chain V regions were found. Both of the new VK genes involved code frequently for anti-phOx antibodies in BALB/c mice but then with different VH genes. We tested five 186.2-coded antibodies for cross-reactions. Four antibodies were specific, one bound only to NNP, one only to phOx and two only to GT (GAT). The fifth antibody (anti-phOx) bound also to NNP, GAT and ABA-HOP though probably with a low affinity. This is the first demonstration that one V gene can code for three different antibody specificities. It emphasizes the role of the combinatorial element in antibody diversity.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Base Sequence; Genes, Immunoglobulin; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nitrophenols; Oxazolone; Peptides; Phenylacetates; Polymers; RNA, Messenger

We determined the half-lives of several sets of murine monoclonal antibodies spanning all immunoglobulin isotypes in the serum. The antibodies in each set possess the same V region. With this approach, the differences in half-life observed between the different isotypes are independent of the V region carried by the monoclonal antibodies and therefore must relate to each other in the same way as the half-lives of each class of serum immunoglobulins. The half-life of a monoclonal antibody of the gamma 2a isotype is identical to the average half-life of serum IgG2a as previously determined (6-8 days; P. Vieira and K. Rajewsky, Eur. J. Immunol. 1986. 16:871). Therefore, the half-lives determined with monoclonal antibodies possessing the same V region represent the half-life of the serum immunoglobulins. In this way we calculated the half-life of IgM as 2 days, IgG3 and IgG1 as 6-8 days, IgG2b has a half-life of 4-6 days. IgE has a half-life of 12 h. A polymeric form of IgA was found to be eliminated from the serum with a half-life of 17-22 h.

Topics: Aging; Animals; Antibodies, Monoclonal; Female; Half-Life; Haptens; Immunoglobulin G; Immunoglobulin Idiotypes; Immunoglobulin Isotypes; Immunoglobulins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Nitrophenols; Phenylacetates

Transfer of peritoneal cells from adult donors into newborn, allotype-congenic mice led to colonization of the recipient mice by donor-derived B lymphocytes expressing the Ly-1 surface marker (Ly-1 B cells). These cells not only persisted in the recipient mice for at least 5 months, but also increased in number. In contrast, bone marrow-derived stem cells did not or scarcely give rise to B cells after intraperitoneal injection into congenic newborn recipients under the same experimental conditions. Approximately half of the IgM in the serum of peritoneal cell-recipients was produced by donor-derived Ly-1 B cells, suggesting that high levels of serum IgM in a normal mouse are produced by this B cell subpopulation. The transferred Ly-1 B cells were able to respond in a normal fashion to alpha (1----3)dextran, but they did not participate in thymus-dependent and -independent (TI II) immune responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). In neither the immune response to alpha (1----3)dextran nor that to NP were we able to detect an influence of the transferred Ly-1 B cells on the selection of the idiotypic repertoire in the recipient mice.

Topics: Animals; Animals, Newborn; Antigens, Ly; B-Lymphocytes; Cell Division; Dextrans; Immunoglobulin lambda-Chains; Immunoglobulin M; Mice; Nitrophenols; Peritoneal Cavity; Phenylacetates; Spleen

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Enzyme-Linked Immunosorbent Assay; Epitopes; Hypersensitivity, Delayed; Immune Tolerance; Mice; Neoplasms, Experimental; Nitrophenols; Phenylacetates; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

The IJ genetic restrictions of suppressor T (Ts) cells are controlled by H-2-related determinants that are expressed on antigen-presenting cells. This has led to the hypothesis that Ts cells carry receptors for a self H-2-related ligand that is expressed on specialized antigen-presenting cells. We refer to this H-2-related ligand as the IJ interacting molecule. This report evaluates the ability of rabbit antibodies directed against idiotypes on monoclonal anti-IJ antibodies (the latter are presumably reactive with the Ts cell receptor) to bind IJ interacting molecule and to inhibit antigen presentation to Ts cells. Such anti-idiotypic reagents were prepared against T cell-reactive monoclonal anti-IJk and anti-IJd antibodies. The F(ab')2 fragments of these anti-idiotypic reagents blocked Ts cell induction. The inhibition was haplotype specific and mapped to the IJ region. The anti-idiotypic antibodies blocked the generation of Ts1, Ts2, and Ts3 cells. The cellular target of the blocking activity mediated by these anti-idiotypic antibodies is a macrophage. This was shown by using a cloned macrophage hybridoma line for both Ts induction and absorption of antibody activity. The combined data support the concept that macrophages express IJ interacting determinants that are responsible for Ts cell induction.

Topics: Animals; Antigen-Antibody Reactions; Antigen-Presenting Cells; Antigens, Surface; Cell Line; Haplotypes; Histocompatibility Antigens Class II; Immune Tolerance; Immunoglobulin Idiotypes; Isoantibodies; Macrophages; Major Histocompatibility Complex; Mice; Nitrophenols; Phenylacetates; Receptors, Immunologic; T-Lymphocytes, Regulatory

Age-related alterations of antigen-specific T cell-mediated suppression have been examined in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. Inducer suppressor T cells (Tsi) were activated in mice at the age of 3 mo (young) or 18 mo (old) by i.v. injection of NP-conjugated syngeneic spleen cells (SC). Spleen cells from the NP-SC-injected mice were subcultured in vitro with spleen cells from normal young or old mice to generate transducer suppressor T cells (Tst). Four days later subcultured cells were added to responder cell cultures 1 day before the PFC assays to trigger effector suppressor T cells (Tse). Responder cell cultures, containing NP-conjugated horse red blood cells (HRBC) and spleen cells from HRBC-primed young or old mice, were assayed on day 4 for anti-NP and anti-HRBC PFC. Suppression was found to be antigen specific and age restricted. NP-specific suppressor cells are easily induced in subculture if the Tsi and Tst cell populations are both derived from young or old mice. Conversely, if Tsi cells from young or old mice are subcultured with Tst cells from mice of a different age, suppression of the anti-NP PFC response is hardly observed. Age restriction was also found to operate in the interactions between subcultured and responder cell populations, indicating that age-matching is required for effective triggering of Tse cells by Tst cells. These results altogether suggest that aging may affect the recognition repertoire expressed in suppressor T cell subsets. Moreover, the finding that suppression is less efficient when exerted on responder spleen cells from old than from young mice provides an explanation for the increased frequency of autoimmune disorders in aging.

Topics: Aging; Animals; Antigens; Autoimmune Diseases; Female; Immune Tolerance; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Nitrophenols; Phenylacetates; Spleen; T-Lymphocytes, Regulatory

Change in the specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (Abs) with time after immunization was studied. The early anti-NP Abs was specific to the ionized (phenolate) form of NP. The specificity changed with time and the late Abs became able to bind to the protonated (phenolic) form as well as the phenolate form of NP. The nucleotide sequences of mRNA coding for variable regions of heavy and light chains suggested that somatic hypermutation contributed to this change of the specificity.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Base Sequence; Female; Haptens; Hydrogen-Ion Concentration; Immunization; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Kinetics; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Nitrophenols; Phenylacetates; RNA, Messenger

Previous work has shown that the expression of a predominant family of idiotypic determinants (NPb) in the in vitro response to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten is dependent on helper activity provided by Ly-1- and Ig-bearing B cells called BH. The ability of these BH cells to perform this idiotype-specific, genetically controlled helper function is related to the NPb idiotype specificity of their cell surface receptors. However, the means by which BH cells communicate with and stimulate NPb idiotypic B cell subsets is unknown. In this paper, an Ly-1- and immunoglobulin-bearing B helper cell hybridoma is described. Supernatants from the hybridoma or its subclones were shown to specifically help the response of NPb idiotypic PFC to NP-Ficoll when added to responder cell cultures depleted of Thy-1 and Ly-1 regulatory cell populations. Under these experimental conditions hybridoma supernatants functioned in much the same fashion as populations of Ly-1- and Ig-bearing BH helper populations described previously. NPb idiotype-specific helper activity was mediated by two separable activities elaborated by the hybridoma, an anti-NPb idiotype antibody and a non-Ig (lymphokine) activity. It was shown that both the Ig and the lymphokine components were required for helper activity. Kinetics experiments showed that the anti-idiotype antibody must be added early in the response to NP-Ficoll, whereas the lymphokine fraction could be added at least as late as day 3 of a 4-day culture in order to observe NPb idiotype-specific help. The data suggest that Ly-1 B cell hybridomas may affect the responsiveness of B cell subsets initially by interaction of anti-idiotype antibody with NPb idiotypic B cell surface receptors, followed by growth or maturation signals mediated by non-Ig lymphokine(s). The possibility that the helper activity of these Ly-1 B cell hybridomas represents the combined effects of an idiotype-specific network system and nonspecific growth or maturation factor activity in direct B cell-B cell interactions is discussed.

Topics: Animals; Antigens, Ly; B-Lymphocytes; Immunoglobulin Idiotypes; Lymphocyte Cooperation; Lymphokines; Mice; Nitrophenols; Phenylacetates; Time Factors

Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Cell Line; Complement Activating Enzymes; Complement C1; Complement C1q; Complement System Proteins; DNA, Recombinant; Electrophoresis, Polyacrylamide Gel; Genes, Immunoglobulin; Glycosylation; Haptens; Hemolysis; Humans; Immunoglobulins; Mice; Nitrophenols; Phenylacetates; Plasmacytoma; Plasmids; Transfection; Tumor Cells, Cultured

The antigen-binding selectivity of 2 sets of anti-DNA antibodies from autoimmune mice and from normal mice was examined. Eighteen affinity-purified anti-DNA auto-antibodies from MRL-lpr/lpr mice were examined for binding to the haptens azobenzenearsonate, phosphorylcholine, (4-hydroxy-3-nitrophenyl)acetyl and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP). Five of these autoantibodies bound to NIP-protein conjugates. In contrast, none of 12 monoclonal antibodies to single-stranded DNA or left-handed Z-DNA induced by immunization of BALB/c and C57BL/6 mice with nucleic acid antigens reacted with the tested haptens. In a reciprocal test of the relationship between anti-DNA and anti-NIP binding, we examined 24 monoclonal antibodies to NIP, from various strains of mice, for binding to DNA. One such antibody from a BALB/c mouse also bound to DNA. These results are discussed in the context of the mechanisms underlying autoantibody hyperproduction.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Autoantibodies; Cross Reactions; DNA; DNA, Single-Stranded; Haptens; Immunization; Immunoglobulin Isotypes; Lupus Erythematosus, Systemic; Mice; Mice, Mutant Strains; Nitrohydroxyiodophenylacetate; Nitrophenols; p-Azobenzenearsonate; Phenylacetates; Phosphorylcholine

Changes with time in specificity and affinity of anti-NP antibodies in C57BL/6 mice after immunization with NP22-chicken gamma-globulin (CGG) were studied by comparing the abilities of the antibodies to bind to NP3-bovine serum albumin (NP3-BSA) at pH 5 and 8. Early anti-NP antibodies (on day 14 after immunization) bound to NP3-BSA at pH 8, but not pH 5. This pH-dependence of binding was explained in terms of the low affinity of the antibody to the phenolic form of NP on the basis of results of fluorescence quenching titration of a monoclonal anti-NP antibody that showed similar specificity to that of the early anti-NP antibodies. Since NP on the CGG molecule ionized with an apparent pK of about 7.4, more than half the NP should be in the unionized (phenolic) form under the immunization conditions. However, early anti-NP antibodies bound preferentially to the ionized (phenolate) form of NP, which was a minor form at neutral pH, whereas later anti-NP antibodies showed ability to bind to both the phenolate and phenolic forms of NP. This change in specificity with time was observed on immunization with T cell-dependent (TD) antigens such as NP-CGG and NP keyhole limpet hemocyanin (KLH), but not with a T cell-independent (TI) antigen such as NP-Ficoll. The heavy (H) chains from the two monoclonal antibodies 3G6 and 3C6, which bound to the phenolate form and both the phenolate and phenolic forms, respectively, were recombined with lambda 1 chains (L3G6 and L3C6) from these antibodies as well as a lambda 1 chain (LHOPC-1) with the amino acid sequence of the germline. Ability to bind to the phenolate form of NP was recovered in all the reconstituted IgGs, while ability to bind to both the phenolate and phenolic form of NP was observed only with IgG reconstituted from H3C6 and L3C6. These results suggest that the specificity corresponding to early anti-NP antibodies were generated even by lambda 1 chains of a germline sequence, but that of late anti-NP antibodies was expressed only by an appropriate pair of H and L chains. The contribution of amino acid substitution by somatic point mutation to the change of specificity with time was discussed.

Topics: Animals; Antibody Affinity; Antibody Specificity; Female; Hydrogen-Ion Concentration; Immune Sera; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Time Factors

A nylon wool-adherent, B cell-enriched population is required during the in vitro induction of third order effector suppressor T cells (Ts3). This B cell population expresses IgM and IgD and is devoid of conventional T cell markers such as Thy-1, L3T4, and Lyt-1. Treatment of the B cell population with anti-NP antibodies expressing the NPb idiotype and complement specifically eliminated the ability to generate Ts cell activity, suggesting that the critical B cells expressed anti-idiotypic receptors. To independently verify the role of anti-idiotypic B cells in the generation of Ts cells, B cells were panned on antibody-coated plates. The results demonstrated that only NPb idiotype-binding B cells could induce effector suppressor cells from naive T cell populations. The combined data demonstrate the role of Ig network interactions in the generation of Ts cells.

Topics: Animals; B-Lymphocytes; Cell Adhesion; Epitopes; Hemolytic Plaque Technique; Immunoglobulin Heavy Chains; Immunoglobulin Idiotypes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes, Regulatory

The control of immunoglobulin class switching appears to involve T cell-derived lymphokines. Such lymphokines have been shown to affect isotype expression in polyclonally activated B cells. We show in this paper that the same lymphokines similarly control isotype expression in an antigen-specific response acting in concert with a "T cell independent" antigen. In this situation, B cell growth factor II (BCGF II) enhances the production of antigen-specific IgM antibodies, whereas the production of antigen-specific IgG1 antibodies is only observed in the presence of B cell differentiation factor gamma (BCDF gamma). These results suggest that these lymphokines (and perhaps additional ones) are involved in the control of isotype expression in antigen-specific responses.

Topics: Animals; B-Lymphocytes; Epitopes; Growth Substances; Haptens; Immunoglobulin Allotypes; Immunoglobulin G; Immunoglobulin M; Interleukin-2; Interleukin-4; Lymphocyte Activation; Lymphokines; Mice; Nitrophenols; Phenylacetates; Recombinant Proteins

The specificity of antibody responses is dependent on the extent to which a given antigen selectively stimulates cells from within a diverse B cell repertoire. Previous studies have shown that the triggering of B cells by T cell-dependent antigens is a highly discriminatory process, and that tolerance induction of immature B cells by antigen is equally discriminatory. This symmetry in the requirements for stimulation and tolerance induction could provide a basis for the capacity of antibody responses to discriminate among foreign antigens and yet minimize self recognition. The extent to which this potential for discriminate recognition is applicable to the mature immune system remains controversial, because B cells reactive to self antigens have been identified and, in addition, several investigators have identified heteroclitic immune responses, such as the response to NP of Ighb mice, wherein antibodies are found with higher affinities for analogues of the immunogen than for the immunogen itself. To further investigate the capacity of B cells to discriminate among closely related antigenic determinants, we analyzed the fine specificity and idiotypic distribution of monoclonal antibodies derived from both splenic B cells and immature sIg- bone marrow B cell precursors stimulated in fragment culture with NP-Hy and its structural analogues NIP-Hy and NNP-Hy. The results indicate that the majority of responsive B cells discriminate among these haptenic determinants; however, lambda-bearing B cells responsive to the NP and NIP determinants represent a highly overlapping set of clonotypes. Comparison of the responses to NP-Hy and NIP-Hy of splenic vs sIg- precursors of this clonotype family suggests that the T cell-dependent stimulation of both mature and immature B cells by antigen is highly affinity dependent. Significantly, the affinity thresholds for both stimulation and tolerance induction of immature B cells appears to be higher than that required for the stimulation of mature splenic B cells. Such a disparity in the requisites for triggering mature vs immature B cells could readily account for the presence of low-affinity self-reactive B cells in the mature B cell pools of normal individuals.

Topics: Animals; Antibody Affinity; Antibody Specificity; Arthropod Proteins; B-Lymphocytes; Bone Marrow; Cell Differentiation; Epitopes; Haptens; Immune Tolerance; Immunity, Innate; Immunoglobulin Idiotypes; Immunoglobulin lambda-Chains; Lectins; Leukocyte Count; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell; Stem Cells

Four murine monoclonal IgE antibodies specific for the hapten, 4-hydroxy-3-nitrophenylacetyl (NP), had been previously found to be heteroclitic in nature in that they bound the crossreacting hapten, 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP), with greater affinity than NP. The influence of antibody affinity on the results of two commonly used assays for IgE, namely the radioallergosorbent test (RAST) and histamine release from rat peritoneal mast cells, was studied using these antibodies. In general, in agreement with previous reports, it was found that affinity influences both RAST and histamine release; however, the affinity constants deduced from equilibrium dialysis measurements for the reactions with monovalent haptens were not directly related to the activities of the antibodies as reflected in assays using multivalent hapten protein conjugates.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Epitopes; Female; Haptens; Histamine Release; Immunoglobulin E; Nitrohydroxyiodophenylacetate; Nitrophenols; Phenylacetates; Radioallergosorbent Test; Radioimmunoassay; Rats; Rats, Inbred Strains

T cell clones specific for IgG2a of the alpha allotype have been isolated from C57BL/6J mice. Antigenic determinants recognized by these clones were localized by using a panel of hybrid IgG2b-IgG2a myeloma proteins. These experiments provide evidence for two distinct antigenic sites, one located in a segment encompassing the hinge region and most of the CH2 domain, and the other in a segment spanning the CH3 domain and the C-terminal eight residues of the CH2 domain. As judged by their failure to respond in the presence of B6.C-H-2bm12 spleen cells, all the clones recognize determinants created, in part, by the I-A beta chain. A strong proliferative response was observed in the presence of spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP, and BALB.B. Experiments testing reactivity directed toward spleen cells from appropriate allotype-congenic mouse strains demonstrated that this response was controlled by Igh-linked genes. These results clearly indicated 1) that Igh-1a-specific T cell clones are stimulated by endogenously synthesized IgG2a, and 2) these T cells recognize shared determinants expressed on IgG2a molecules of various strains. These experiments thus provide strong evidence for presentation of self antigens under normal physiological conditions.

Topics: Animals; Antigen-Presenting Cells; Clone Cells; Epitopes; Female; H-2 Antigens; Immunoglobulin Allotypes; Immunoglobulin G; Immunoglobulin Heavy Chains; Lymphocyte Activation; Male; Mice; Mice, Inbred A; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Mutant Strains; Nitrophenols; Phenylacetates; T-Lymphocytes

The monoclonal anti-idiotypic antibody (mAb2) 87.92.6 directed against the 9B.G5 antibody specific for the virus neutralizing epitope on the mammalian reovirus type 3 hemagglutinin was previously demonstrated to express an internal image of the receptor binding epitope of the reovirus type 3. Furthermore, this mAb2 has autoimmune reactivity to the cell surface receptor of the reovirus. The nucleotide and deduced amino acid sequences of the 87.92.6 mAb2 heavy and light chains are described in this report. The sequence analysis reveals that the same heavy chain variable and joining (VH and JH) gene segments are used by the 87.92.6 anti-idiotypic mAb2 and by the dominant idiotypes of the BALB/c anti-GAT (cGAT) and anti-NP (NPa) responses. [GAT; random polymer that is 60% glutamic acid, 30% alanine, and 10% tyrosine. NP; (4-hydroxy-3-nitrophenyl)-acetyl.] Despite extensive homology at the level of the heavy chain variable regions, the NPa positive BALB/c anti-NP monoclonal antibody 17.2.25 binds neither 9B.G5 nor the cellular receptor for the hemagglutinin. Amino acid sequence comparison between the viral hemagglutinin and the 87.92.6 mAb2 light chain "internal image," reveals an area of significant homology indicating that antigen mimicry by antibodies may be achieved by sharing primary structure.

Topics: Amino Acid Sequence; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Base Sequence; Cloning, Molecular; Epitopes; Hemagglutinins, Viral; Immunoglobulin Idiotypes; Immunoglobulin Variable Region; Mice; Nitrophenols; Peptides; Phenylacetates; Polymers; Reoviridae; RNA, Messenger

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.

Topics: Aging; Animals; Antibodies, Monoclonal; B-Lymphocytes; Epitopes; Immunization; Immunoglobulin Idiotypes; Male; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Spleen; Stem Cells

The nucleotide sequences of the variable regions of lambda 1 chain bearing anti-NP antibodies from the secondary response of C57BL/6 mice were determined. The data indicate that the V186.2 VH gene which dominates the primary anti-NP response is expressed in nine out of 10 secondary response antibodies and is extensively mutated. In the V lambda 1 regions somatic mutations are less frequent. While point mutations predominate, there is suggestive evidence for two conversion events, one involving a one-codon deletion. Most, but not all, secondary response antibodies have a higher affinity (up to 10-fold) for the hapten than is seen in the primary response. The increase in affinity correlates with 'parallel' mutations in CDRs of H and L chains, likely to play a role in hapten binding. The analysis of VDJH rearrangements demonstrates that the secondary response lambda 1 chain-bearing antibodies are produced by a diverse set of B cell clones, which are only rarely expressed in primary responses. These clones are characterized by N-sequence-mediated heterogeneity in the 3' half of CDR3, where the germ line sequence of the D element DFl16.1 predominates in primary response antibodies. The antibodies analyzed in this and in previous work were isolated from idiotypically suppressed mice in order to evaluate whether, intraclonally, idiotype suppression selects antibody mutants into the memory pool, through suppression of the wild-type. A selection of this type was not detectable. However, idiotype suppression may control the pattern of clonotypes expressed in the primary versus the secondary response.

Topics: Animals; Antibody Formation; Base Sequence; Cell Line; Cloning, Molecular; Genes; Haptens; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Immunologic Memory; Liver; Mice; Mice, Inbred C57BL; Mutation; Nitrophenols; Phenylacetates; Poly A; RNA; RNA, Messenger

Hapten-coupled splenic adherent cells or resident peritoneal cells from autoimmune B6.lpr mice that are over 5 mo of age fail to induce first-order inducer suppressor T cells (Ts1). However, the same population of hapten-coupled cells can induce both delayed-type hypersensitivity responses and third-order effector suppressor T cells (Ts3). Thus, splenic and peritoneal antigen-presenting cells from B6.lpr mice display a defined defect in the ability to induce certain suppressor T cell responses. The cellular defect in Ts1 induction is controlled by the lpr gene, since age-matched congenic B6 mice do not display this defect. The splenic adherent cell defect is temporarily correlated with the autoimmunity that develops in B6.lpr animals. The antigen-presenting defect in the B6.lpr splenic adherent population for Ts1 induction is reversible by culturing the cells in interferon-gamma. The results are discussed as an illustration of the relationship between experimental models of autoimmunity and defects in a suppressor T cell cascade.

Topics: Aging; Animals; Antigen-Presenting Cells; Cell Adhesion; Cells, Cultured; Hypersensitivity, Delayed; Interferon-gamma; Kinetics; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Spleen; T-Lymphocytes, Regulatory

Idiotope-specific immunoenhancement or suppression was induced in C57BL/6 mice by the injection of physiological amounts (100 ng-10 micrograms) of monoclonal anti-idiotope antibody. As previously described, nanogram doses enhanced idiotope expression while a 10-micrograms dose of anti-idiotope antibody induced the activation of a population of Thy 1.2+, Lyt 1-, 2+ suppressors. Both positive and negative regulatory activities were confined to the non-mu, idiotope+ compartment of the plaque-forming cell response. Administration of intermediate doses of anti-idiotope antibody resulted in an immune state indistinguishable from that of naive mice. This apparently normal response was in fact the product of a simultaneous activation of balanced enhancing and suppressive activities. When treated with anti-Lyt 2 or Lyt 1 and complement, spleen cell populations taken from such phenotypically "naive" mice revealed latent idiotope-specific immunoenhancement or suppression, demonstrating the components of a functional regulatory equilibrium.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Chickens; Female; Ficoll; Hemolytic Plaque Technique; Immunoglobulin Idiotypes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Nitrophenols; Phenotype; Phenylacetates; Spleen; T-Lymphocytes, Regulatory

Several commonly used strains of laboratory mice from Charles River Laboratories were found to produce extremely low or undetectable levels of serum immunoglobulins bearing lambda 1 light chain (lambda 1 Ig). Individual CF-1, CD-1, and CFW random-bred mice were tested for serum lambda 1 levels, lambda 1-specific anti-NP responses, and genomic polymorphisms at the lambda 1 locus. In all cases, a complete correlation among these parameters was observed. The results indicated that nearly all CFW, greater than 70% of CD-1 but none of the CF-1 mice produced low levels of lambda 1 light chain. The low lambda 1 Ig production is due to a genetic defect either similar or identical to that observed in SJL mice. The data suggest that the lambda 1 locus of CD-1, CFW, and SJL mice are derived from a common ancestor. We also surveyed lambda 1 Ig production in a series of wild mice. Mice producing low lambda 1 Ig were frequently observed. The wild mice with low lambda 1 Ig levels were captured in diverse geographic areas, including Europe, Middle East, Asia, and South America. Preliminary study suggests that the defect in the wild mice is different from that of SJL, CD-1, or CFW mice and implies that other mechanisms regulate lambda 1 Ig production in wild mice.

Topics: Animal Population Groups; Animals; Animals, Wild; Cloning, Molecular; Genotype; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Species Specificity

To assess the significance of somatic point mutation in the hyperimmune response to the hapten NP, an in vivo enrichment procedure was followed. Mice that expressed high titers of B1-8 idiotopic determinants were selected as donors for serial transfer of small numbers of immune spleen cells into syngeneic irradiated recipient mice. Cells expressing B1-8 idiotopic determinants were chosen for enrichment because B1-8 cross-reactive determinants constitute a significant portion of the primary response. Furthermore, B1-8 is a monoclonal antibody derived from a primary response to NP, and its heavy and light chains are unmutated products of the germ-line genes VH186.2 and VL lambda 1, respectively. The germ-line sequence is thus available for comparison with the somatic mutants that arise during enrichment and hyperimmunization. The data show that serial transfer of spleen cells from mice with a high titer of idiotypic determinants results in a dramatic decrease in the titers of antibodies that bind antigen. Three lines of evidence indicate that progeny cells from the initial lambda-positive, idiotype-bearing, antigen-binding cells are successfully transferred and expanded during successive adoptive transfers. First, the proportion of lambda-bearing antibodies relative to NP-specific lambda-bearing antibodies increases with transfer, which is consistent with mutation away from antigen binding. Second, analysis of serum antibodies and hybridoma proteins derived from transfer-recipient mice confirm the presence of idiotype-positive antibodies that do not bind antigen. Third, RNA dot blot analysis of hybridomas constructed from a recipient mouse in the fourth transfer indicates a high frequency of expression of the VH gene predominantly used in the NP response. Many of the antibodies expressed by these hybridomas not only do not bind antigen, but have also lost the determinants recognized by the anti-idiotypic reagents. Most of these VH-positive hybridomas express lambda L chain. The most likely interpretation of the data is that somatic mutation is occurring during the hyperimmune response. Because we selected donor mice that expressed a high titer of idiotype-positive, antigen-specific antibody and immunized the recipient mice, we expected to observe a selective expansion of somatic variants that bound antigen. This was not the case. The observed loss of antigen binding suggests that the majority of mutations arising result in antibodies with lower affinity for

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody Specificity; Carrier Proteins; Chickens; Haptens; Hybridomas; Immunization, Passive; Immunoglobulin Idiotypes; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

The diversity of lambda variable (V lambda) domains is extremely restricted when compared to those of VH and V kappa. In addition, each V lambda domain is determined by the C lambda domain. For these reasons, the lambda system is an excellent model for the study of the associated VH region repertoire. The study of V lambda domain diversity has been limited by the small contribution (approximately 5%) of lambda-bearing proteins to the total Ig pool. We now show that treatment of BALB/c mice with rabbit anti-lambda 1 antibodies coupled to lipopolysaccharide induces a production of polyclonal lambda 1 light chain-bearing Ig whereas, conversely, treatment with rabbit anti-lambda 2 antibodies induces a production of polyclonal lambda 2 + lambda 3 light chain-bearing Ig. The antigenic specificities of these two distinct lambda populations were then studied using B1355 dextran, (4-hydroxy-3-nitrophenyl)acetyl (NP) and 2,4-dinitrophenyl (DNP) as antigens. The anti-alpha(1-3)dextran antibody specificity was found to be mediated exclusively by antibodies bearing the lambda 1 isotype whereas the anti-NP and anti-DNP antibody specificities are mediated by both the lambda 1 and lambda 2 + lambda 3 isotypes. In addition, various mouse strains with the VHa or VHb allotypic haplotype and the rlo lambda 1 or r+ lambda 1 phenotype were treated with rabbit anti-lambda 1 antibodies. The lambda 1 anti-NP and anti-DNP antibody specificities were similar in all strains whereas the lambda 1 anti-alpha(1-3)dextran specificity was linked to the presence of the VHa allotypic haplotype. The mouse strains with the rlo lambda 1 or r+ lambda 1 phenotype did not differ in terms of their patterns of lambda 1 antibody specificities.

Topics: Animals; Antibodies, Monoclonal; Antibody Diversity; Antibody Specificity; Dinitrobenzenes; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred Strains; Nitrophenols; Phenylacetates

Inducer/helper T cells recognize nominal antigen in association with Ia on the surface of the antigen-presenting cell (APC). Recent studies have shown that B cells can effectively function as APC. In the present study we have assessed the ability of cloned inducer T cells to discriminate between activated B cells or splenic macrophages as APC. We found that most of the clones tested demonstrated an equivalent response to antigen presented by activated B cells or splenic adherent cells. Some clones were very efficiently stimulated by antigen presented by activated B cells, whereas other clones failed to respond or responded very poorly when activated B cells were used to present antigen. We attempted to determine the mechanism responsible for the inability of certain clones to proliferate in response to antigen presented by activated B cells.

Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Clone Cells; Haptens; Histocompatibility Antigens Class II; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Nitrophenols; Phenylacetates; T-Lymphocytes

The mechanism of B cell suppression by a T cell hybridoma-derived monoclonal effector suppressor factor (TsF3) was studied in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. The NP-specific effector suppressor cells that produce TsF3 are Lyt-1-, 2+, I-J+, NP-binding T cells and are induced by immunization with NP conjugates. Monoclonal TsF3 inhibits both T cell activity as measured by suppression of contact sensitivity responses and B cell function as measured by suppression of antibody production to both T-independent and T-dependent antigens. The present studies were designed to specifically investigate the mechanisms and genetic restrictions that govern the interactions between TsF3 and its target cells in the plaque-forming cell (PFC) response. The results show that the target of TsF3 is a splenic adherent cell. Suppression will occur only if the restriction specificity of the TsF3 matches the H-2 genotype of the adherent population. Once this TsF3-adherent cell interaction has occurred, suppression of NP-specific B cells can occur across an H-2 barrier. The data also demonstrate that Igh-linked gene products do not appear to play a part in the TsF3-mediated suppression of in vitro PFC responses, which contrasts with the requirements for regulation of T cell-mediated contact sensitivity responses.

Topics: Animals; B-Lymphocytes; Epitopes; H-2 Antigens; Hemolytic Plaque Technique; Hybridomas; Immunoglobulin Heavy Chains; Kinetics; Lymphocyte Activation; Lymphokines; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Spleen; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

Hybridomas generated from C57BL/6 mice immunized with the hapten NP coupled to ficoll, a T-cell-independent carrier, produce monoclonal antibodies that use a large repertoire of VH regions and light chains. This contrasts with the homogeneity of the strain-specific response to NP observed with T-cell-dependent carriers, where most of the antibodies use a single VH region, V186.2, in combination with the lambda-1 light chain. There is no evidence for somatic mutation in any of the sequenced regions of the antibodies generated by NP-ficoll. Thus T cell participation is required for the homogeneity of the strain-specific hapten response, and probably for somatic mutation as well.

Topics: Animals; Antibodies, Monoclonal; Base Sequence; Chickens; Ficoll; gamma-Globulins; Hybridomas; Immunoglobulin Heavy Chains; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.

Topics: Animals; Antibody-Producing Cells; B-Lymphocytes; Epitopes; Hemolytic Plaque Technique; Immunoglobulin Heavy Chains; Immunoglobulin Idiotypes; Lymphocyte Activation; Lymphocyte Cooperation; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes, Regulatory

Eight monoclonal antibodies from the primary response of C57BL/6 mice against the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were isolated. The antibodies carry lambda 1 light chains and have similar affinities for the immunizing hapten. Sequence analysis at the level of mRNA reveals that all antibodies express the VH gene 186.2 and all but one the DFl 16.1 gene segment. The J segment of the heavy chain is JH2 in six cases and JH4 in two. Somatic point mutations are scarcely detectable in the antibodies, but there is extensive sequence variability at the boundaries of the D gene segment, mainly at its 5' end. However, seven of eight antibodies express tyrosine in position 99 of the heavy chain, encoded either by the 5' codon of DFl 16.1 or by presumed N sequences. In the former case, the tyrosine is the first of a stretch of three (positions 99-101). In the latter, a similar stretch (positions 99, 101, 102) is interrupted by aspartic acid, asparagine or cysteine in position 100. These variations profoundly affect idiotypic specificity. Six of the eight monoclonal antibodies came from mice neonatally suppressed by an anti-idiotope antibody whose target idiotope is regularly expressed in primary anti-NP responses and depends upon a non-germ-line-encoded aspartic acid in position 100 of the heavy chain. The sequence data show that the mice circumvent suppression by expressing antibodies which lack this aspartic acid but are otherwise structurally very similar to anti-NP antibodies from normal animals. Since suppression in the animals is partly controlled by regulatory T cells, we conclude that these T cells are highly restricted in their specificity in that they preferentially see a determinant which also depends upon the aspartic acid in position 100. The data suggest that the VH to D boundary serves as a target of idiotypic selection.

Topics: Amino Acid Sequence; Animals; Animals, Newborn; Antibodies, Monoclonal; Antibody Affinity; Base Sequence; Genes; Haptens; Immune Tolerance; Immunoglobulin Idiotypes; Mice; Mice, Inbred C57BL; Mutation; Nitrophenols; Phenylacetates; RNA, Messenger; T-Lymphocytes

To evaluate the contribution of environmental regulatory mechanisms in fashioning the primary B cell repertoire, we have compared the repertoire of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific primary splenic B cells with that of precursor cells present as surface immunoglobulin-negative (sIg-) cells in adult bone marrow of C.B20 (Ighb) mice. Previous analyses using a variety of antigens have led to the conclusion that the antibody repertoire expressed in the spleen is similar to that expressed in newly generated B cell precursors with respect to both repertoire diversity and the representation of various predominant clonotypes. However, in the response to NP of C.B20 precursor cells, two marked disparities have been identified between the repertoire of sIg- bone marrow cells vs splenic precursor cells. The first concerns precursor cells that give rise to lambda-bearing NP-specific antibodies with heteroclitic fine specificity. Such antibodies normally dominate the primary response of Ighb mice; however, the representation of precursor cells giving rise to lambda-bearing antibodies is disproportionately low in the sIg- bone marrow cell population of C.B20 mice. Thus, during the maturation of these cells, post-sIg receptor expression, there is an apparent increase in the proportionate representation of lambda-bearing NP-specific cells. The second disparity concerns precursor cells whose antibody products bear kappa-light chains and exhibit high affinity and homoclitic binding for the NP haptenic determinant. Such precursor cells are poorly represented in the spleen, but represent a sizeable proportion of the sIg- NP-specific precursor cell population. Thus, there seems to be a selective elimination of high affinity, kappa-homoclitic anti-NP antibody-bearing cells as they acquire their sIg receptors. The elimination of this cell population could partially account for the dominance of lambda-heteroclitic antibodies in the serum responses to NP of C.B20 mice.

Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; B-Lymphocytes; Bone Marrow Cells; Cell Separation; Female; Haptens; Hematopoietic Stem Cells; Leukocyte Count; Male; Mice; Mice, Inbred BALB C; Nitrophenols; Phenotype; Phenylacetates; Receptors, Antigen, B-Cell; Spleen

The hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), when conjugated to carrier proteins, elicits a characteristic idiotypic response (NPb) in C57BL/6 mice. The response can be divided serologically into two distinct NPb-positive groups of antibodies. The first group consists of four crossreacting subgroups (I-IV), the second of two subgroups (V, VI). Some antibodies of subgroups I and II have been shown to express the unmutated heavy chain variable region (VH) germline gene 186.2. Antibodies of subgroups V and VI crossreact extensively with the NPa-positive antibodies of BALB/c mice. We sequenced heavy chain complementary DNA from eight hybridomas producing anti-NP antibodies. Six of these belong to subgroups V and VI, and two were NPa-positive hybridomas of BALB/c origin. All sequences were homologous to each other, and differed by approximately 80 basepairs from the 186.2 C57BL/6 germline VH gene. From our sequence and Southern blot analyses we suggest: (a) the NPb idiotypic response is the product of several VH germline genes, (b) some of these genes are very homologous to the gene coding for the BALB/c NPa idiotype, and might represent the C57BL/6 allelic forms of this gene, (c) the diversity regions of NPb and NPa-positive antibodies are diverse in length and amino acid composition, except for the first residue, which is always tyrosine, (d) all four heavy chain joining region gene segments are expressed without mutation. We discuss our data in terms of diversity in the germline VH gene repertoire, as well as diversity created by gene segment-joining events and somatic mutation.

Topics: Animals; Antibodies, Monoclonal; Base Sequence; DNA; Female; Genes; Haptens; Hybridomas; Immunoglobulin Heavy Chains; Immunoglobulin Idiotypes; Immunoglobulin Variable Region; Mice; Mice, Inbred Strains; Nitrophenols; Phenylacetates

The primary anti-(4-hydroxy-3-nitrophenyl)acetyl (anti-NP) antibody response of C57BL/6 mice was analyzed by several methods. Serum analyses by solid-phase radioimmunoassay (RIA) showed that stimulation with the thymus-independent (TI) type 1 antigen NP-LPS results in an anti-NP antibody response dominated by kappa (kappa) light chain bearing antibodies, whilst responses to NP-Ficoll (a TI-2 antigen) and NP-KLH (a thymus dependent antigen) are dominated by lambda (lambda) 1 light chain bearing antibodies. However, in all these responses NP-specific plaque-forming cells (PFCs) were predominantly heteroclitic, and inhibitable by anti-lambda antiserum. In addition, kappa-bearing IgM-producing hybridomas obtained by fusion of spleen cells from NP-LPS-immunized mice, although producing NP-specific antibodies detectable by RIA, were unable to produce NP-specific plaques. Direct determination of the affinity of 5 of those hybridomas by fluorometric titrations showed that their affinity is indeed lower than 10(-5) M. These results suggest that most NP-specific antibodies stimulated by NP-LPS are of too low affinity to be detected in a direct PFC assay, with the exception of a population of lambda-bearing antibodies. Therefore, the differential expression of kappa- or lambda-bearing antibodies in primary responses to the hapten NP presented on different carriers may be due to different affinity requirements for B-cell triggering via different activation pathways.

Topics: Animals; Antibody Affinity; Haptens; Hemolysis; Hemolytic Plaque Technique; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Radioimmunoassay

In the response to NP-lipopolysaccharide or NP-Ficoll predominantly anti-NP antibodies of the IgM class are produced in mice with lower amounts of IgG3 and IgG2b but little or no IgG1 and IgG2a. In contrast, in the primary T-dependent response to NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin high amounts of all IgG isotypes are induced. To investigate whether isotype-specific T cells are responsible for these differences we carried out cell transfer experiments using carrier-specific T cell lines. Two such lines were established and one of the two could be cloned. Upon activation by antigen the T cell lines induced unprimed syngeneic splenic B cells to proliferate and differentiate into antibody-secreting cells in vitro in an antigen-nonspecific way. Antigen-specific activation of unprimed B cells in a cell transfer system in vivo showed that high concentrations of hapten-specific antibodies of all IgG isotypes are induced through both carrier-specific T helper lines. The isotypic pattern of these antibodies is similar to that produced via heterogeneous splenic T cells in the cell transfer system, or in normal animals on immunization with the same antigen. These results suggest that isotype-specific T cells are not required for the production of IgG isotypes in a primary anti-NP response and thus not responsible for the differences seen in isotypic patterns between T-dependent and T-independent responses.

Topics: Animals; Antigens; Antigens, T-Independent; Carrier Proteins; Clone Cells; Epitopes; Hemocyanins; Immunization, Passive; Immunoglobulin Allotypes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Nitrophenols; Phenylacetates; T-Lymphocytes, Helper-Inducer

A helper cell population with phenotypic characteristics of both B and T cells is described. This helper population, called BH, is present in normal unprimed C57BL/6 mice and preferentially helps the expression of NPb idiotype-bearing plaque-forming B cells in the absence of T helper cells. Its surface phenotype is Lyt-1.2+, Ig+, Lyb-3+, Thy-1.2-, Lyt-2.2-. The helper activity of the BH population is IgH restricted and BH cells selectively bind NPb idiotypic determinants. Collectively the data demonstrate that this unique subpopulation can regulate the response of antibody-secreting B cells through specific recognition of idiotypic determinants.

Topics: Animals; Antibody-Producing Cells; Antigens, Ly; Antilymphocyte Serum; B-Lymphocytes; Epitopes; Histocompatibility Antigens Class II; Immunoglobulin Heavy Chains; Immunoglobulin Idiotypes; Lymphocyte Cooperation; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenotype; Phenylacetates; Receptors, Antigen, B-Cell; T-Lymphocytes, Helper-Inducer

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.

Topics: Animals; Antigens, Ly; B-Lymphocytes; Binding Sites, Antibody; Epitopes; Hemocyanins; Histocompatibility Antigens Class II; Immunoglobulin Idiotypes; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenotype; Phenylacetates; T-Lymphocytes, Regulatory

The carrier requirements for the induction of helper and suppressor T (Ts) cells were compared. Although H-2-linked Ir genes control the development of helper T cells and hapten-specific B cells, they do not influence Ts3 generation. That is, GL phi nonresponder C57BL/6 mice can generate NP-specific Ts3 cells after priming with NP-GL phi. The Ts3 cells generated under these conditions are functionally and phenotypically identical to the NP-specific Ts3 cells previously characterized. Furthermore, these Ts3 populations can be specifically depleted with a monoclonal anti-idiotope antibody prepared against monoclonal anti-NP antibodies. By using related polymers, carrier effects on Ts3 induction were noted. NP-D-GL and NP-Ficoll failed to induce Ts3 cells, whereas NP-L-GL induced this suppressor subset. The data demonstrate that Ts3 induction is independent of the carrier requirements involved in helper T cell induction and is not dependent upon B cell priming. The implications of these results with regard to the mechanisms of Ts3 induction are discussed.

Topics: Animals; Antigens; Carrier Proteins; Epitopes; Haptens; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Nude; Nitrophenols; Phenotype; Phenylacetates; Polyglutamic Acid; Polylysine; T-Lymphocytes, Regulatory

Previous studies have shown that dextran B1355 (DEX)- and (4-hydroxy-3-nitrophenyl) acetyl (NP)-coupled antigens triggered, respectively, BALB/c and C57BL/6 (B6) lymphocytes in which the V lambda 1 gene and a specific VH gene (VHDEX and VHNPb) have functionally rearranged. In this paper, we studied whether the closely-related V lambda 2 gene can be utilized in association with these VH genes to generate antigen-specific lymphocytes. We found that the VHDEX gene was restrictedly utilized by the V1 lambda 1 gene to generate anti-DEX lymphocytes, and in contrast, both the V lambda 1 and V lambda 2 genes were utilized together with a VHNPb germline gene to form anti-NP lymphocytes. Southern blot and DNA sequencing of an anti-NP hybridoma confirmed that the germline form of the (186-2) VHNPb gene can be used in association with either the V lambda 1 or V lambda 2 genes.

Topics: Animals; Antibody Diversity; Dextrans; Epitopes; Guinea Pigs; Hybridomas; Immunoglobulin J-Chains; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (gamma, kappa) tumour cells with spleen cells from (BALB/c X C57B1/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-epsilon-3.57, NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31 all possessed lambda 1 (or possibly lambda 3) light chains; and that NP-epsilon-3.57 possessed lambda 2 light chains; NP-epsilon-95.31 also expressed the P3-X20 derived, MOPC-21 kappa light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-epsilon-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Carbohydrates; Electrophoresis, Polyacrylamide Gel; Haptens; Histamine Release; Hybridomas; Immunoglobulin E; Mice; Mice, Inbred Strains; Nitrophenols; Passive Cutaneous Anaphylaxis; Phenylacetates; Radioallergosorbent Test; Rats

When the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) is presented on different carrier molecules, different anti-NP antibody responses are stimulated. On stimulation with NP-lipopolysaccharide (LPS) [T-independent type 1 (TI-1) antigen] kappa + antibodies are the major population, whereas on stimulation with NP-Ficoll [T-independent type 2 (TI-2) antigen], NP-keyhole limpet hemocyanin (KLH) or NP-chicken gamma globulin (CG) [T-dependent (TD) antigens], lambda 1+ antibodies dominate. The relative contribution of idiotopes Ac38 or Ac146 to the lambda 1+ anti-NP response was also different on comparison of TI-1 with TI-2 or TD anti-NP responses. We investigated whether light chain- or idiotype-specific T cells are responsible for these differences. Analysis of the anti-NP response of nude mice after immunization with NP-Ficoll showed lambda 1 dominance. Likewise primary adoptive transfer experiments using carrier-specific T cell lines to reconstitute the TD anti-NP response to NP-KLH or NP-CG, showed that help from carrier-specific T cells alone is capable of stimulating the characteristic lambda 1 dominant response. No significant difference could be found in the levels of Ac38 and Ac146 idiotope expression between mice reconstituted with splenic T cells and those reconstituted with T cell lines. These results suggest that light chain- or idiotype-specific T cells are required neither for the production of lambda 1 light chain dominance, nor for the appearance of idiotopes characteristic of the primary anti-NP response. The possible reasons for differences seen in both light chain and idiotope expression between primary anti-NP responses to the TI-1 antigen NP-LPS and those to TD or TI-2 antigens are discussed.

Topics: Animals; Antibody-Producing Cells; Carrier Proteins; Clone Cells; Ficoll; Immunization, Passive; Immunoglobulin Idiotypes; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred C57BL; Mice, Nude; Nitrophenols; Phenylacetates; T-Lymphocytes, Helper-Inducer

A mechanism responsible for the induction of NP-specific first order (inducer) suppressor cells (TS1) is described. TS1 cells are induced by i.v. administration of hapten-coupled splenic cells. Their activity is assessed by the adoptive transfer of NP-specific suppression during the afferent phase of the contact sensitivity response. NP-coupled firmly adherent, FcR+, I-A-bearing macrophages induce TS1. The antigen-presenting cells required for TS1 induction lack the Thy-1 and Lyt-1 markers, and are resistant to 500 R irradiation and to cyclophosphamide treatment. NP-coupled dendritic cells fail to induce TS1 activity. The induction of TS1 cells is genetically restricted by genes that map in the I-J region of the H-2 complex. The NP-coupled antigen-presenting cells must share at least one I-J allele with the TS1 donor for effective induction of TS1 activity. To minimize allogeneic effects in these studies, the activity of the TS1 population was assessed by adoptive transfer into syngeneic recipients. The present results are compared with the mechanisms required for the induction of second and third order suppressor cells.

Topics: Animals; Cell Adhesion; Cyclophosphamide; Genes, MHC Class II; Histocompatibility Antigens Class II; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Nitrophenols; Phenotype; Phenylacetates; Spleen; T-Lymphocytes, Regulatory

A T-cell hydridoma, 7C3-13-Ag6, which produces a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T-cell factor associated with an I-J determinant, was utilized to study the hapten-binding receptor of T-cells. This hybridoma had been shown to express NP-binding receptor molecules on the cell surface with heteroclitic fine specificity for a cross-reactive hapten, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP). The stoichiometric analysis of the hapten binding by 7C3-13-Ag6 cells was performed by the measurement of direct binding of highly radioactive haptens to the cell surface. The affinity constant (Ka) of the receptor for N125IP-epsilon-aminocaproic acid (N125IP-cap), as calculated from a Hill plot, was 5.75 X 10(7) M-1 [Hill coefficient (a) = 0.86; expression of receptor sites per cell = approximately 1 X 10(3) on average]. The receptor molecule was specifically affinity labeled with photoreactive nitroaryl azide derivatives of N125IP (510-570 Ci/mmole). The specificity of photoaffinity labeling was demonstrated both by competitive inhibition of labeling with NIP- or NP-cap and by differential photoaffinity labeling based on the reversibility of hapten-receptor interaction. The gel electrophoretic analysis of the photoaffinity-labeled molecule indicated that the hapten-binding receptor of 7C3-13-Ag6 has a mol. wt of 28,000 +/- 3000 and an isoelectric point of 5.6-5.7. No immunoglobulin determinants were detected on the molecule. A comparative immunoprecipitation analysis of the membrane lysate of 7C3-13-Ag6 with monoclonal anti-I-J reagents identified a separate I-J molecule of 25,000 +/- 1000 mol. wt that is distinct from the photoaffinity-labeled hapten-binding molecule.

Topics: Affinity Labels; Binding Sites; Fluorescence; Haptens; Hybridomas; Methods; Nitrohydroxyiodophenylacetate; Nitrophenols; Phenylacetates; Photolysis; Receptors, Immunologic; T-Lymphocytes; T-Lymphocytes, Regulatory

There is considerable confusion over whether the antigen-specific T suppressor factors (TsF) described by different authors are indeed equivalent. This paper investigates whether monoclonal TsF3, obtained from hybridomas derived from mice injected subcutaneously with NP derived spleen cells, is functionally equivalent to the conventional T suppressor factor, produced by mice injected intravenously with chemically reactive, water soluble haptene (picrylsulphonic acid and oxazolone thioglycolic acid). Comparison of monoclonal anti-NP TsF3 with conventional anti-picryl and anti-oxazolone T suppressor factor showed that both armed the non-specific T acceptor cell (Tacc) which was sensitive to cyclophosphamide and adult thymectomy. Moreover, non-specific inhibitor (nsINH) of the transfer of contact sensitivity was released when antigen, together with major histocompatibility complex products (MHC), reacted with conventional or monoclonal TsF on the surface of the non-specific T acceptor cell. The interaction of monoclonal TsF3 with antigen, which led to the release of NsINH, required the presence of MHC and was I-J restricted. However, there was no Igh-1 restriction. The equivalence of conventional anti-picryl and anti-oxazolone TsF has been demonstrated by arming the Tacc with a mixture of these two suppressor factors, and then triggering the release of nsINH with the mixed haptene 'picryl-oxazolone-lysine' which crosslinks separate molecules of TsF. A similar equivalence of conventional anti-oxazolone TsF and monoclonal anti-NP TsF3 was demonstrated using the mixed hapten 'NP-oxazolone-lysine' to trigger the release of nsINH. It was concluded that monoclonal TsF3 and conventional TsF were equivalent, and that both had an indirect mode of action through the non-specific T acceptor cell which led to the production of non-specific inhibitor.

Topics: Animals; Cyclophosphamide; Dermatitis, Contact; Haptens; Hybridomas; Immunization, Passive; Lymphokines; Major Histocompatibility Complex; Mice; Mice, Inbred Strains; Nitrophenols; Oxazolone; Phenylacetates; Picryl Chloride; Suppressor Factors, Immunologic; T-Lymphocytes; Thymectomy

A high proportion (greater than 40%) of lambda-anti-NP antibodies were induced after the administration of hapten conjugates of the relatively T-independent antigen NP-Ficoll. In 11 of 12 strains, lambda 1 anti-NP antibodies were the predominant isotype. In lambda 1-defective SJL mice, lambda 2,3 anti-NP antibodies were the major species after NP-Ficoll immunization. In contrast, the ability to elicit a high proportion of lambda-anti-NP antibodies with the T-dependent conjugate of ovalbumin, NP-OVA, varied among mouse strains. Igh-1b-bearing mice were high producers of lambda 1 anti-NP antibodies (greater than 70% of the response); DBA/2 and BALB/c mice were moderate (40 to 50%) lambda 1 anti-NP producers, and A.TL, AKR, NZB, and C3H mice were low lambda 1 anti-NP producers (less than 10%) after primary NP-OVA immunization. In the latter group, NP-OVA preferentially elicits kappa-bearing anti-NP antibodies. The parameters that influence the distribution of light chain isotypes were investigated. The preferential induction of lambda-anti-NP antibodies with NP-Ficoll was a) partially influenced by Igh-linked genes, b) adjuvant independent, and c) maintained on prolonged immunization. In contrast, induction of a high proportion of kappa-anti-NP antibodies by NP-OVA is (a) strictly regulated by Igh-linked genes and (b) enhanced after hyperimmunization. The immunochemical, genetic, and cellular bases for these observations are discussed.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Anti-Idiotypic; Antigens, T-Independent; Ficoll; Guinea Pigs; Immunoglobulin Allotypes; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred A; Mice, Inbred AKR; Mice, Inbred BALB C; Mice, Inbred DBA; Mice, Inbred NZB; Nitrophenols; Ovalbumin; Phenylacetates

Hapten specific T cell proliferation was induced in several strains of mice. When lymph node T cells from 4-hydroxy-3-nitrophenyl acetyl-keyhole lympet hemocyanin (NP-KLH)-primed mice were stimulated in vitro with NP-polymer glutamic acid-lysine-phenyl alanine (NP-GL phi) or NP-ovalbumin (NP-OVA), they displayed a good level of proliferative responses. It was observed that NP-GL phi could induce NP-hapten specific proliferation even with NP-KLH lymphocytes from GL phi nonresponder strains. NP-KLH primed lymphocytes from C57BL/6 (H-2b, Igh-1b), CKB (H-2k, Igh-1b), CWB (H-2b, Igh-1b), and B10.BR (H-2k, Igh-1b) mice showed good proliferative responses to both 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl-GL phi and NIP-OVA antigens. However, NP-KLH primed lymphocytes from C3H/He (H-2k, Igh-1j) and C3H. SW (H-2b, Igh-1j) mice displayed poor proliferative responses to NIP-GL phi and NIP-OVA antigen. These results suggested that the gene coding for the NIP-cross-reaction might be mapped in the Ig heavy-chain linked locus.

Topics: Animals; Cross Reactions; Haptens; Immunoglobulin Allotypes; Immunoglobulin Heavy Chains; In Vitro Techniques; Lymphocyte Activation; Mice; Mice, Inbred Strains; Nitrohydroxyiodophenylacetate; Nitrophenols; Phenylacetates; T-Lymphocytes

Previous work has shown that the injection of antiidiotope antibodies specific for idiotopes of the germline-encoded anti-(4-hydroxy-3-nitro-phenyl) acetyl (NP) antibody B1-8 enhanced or suppressed the expression of B1-8 idiotopes in subsequent humoral anti-NP responses, depending on the dose and perhaps also the isotype of the injected antibody. To formally answer the question of whether the isotype of an antiidiotope determines its effector function in this type of idiotypic control, we have performed regulatory experiments with isotype switch variants selected from two hybridomas secreting anti-B1-8 idiotopes of CBA (Ighj) and C57BL/6 (Ighb) origin. The antibodies of each variant family differ from each other only in the constant region of the heavy chain. The results show that, irrespective of whether an antiidiotope antibody belongs to the IgG1, IgG2b, IgG2a, or IgE class, a 10-ng dose enhances idiotope expression whereas a dose of 10 micrograms exerts a suppressive effect. It emerges from the present and parallel data that the expression of antibody V regions resembling idiotypically that of antibody B1-8 can be enhanced and suppressed by any of four antiidiotope antibodies that recognize distinct idiotopes on those V regions. This suggests that the initial step in the regulatory process induced by an antiidiotope is its binding to antibody V regions carrying the target idiotope. The antiidiotopes preferentially regulate the expression of antibodies that coexpress with the target idiotope other B1-8 idiotopes, despite the fact that some B1-8 idiotopes are also expressed independently of each other in anti-NP responses of idiotypically unmanipulated mice. This finding may reflect high affinity binding of the antiidiotopes to the target against which they were originally raised (i.e., antibody B1-8) or, more likely, a preferential recognition of B1-8-like V regions by regulatory T cells.

Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Binding Sites, Antibody; Binding, Competitive; Genetic Variation; Immunoglobulin Allotypes; Immunoglobulin E; Immunoglobulin G; Immunoglobulin Idiotypes; Immunosuppression Therapy; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

Dorf and colleagues (1-4) found that the contact sensitivity (CS) primed with (4-hydroxy-3-nitrophenyl)acetyl (NP) could be elicited as easily with the iodoanalog (NIP) as with NP when studied in Igh-1b mice but could only be elicited with NP, not NIP, in Igh-1j mice. Since this fine-specificity was parallel to the fine-specificity of anti-NP antibodies in the two types of mice and since anti-NP antibodies of Igh-1b mice are controlled by gene Igh-NPb the authors concluded that CS also was controlled by the Igh-NPb gene. The aim of this study was to confirm their findings with a more quantitative method (5). We confirmed equality of NP and NIP as elicitors of NP-primed CS in Igh-1b mice when the priming antigen was given subcutaneously into non-cyclophosphamide-treated mice (their method). We also found that this priming induced an anti-NP antibody response detectable at the time of challenge. Most experiments were carried out with a method that does not induce a detectable antibody response (pretreatment of mice with 200 mg/kg of cyclophosphamide; application of the sensitizing compound on skin). Since the NP-primed (and NBrP-primed) CS reactions exhibited "expected specificities," the immunizing compound was clearly the most efficient elicitor (relative efficiencies of homologs varied from 2 to 4). The Igh-NPb gene appears not to have a role in "antibody-free" reactions.

Topics: Animals; Cross Reactions; Cyclophosphamide; Dermatitis, Contact; Epitopes; Hypersensitivity, Delayed; Immunity, Cellular; Mice; Nitrophenols; Phenylacetates

The ability of two cloned T cell hybridomas and their products to specifically suppress the in vitro plaque-forming cell (PFC) response to the 4-hydroxy-3-nitrophenyl acetyl hapten (NP) was studied. Supernatant from one hybridoma (TS1) was shown to suppress in the induction but not the effector phase of the immune response. Supernatant from the TS1 hybridoma was capable of inducing second-order (TS2) effector-phase suppressor cells in vitro but did not suppress the response of anti-I-J plus C-treated responder cells. In contrast, supernatant from a second hybridoma (TS3) was capable of suppressing PFC responses when added either in the induction or the effector phase of the response. TS3 supernatant was unable to induce effector-phase suppressor cells but was capable of suppressing the response of anti-I-J plus C-treated responder cells. In addition, specific suppressor factors isolated from supernatants of the TS1 and TS3 hybridomas were shown to bind to NP, bear NPb idiotypic and I-J-encoded but not immunoglobulin-constant region determinants. The factor secreted by the TS3 hybridoma appears to act directly on B cell targets. Mild reduction of this factor results in two separable moieties, only one of which binds NP. Reconstitution experiments suggest that both chains are required for function. The collective data indicate that these hybridomas represent cells from first- and third-order suppressor T cell populations described previously in contact sensitivity and in vitro PFC systems. The implications of the ability of these hybridoma products to affect both T and B cell-mediated immune responses are discussed.

Topics: Animals; Antibody-Producing Cells; Antigens, T-Independent; Complement System Proteins; Epitopes; Hemolytic Plaque Technique; Histocompatibility Antigens Class II; Hybridomas; Kinetics; Lymphokines; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Rabbits; Suppressor Factors, Immunologic; T-Lymphocytes

In mice, granuloma formation after Schistosomiasis mansoni infection is known to be a T cell-dependent response to schistosome eggs that peaks at 6 to 8 wk after infection (early) then regresses to a minimum by 20 to 32 wk (late). This decline in host responsiveness, termed modulation, has been attributed to T cell-mediated suppression. We now have investigated the macrophages from either Early or Late schistosome granulomas phenotypically and found no difference in expression of I-A subregion encoded antigens. We also tested granuloma macrophage (Gm) populations in their capacity to induce positive and negative antigen-specific immune responses to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). As few as 10(3) to 10(4) NP-coupled Early or Late Gm given s.c. were equally capable of inducing MHC-restricted NP-specific delayed-type hypersensitivity (DTH) responses and this DTH-inducing capability was equivalent to that of splenic adherent cell (SAC) controls. Further, both Early and Late Gm were able to generate NP-specific induction-phase suppressor cells (Ts1) when as few as 10(2) NP-coupled Gm were given i.v., the same as NP-SAC controls. Finally, NP-specific effector-phase suppressor cells (Ts3) were equally induced by Early Gm, Late Gm, or SAC controls. Therefore, macrophages derived from Early or Late schistosome granulomas or normal spleens are apparently phenotypically indistinguishable and equally capable, in extremely small quantities, of inducing NP-specific DTH, Ts1, and Ts3 immune responses.

Topics: Animals; Epitopes; Female; Granuloma; Histocompatibility Antigens Class II; Hypersensitivity, Delayed; Immunity, Cellular; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C3H; Nitrophenols; Phenylacetates; Schistosoma mansoni; Schistosomiasis; T-Lymphocytes; T-Lymphocytes, Regulatory

The Ts3 subset of suppressor cells is generated after antigen priming, but, in order to express suppressor activity these cells require an additional activation step involving triggering with specific suppressor factors (TsF2). This report characterizes two cloned hybridoma cell lines (pTs3 hybridomas) that represent this stage of Ts3 cell differentiation. These hybridoma cells could be specifically activated with TsF2 to release another antigen-specific suppressor factor (TsF3) within 6 h. The inducible feature of these cells permitted analysis of the signals necessary for Ts3 activation. Antigen was not required for activation. Only TsF2 factors derived from antiidiotypic second-order suppressor cells could activate pTs3 hybridoma cells. There were stringent genetic restrictions on the ability of Ts2 to activate pTs3 cells. Triggering of pTs3 required corecognition of two determinants on the TsF2 molecular complex, i.e., the I-J and Igh-related idiotypic determinants. Thus, although pTs3 cells could absorb TsF2 from an I-J-mismatched source, these pTs3 were not activated by the allogeneic TsF2. For activation to occur, the H-2 (I-J) and Igh complexes of the TsF2 donor had to match those of the strain from which the pTs3 cells were derived. Mixing two distinct TsF2, one derived from an H-2-matched source and the other from an Igh-matched source, failed to activate pTs3 cells. Once activated, the pTs3 cells released a suppressive material that was indistinguishable from the TsF3 factors previously characterized in this system. Finally, the activation of the pTs3 cells apparently does not induce the de novo synthesis of TsF3 since the suppressive activity could be extracted from nonactivated pTs3 cells. Thus, the inducible pTs3 hybridomas represent a mature stage in the differentiation cycle of Ts3 cells and provide a means for studying the nature of the specific signals required for Ts3 activation.

Topics: Animals; Epitopes; Hybridomas; Kinetics; Lymphocyte Activation; Lymphokines; Mice; Mice, Inbred AKR; Mice, Inbred C3H; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Species Specificity; Suppressor Factors, Immunologic; T-Lymphocytes; T-Lymphocytes, Regulatory

BALB/c Lyt-1+ cells that proliferate in response to NP-modified BALB/c Ig fail to respond to BALB/c Ig modified by a different hapten, TNP. By contrast, when NP-modified BALB/c Ig is used as the immunogen in C.B-20 mice, a strain congenic with BALB/c but expressing the Ighb allotype of C57BL/6 (B6), a partial cross-reaction between NP- and TNP-BALB/c Ig is observed. Similarly, strains expressing different Igh haplotypes also show distinct reactivities toward NP-B6 Ig and NP-modified myeloma proteins. Thus, the proliferative response to NP-modified Ig is regulated by Igh-linked genes. In this paper, we also characterize the T cell proliferative response to IgG2a of the b allotype. We show that allotypic determinants on this molecule are recognized in association with self MHC-encoded gene products and that responsiveness is controlled by MHC-linked Ir genes. Thus, products of MHC-linked genes also influence the activity of Ig-recognizing T cells. Taken together, experiments described in this report suggest that T cells directed against immunogenic determinants on antibody molecules are governed by the same rules as T cells that respond to conventional antigens.

Topics: Animals; Antigens, Ly; Epitopes; Genes, MHC Class II; Immunoglobulin Allotypes; Immunoglobulin G; Immunoglobulin Heavy Chains; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Myeloma Proteins; Nitrophenols; Phenotype; Phenylacetates; T-Lymphocytes; Trinitrobenzenesulfonic Acid

In vivo and in vitro approaches for measuring DTH to NP and the cross-reactive hapten, NIP, were taken. Mice were immunized subcutaneously with NP-OVA, NP-BGG or NP-CGG in CFA or NP-spleen cells, challenged intradermally with NP or NIP-coupled to a heterologous carrier, and footpad or ear swelling determined 4, 24, and 48 h later. Alternatively, draining LNC were removed and challenged in vitro with either haptenated protein or haptenated, irradiated, syngeneic spleen cells to induce lymphotoxin (LT) production or proliferation. Our results show that although carrier-specific DTH responses are easily elicited both in vivo and in vitro, NP-specific DTH effector cells cannot be evoked by conventional immunization regimens. This failure to induce hapten-specific DTH is not due to suppressor mechanisms. Attempts to induce LT production and T cell proliferation by re-exposure to NP were unsuccessful. Immunization with NP-coupled protein in CFA does elicit an intense Arthus reaction when mice are challenged with the hapten 8 days later. The antibody-mediated nature of this hapten-specific response is indicated by the kinetics of the reaction, which peaks 4 hr after challenge, intensely positive ELISA of circulating anti-NP antibodies, sensitivity to pretreatment with a high dose of cyclophosphamide, and the ability to transfer the reaction to naive recipients with serum. This early response is highly cross-reactive with NIP and is not restricted to mice of the igh-1b allotype.

Topics: Animals; Arthus Reaction; Carrier Proteins; Cattle; Chickens; Cyclophosphamide; Epitopes; Female; gamma-Globulins; Hypersensitivity, Delayed; Immunization, Passive; Lymphotoxin-alpha; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred CBA; Nitrophenols; Ovalbumin; Phenylacetates

We have analyzed a panel of T cell clones that corecognize defined epitopes of the insulin molecule in association with Ia for their patterns of recognition of alloantigens. A striking correlation is observed between recognition of the I-Ab gene product and cow insulin alpha loop and recognition of I-Eu of the PL/J haplotype. These results are consistent with the notion that reactions to foreign major histocompatibility complex (MHC) products reflect molecular mimicry by foreign class II antigens of 'physiologic' complexes formed by autologous class II MHC molecules and antigen.

Topics: Animals; Antibodies, Monoclonal; Antigens; Clone Cells; Epitopes; Female; Genes, MHC Class II; Insulin; Lymphocyte Activation; Major Histocompatibility Complex; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Nitrophenols; Phenylacetates; T-Lymphocytes

Topics: Animals; Antibodies, Monoclonal; Antibody Diversity; Epitopes; Gene Expression Regulation; Genes; Haptens; Immunoglobulin Idiotypes; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutation; Nitrophenols; Phenylacetates; Recombination, Genetic

The (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific T suppressor cell hybridoma 7C3-13 was established by fusing splenic B10.BR T cells enriched on NP-coated petri dishes with the AKR thymoma BW5147. 7C3-13 was selected by anti-NPb idiotypic and anti-I-Jk antibodies in microcytotoxicity tests. The hybridoma expressed H-2k, I-Jk, Qa-1, Thy-1.1 as well as idiotypic (binding site-related) and framework Ig VH determinants, while it was negative for I-A, I-E/C, Thy-1.2, Lyt-1, Lyt-2 and Ig constant region determinants. Hapten-binding receptor material could be isolated from 7C3-13 cells on NP-coupled nylon nets and functionally active T suppressor factor (TsF) could be extracted from the hybridoma. Both types of soluble molecules express NPb idiotype, but the TsF carries I-J determinants in addition while the isolated receptors do not. The molecular weight of the isolated receptor material is 80 000, that of the TsF activity is 27 000 and 57 000-64 000, respectively. We thus were able to show that NP-binding molecules can be obtained in the form of cellular surface receptors, isolated receptor material and extracted TsF from one and the same, monoclonal, cell source.

Topics: Animals; Antigens, Surface; Haptens; Hybridomas; Immunoglobulin Idiotypes; Lymphokines; Mice; Molecular Weight; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory; Thymoma; Thymus Neoplasms

Covalently, cross-linked immune complexes were prepared with multivalent 2-nitro-4-azidophenyl X human serum albumin (NAP X HSA) and antibodies to NAP at five times antigen excess. After purification with gel filtration, affinity chromatography with antigen-agarose column, and addition of the hapten, 9.5% of the antibodies dissociated from the complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. After injection of these cross-linked immune complexes into mice, glomeruli stained for the complexes by immunofluorescence microscopy for only a few hours and electron-dense deposits were not detected. In contrast, when the same immune complexes with comparable lattice but without covalent cross-linking were administered to a second group of mice, the initial deposition by immunofluorescence was comparable and then increased to extensive deposits that persisted to 96 h. In this second group of mice extensive electron-dense deposits evolved. These observations supported the conclusion that the immune complexes initially deposited from circulation must undergo rearrangement to persist and to form electron-dense deposits in glomeruli. The covalently cross-linked immune complexes existed in glomeruli only for a short period of time since these complexes could not rearrange.

Topics: Animals; Antigen-Antibody Complex; Binding Sites, Antibody; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Antibody Technique; Humans; Kidney Glomerulus; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Rabbits; Serum Albumin

Experiments presented in this report demonstrate that specificity of the Ly1+ T cell proliferative response to NP-modified Ig is controlled by Igh-C-linked genes. In addition, we describe the mechanism whereby Igh-C-encoded molecules influence Ly1+ T cell activity. We show that Igh-C-linked control of T cell responses to NP-modified Ig is a secondary consequence of naturally acquired tolerance for self Ig. Unresponsiveness to self Ig is not due to a defect expressed functionally at the level of the antigen-presenting cell, nor is it associated with active suppression. These results suggest that tolerance for self Ig at the level of the Ly1+ T cell is due to functional deletion of Ly1+ T cell clones specific for self Ig. The possibility is considered that regulatory effects mediated by passively administered antibodies may in part be due to induction of Ly1+ T cell tolerance for self Ig.

Topics: Animals; Antigens, Ly; Immune Tolerance; Immunoglobulin Heavy Chains; Immunoglobulins; Lymphocyte Activation; Major Histocompatibility Complex; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; T-Lymphocytes; Trinitrobenzenes

The response of C57BL/6 and BALB/c mice to immunization with proteins coupled to (4-hydroxy-3-nitrophenyl)acetyl (NP) is dominated by distinctly different sets of antibodies. The VH gene family previously shown to be involved in the C57BL/6 response has now been shown to have highly homologous counterparts in BALB/c but of five sequenced BALB/c VH regions, none appeared likely to be able to encode an NP-binding protein. The active VH region from a BALB/c hybridoma making a characteristic anti-NP antibody was recovered and sequenced and shown to be quite different from the VH gene family involved in the C57BL/6 response. Comparison of the variation of the closely related VH regions between the two mouse strains showed that there are separate types of evolutionary pressures on the framework and complementarity-determining regions. The molecular basis for strain-specific immune responses appears to be that the structural divergence of VH regions between mouse strains is great enough that different strains use different VH regions for making the predominant class of antibodies to a specific hapten.

Topics: Amino Acid Sequence; Animals; Base Sequence; Biological Evolution; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Nucleic Acid Hybridization; Phenylacetates; Polymorphism, Genetic

Hapten-reactive inducer T cell clones can be divided into two groups based on their activation specificity. The first and largest group is conjugate specific. These clones are activated only by hapten coupled to the same carrier protein used for in vitro selection. The second group, which is quite rare, is hapten specific. Clones of this type are activated by hapten coupled to all foreign and autologus proteins tested. Both types of clones corecognize soluble antigen in association with products of the I-A locus. The hapten-specific cells were used to analyze the molecular basis of I-A vs. I-E gene control. The physiologic significance of hapten- and carrier-specific inducer T cells in the response to foreign antigens and autoantigens is discussed.

Topics: Animals; Clone Cells; Haptens; Histocompatibility Antigens Class II; Lymphocyte Activation; Major Histocompatibility Complex; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes; Trinitrobenzenes

Direct binding of 125I-labeled rabbit anti-NPb idiotype antibodies (RaId) was used to quantitate the expression by immune spleen and thymus cells of NPbId, the characteristic Id of the lambda 1-containing antibodies made by C57BL/6 (B6) mice to the (4-hydroxy-3-nitrophenyl)acetyl (NP) group. Direct binding of RaId by B and T cell preparations reached a maximum of 12 ng RaId per 10(8) cells at 7 days after immunization. Spleen T cell preparations maintained similar levels of binding after positive selection for Thy-1.2+ cells and overnight culture. RaId binding was also demonstrated for immune B6 thymus cells and for spleen and thymus cells of immune SJL mice, which have the appropriate heavy chain allotype for NPbId expression but have only barely detectable serum Id. However, the NPbId of T and B cell preparations were indistinguishable by (a) the susceptibility of RaId binding by the cells to inhibition by hapten or by antibodies to the variable regions of lambda light chains (anti-V lambda) and by (b) the ability of anti-V lambda and of monoclonal antibodies to the constant region of lambda 1 chains (anti-C lambda 1) to immunoprecipitate antigen (NP10-bovine serum albumin)-binding proteins from detergent extracts of isotopically labeled cells. The results strongly imply that virtually all of the NPbId of T cell preparations is due to conventional NPbId antibody that is tightly bound to T cells. The results do not, however, exclude the possibility that the T cell preparations contain a trace amount (less than or equal to 1 ng/10(8) cells) of unusual NPbId-like molecules that lack lambda chains.

Topics: Animals; B-Lymphocytes; Binding Sites, Antibody; Binding, Competitive; Immunoglobulin Idiotypes; Kinetics; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenotype; Phenylacetates; Rabbits; Receptors, Antigen; Spleen; T-Lymphocytes; Thymus Gland

Topics: 2,4-Dinitrophenol; Animals; Antibodies, Monoclonal; Antibody Formation; B-Lymphocytes; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Dinitrophenols; Gene Expression Regulation; Haptens; Immunoglobulins; Mice; Nitrophenols; Phenylacetates; Phosphorylcholine; Receptors, Immunologic; Spleen

The involvement of a third-order suppressor T cell population (Ts3) in the suppression of in vitro PFC responses was analyzed. It was shown that Ts2 effector-phase suppressor cells, induced by the i.v. injection of NP-coupled syngeneic spleen cells, require a third suppressor T cell population to effect NPb idiotype-specific suppression of an in vitro B cell response. This Ts3 population was shown to be present in NP-primed but not unprimed donors. The Ts3 population specifically binds NP and is Lyt-1-, Lyt-2+, I-J+ and bears NPb idiotypic determinants. The involvement of the Ts3 population in a suppressor pathway that requires recognition of idiotypic determinants is discussed.

Topics: Animals; Antibody-Producing Cells; Epitopes; Erythrocytes; Guinea Pigs; Haptens; Hemolytic Plaque Technique; Histocompatibility Antigens Class II; Immunoglobulin Idiotypes; Male; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Sheep; Spleen; T-Lymphocytes, Regulatory

Topics: Animals; Dermatitis, Contact; Dinitrobenzenes; Epitopes; Glucans; Immune Tolerance; Immunity, Cellular; Immunoglobulin Idiotypes; Kinetics; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Immunologic; T-Lymphocytes, Regulatory

Topics: Animals; Antibodies; Carrier Proteins; Cattle; gamma-Globulins; Immunization; Immunologic Techniques; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrohydroxyiodophenylacetate; Nitrophenols; Phenylacetates; Serum Albumin, Bovine

Topics: Animals; H-2 Antigens; Hypersensitivity, Delayed; Immune Tolerance; Immunoglobulin Variable Region; Lymphokines; Mice; Nitrophenols; p-Azobenzenearsonate; Phenotype; Phenylacetates; Receptors, Antigen, T-Cell; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

We have isolated by fluorescence-activated cell sorting an IgG1 expressing class switch variant (S24-1/47) from the IgG3-producing mouse hybridoma cell line S24/63/63. Both Ig bind the hapten 4-hydroxy-3-nitro-phenylacetyl (NP) with approximately the same affinity and fine specificity and are idiotypically indistinguishable. The apparent m.w. is 50,000 for the gamma 1 and 51,000 for the gamma 3 chain. The genomic DNA of IgG3-producing S24/63/63 cells contains a 14-kb Eco RI restriction fragment with C gamma 3 sequences representing a rearranged C gamma 3 gene. In the class switch variant S24-1/47 the C gamma 3 gene is deleted.

Topics: Animals; Binding Sites; Cell Line; Cell Separation; Clone Cells; Genetic Variation; Goats; Haptens; Hybridomas; Immunoglobulin Constant Regions; Immunoglobulin G; Immunoglobulin Heavy Chains; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Rabbits

Topics: Animals; Cell Line; Galactose; H-2 Antigens; Immunoglobulin D; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Immunoglobulin M; Immunoglobulin Variable Region; Mannose; Mice; Nitrophenols; Oligosaccharides; Phenylacetates; Tunicamycin

In the present analysis we dissect the idiotype repertoire, independently of hapten-binding specificity, by immunizing different strains of mice with cross-linked monoclonal anti-idiotope antibodies against antibody B1-8. B1-8 is a monoclonal antibody with specificity for the hapten (4-hydroxy-3-nitro-phenyl)acetyl (NP) and carries a germ line gene-encoded variable region. The results demonstrate that the expression of B1-8 idiotopes and their association with each other and with NP-binding specificity are strain-specific. Certain idiotopes are expressed on antibodies differing in antigen-binding specificity, whereas one of the idiotopes appears strictly associated with NP-binding antibodies. The genetic analysis provides strong evidence that the strain specificity of the idiotope repertoire is a result of V region polymorphism in the mouse.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Binding Sites; Gene Expression Regulation; Immunization; Immunoglobulin Idiotypes; Mice; Mice, Inbred Strains; Nitrophenols; Phenylacetates; Species Specificity

Topics: Animals; Clone Cells; Haptens; Hybridomas; Immunologic Techniques; Lymphokines; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell; Suppressor Factors, Immunologic; T-Lymphocytes

A comparative antigenic analysis was carried out to determine whether cross-reactivity exists between the major idiotypic responses to (4-hydroxy-3-nitrophenyl)acetyl (NP) in BALB/c and C57BL/6 mice. Extensive cross-reactivity exists between the NPa (BALB/c) and NPb (C57BL/6) allotype-linked idiotypic responses to NP. The cross-reactive determinants of the NPb idiotype are confined to one particular group of NPb-positive monoclonal antibodies. The extent of cross-reactivity between this group of C57BL/6 antibodies and idiotype-positive monoclonal antibodies of BALB/c is so great that they cannot be readily distinguished as NPb- or NPa-positive antibodies with polyclonal anti-idiotypic reagents. That this cross-reactivity is not unique to monoclonal antibodies was confirmed by the demonstration of these cross-reactive determinants in the immunesera of individual BALB/c and C57BL/6 mice. Additionally, evidence was obtained from these experiments and from earlier ones from this laboratory which suggests that the BALB/c idiotypic response to NP-protein conjugate is more homogeneous than the C57BL/6 idiotypic responses.

Topics: Animals; Antibody Specificity; Binding Sites, Antibody; Cross Reactions; Genes; Haptens; Immunoglobulin Idiotypes; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

Twenty-one rabbit serum samples were assayed for IgG antibodies against 3-iodo-5-nitrophenyl-epsilon-amino-n-caproic acid (NIP-cap) by equilibrium dialysis and enzyme-linked immunosorbent assay (ELISA). The results of ELISA, expressed either as absorbances or titers, did not correlate to antibody concentration or average affinity measured by the equilibrium dialysis. Affinity distributions of the antibodies within each sample were determined by equilibrium dialysis in 30 antigen concentrations. Thus, it was possible to study the correlation of ELISA results with separate ranges of antibody affinities. ELISA absorbances measured at a low sample dilution (1:80) correlated with high affinity antibodies (affinity greater than 8 X 10(-6) M-1). Use of high antigen density increased this correlation. End-point titers obtained at a low cut-off absorbance level tended to correlate to a larger range of affinities (affinity greater than 6 X 10(4) M-1). ELISA measures antibodies of different affinities depending on how the results are expressed. This can be utilized in qualitative characterization of the measured antibodies.

Topics: Animals; Antibody Affinity; Cattle; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Immunoglobulin G; Nitrophenols; Phenylacetates; Rabbits; Spectrophotometry

Helper cells, with specificity for the haptens (4-hydroxy-3-nitro-phenyl)acetyl (NP) or (4-hydroxy-5-iodo-3-nitro-phenyl)acetyl (NIP), were raised in B10.BR mice by in vivo priming and in vitro long-term enrichment with hapten-derivatized syngeneic spleen cells. Upon co-culture with the homologous antigen (NP or NIP self), selected helper cells specifically responded by proliferation and by inducing large numbers of B cells to clonal expansion and immunoglobulin secretion. Criss-cross experiments demonstrated the nonheteroclitic nature of antigen recognition by helper cells, as the proliferative and helper cell activities were in every case one order of magnitude higher when confronted with the homologous hapten used for immunization.

Topics: Animals; Binding Sites; Epitopes; Haptens; Lymphocyte Activation; Lymphocyte Cooperation; Mice; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell; T-Lymphocytes

Suppressor factor derived from three different murine T cell hybridomas were characterized . They specifically inhibited 4-hydroxy-3-nitrophenyl acetyl cutaneous sensitivity responses. The factors bind antigen and bear I-J and idiotypic determinants, but lack conventional immunoglobulin constant-region determinants. The factors function during the induction phase of the immune response, by inducing a second population of suppressor cells (Ts(e)). Suppressor factor can inhibit both cellular and plaque-forming cell responses in appropriate strains of mice. These hybridoma suppressor factors directly suppress strains of mice that are Igh-V homologous with the strain producing the factor. Thus, there is an apparent Igh-V restriction in the activity of these factors. However, this is a pseudogenetic restriction because these factors generate second order suppressor cells (Ts(e)) in Igh-incompatible mice, but in order to express the suppressive activity, the cells must be adoptively transferred into recipients that are Igh compatible with the strain producing the suppressor factor. Finally, it was shown that the factor-induced Ts(e) population is under an apparent dual genetic restriction. Thus, Igh and H-2 homology is required in order for the Ts(e) population to express its suppressive activity.

Topics: Antibody Formation; Clone Cells; Epitopes; Haptens; Hybrid Cells; Immune Tolerance; Immunity, Cellular; Nitrophenols; Phenylacetates

We have made several T-T hybridomas which secrete soluble factors capable of suppressing an in vitro antibody response to nitrophenol (NP), but not other unrelated antigens. These factors bind specifically to NP, and express determinants coded for in the I-J region of the mouse major histocompatibility complex. No determinants that cross-react with the constant regions of mouse immunoglobulins are present on the factors. Three sub-clones originating from the same initial culture well of hybridoma cells secrete factors which carry I-J determinants of different haplotypes. One clone expresses I-J determinants derived from the suppressor cell parent, another expresses I-J determinants derived from the tumour cell parent, and a third expresses both. This correlates exactly with I-J determinants expressed on the cell membrane, and suggests the participation of at least two genes in the determination of suppressor-factor structure.

Topics: Animals; Antigens, Surface; Clone Cells; Epitopes; Genes, MHC Class II; Haptens; Hybrid Cells; Karyotyping; Mice; Nitrophenols; Phenylacetates; T-Lymphocytes, Regulatory

The serological characteristics of the antigen receptor on 4-hydroxy-3-nitrophenylacetyl (NP) specific suppressor T cell hybridomas were analyzed. Three T-cell hybrids could be lysed with anti-idiotype and complement. The reactivity pattern observed from a panel of anti-idiotypic reagents indicated that NPb determinants were detected on all three hybrid lines. NP conjugates with bovine serum albumin or caproic acid specifically inhibited the complement-mediated lysis of these cells by both anti-NPb idiotype and anti-I-J antisera. These hapten conjugates failed to block lysis by anti-Thy 1 or anti-H-2K antisera on the same target cell populations. The data indicate that both I-J and Igh variable region gene products are intimately involved in the recognition of antigen by suppressor T cells. Finally, the suppressor cell hybrids produce soluble factors that mediate antigen-specific suppression. The characteristics of the cells and their factors indicate that the hybrids correspond to the Tsi or first-order suppressor cells in the suppressor cell pathway.

Topics: Animals; Cell Line; Epitopes; Haptens; Hybridomas; Immunoglobulin Idiotypes; Isoantibodies; Major Histocompatibility Complex; Mice; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell; T-Lymphocytes, Regulatory

Immunization of C57BL/6 mice with bovine gamma-globulin (BGG) conjugated with (4-hydroxy-3-nitrophenyl) acetyl (NP) induced a population of anti-NP antibodies that bear predominantly lambda light chain, exhibit heteroclitic affinity for heterologous NP derivatives, and share NPb idiotype. The present study analyzes the idiotypes of antibodies induced with BGG conjugated with the iodo-, bromo-, or nitro-NP derivatives (NIP, NBrP, and NNP). NIP-BGG, NBrP-BGG, and NNP-BGG, induce specific antibodies bearing NPb idiotype in C57BL/6 or C57BL/10 congenic mice, but not in many other inbred strains. Furthermore, the quantity of NPb idiotype in immune sera from various mouse strains immunized with NIP-BGG, NBrP-BGG, and NNP-BGG was similar to that in sera from mice immunized with NP-BGG. Anti-idiotypic antisera against C57BL anti-NP, anti-NIP, or anti-NBrP antibodies exhibit extensive idiotype binding of specifically purified B6 anti-NP, -NIP, -NBrP, and -NNP antibodies. These purified antibodies contain a high percentage (greater than 70%) of lambda-chain-bearing molecules. The data indicate that an extensively shared repertoire composed of predominantly lambda-bearing NPb-positive idiotypic antibodies is used in response to NP and its derivatives in C57BL mice.

Topics: Animals; Antibody Specificity; Cattle; Cross Reactions; gamma-Globulins; Guinea Pigs; Immune Sera; Immunoglobulin Idiotypes; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Rabbits

The fine specificity of anti-idiotypic, effector-phase suppressor T cells (Ts2) induced by the intravenous injection of syngeneic spleen cells covalently coupled with the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten was studied in an in vitro plaque-forming cell system. By comparing the ability of these suppressor cells to bind monoclonal anti-NP antibodies that express different levels of serologically detected NPb idiotypic determinants, it was shown that anti-idiotypic suppressor T cells do not recognize the predominant NPb idiotypic determinants that are defined by serologic analysis. The implications for the possible expression and/or recognition of different sets of idiotypic determinants on T and B cells are discussed.

Topics: Animals; Antibodies, Monoclonal; Epitopes; Guinea Pigs; Haptens; Immunoglobulin Idiotypes; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes; T-Lymphocytes, Regulatory

In the current study, we examine the mechanism of suppression of cutaneous sensitivity (CS) responses to 4-hydroxy-3-nitrophenyl acetyl succinimide ester. Intravenous administration of haptenated syngeneic spleen cells induces a state of hapten-specific tolerance involving I-J bearing suppressor T cells that function at either the induction phase or the effector phase of the CS response. The effective phase suppressor cells (Tse) are genetically restricted by both Igh and H-2 region genes. However, a third cell population is also required in he immune lymphocyte population for immune suppression. This third cell population, termed Ts3, is an I-J+, cyclophosphamide-sensitive T cell, as shown by reconstitution experiments. Further, the Tse-Ts3 interaction is restricted by genes in he H-2 and Igh gene complexes. The results are discussed with respect to the pathway of cellular interactions leading to immuno suppression.

Topics: Animals; Antibodies; Antilymphocyte Serum; Cyclophosphamide; H-2 Antigens; Haptens; Hybrid Cells; Hypersensitivity, Delayed; Immune Tolerance; Mice; Nitrophenols; Phenotype; Phenylacetates; T-Lymphocytes; T-Lymphocytes, Regulatory

The ability of T suppressor cells, induced by the intravenous injection of 4-hydroxy-3-nitrophenyl acetyl (NP)-modified syngeneic spleen cells, to affect an ongoing B cell response was studied in vitro. It was found that the expression of NPb idiotype-positive B cells could be selectively inhibited by the addition of antigen-induced suppressor cells in the last 24 h of the in vitro culture. This effector-phase suppression of B cell responses was antigen specific and mediated by an Lyt 1-, Lyt 2+, idiotype-binding, T cell population whose suppressive function was restricted by genes linked to the Igh locus.

Topics: Animals; Antibody Formation; Antigens, Surface; B-Lymphocytes; Cells, Cultured; Haptens; Immune Tolerance; Immunoglobulin Idiotypes; Isoantibodies; Lymphocyte Cooperation; Male; Mice; Nitrophenols; Phenylacetates; T-Lymphocytes; T-Lymphocytes, Regulatory

To examine germ line genes of the heavy chain variable region (VH) that might contribute to formation of antibodies of the NPb family, we have derived cDNA clones from two hybridomas making NPb antibodies. One, B1-8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the VH region, had very different D segments and had identical J segments (J2). A set of closely related germ line VH genes was then cloned from a partial Eco RI library of C57Bl/6 DNA. By comparing the germ line VH regions to the cDNA VH regions, we identified seven potential candidates for encoding the VH regions of NPb antibodies. The seven VH regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1-8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1-8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other VH genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their VH regions from the same germ line gene. Certain of the sequenced VH genes contain crippling mutations; the repertoire of germ line VH genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hydridization.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibody Diversity; Base Sequence; Binding Sites, Antibody; Cloning, Molecular; DNA; Genes, MHC Class II; Haptens; Immunoglobulin gamma-Chains; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Mice; Mutation; Nitrophenols; Phenylacetates

Preinjection of C57BL/6 mice with nano-to microgram amounts of a monoclonal IgG1 antibody directed against a binding site-related idiotope of the anti-NP [(4-hydroxy-3-nitro-phenyl)acetyl] antibody B1-8 results in enhancement or suppression of the corresponding and of another B1-8 idiotope in a subsequent anti-NP response, depending on the dose of the injected anti-idiotope antibody. The enhancing and suppressive effects appear two weeks after anti-idiotope administration and are maximal after 6-8 weeks. They are predominantly expressed at the level of IgG, not IgM, antibodies. Enhancement of idiotype expression, i.e. idiotypic memory, can also be induced by the injection of idiotypic antibody of the IgM class, namely antibody B1-8. This effect might represent one of the general mechanisms by which immunological memory is established.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Antibody Formation; Dose-Response Relationship, Immunologic; Female; Immunoglobulin G; Immunoglobulin Idiotypes; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

Topics: Animals; Antibodies; B-Lymphocytes; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Receptors, Antigen, B-Cell

Hybridoma cell lines secreting antibodies specific to (3-nitro-4-hydroxyphenyl) acetyl (NP) were generated by fusion of NP-immunized SJL spleen cells with the SP2/0 cell line. One hybridoma (N-hybridoma) anti-NP antibody (mu, lambda2) was found to partially inhibit (35-40%) the binding of the predominant idiotype in primary C57BL/6 anti-NP antibodies (NPb). Iodinated hybridoma antibody could be completely bound with anti-idiotypic antiserum made against either specifically purified C57BL/6 anti-NP antibodies, SJL anti-NP antibodies, or N-hybridoma antibody. The idiotypic specificities defined with anti-idiotypic antiserum made against N-hybridoma antibody were termed NP-1 idiotype. Strain distribution and genetic mapping studies indicate that the gene(s) controlling the production of NP-1 idiotype is closely associated with Igh-1b and Igh-1e alleles and mapped within the same chromosomal segment that controls the synthesis of NPb idiotype. However, unlike NPb idiotype, the expression of NP-1 idiotype is not influenced by the gene(s) that control lambda1 chain synthesis. Thus, SJL mice that produce low or undetectable levels of NPb idiotype due to a defect in lambda1 chain production express high levels of NP-1 idiotype. Specifically purified C57BL/6 and SJL anti-NP antibodies fully express NP-1 idiotype, the level of which correlates with the level of lambda2 chain-bearing molecules. Nonetheless, further experiments indicate that lambda1-bearing anti-NP antibodies can express extremely weak NP-1 idiotypic cross-reactivity.

Topics: Animals; Antibody Specificity; Binding Sites, Antibody; Chromosome Mapping; Cross Reactions; Goats; Guinea Pigs; Hybridomas; Immunoglobulin Idiotypes; Immunoglobulin lambda-Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Rabbits

Spleen cells from BALB/c mice were fused with the nonsecreting myeloma line X63.Ag8.6.5.3 seven days after immunization with NP-CG (4-hydroxy-3-nitrophenyl) acetyl-chicken gamma-globulin. The hybrid cell lines obtained were analyzed for heavy and light chain distribution, fine specificity, and idiotype. The majority of monoclonal antibodies possessed either gamma 1 or mu chains. The distribution of L chains among these antibodies was approximately half lambda and half kappa . Thirteen monoclonal antibodies were grown as ascites tumors in mice. Examination of their fine specificity patterns showed that all of the lambda antibodies are heteroclitic and have similar fine specificity patterns. Five of the seven kappa antibodies are also heteroclitic, but their fine specificity patterns are more heterogeneous than those of the lambda antibodies. Polyspecific anti-idiotypic sera directed against pooled primary serum antibody (R a-NPa) or against individual monoclonals were used for idiotypic characterization of the monoclonal antibodies. The Ra-NP bound all of the lambda antibodies but none of the kappa antibodies suggesting that the kappa antibodies may be much more heterogeneous and were therefore not recognized in the presence of the more homogeneous lambda antibodies. Further idiotypic analysis demonstrated that the lambda antibodies, although no two are identical, are a very homogeneous group of antibodies which cross-react with one another but not with the kappa antibodies. Some, but not all, of the kappa antibodies cross-react with each other although none are cross-reactive with the lambda antibodies. Because the lambda-associated idiotype is recognized by the R a-NPa and its characteristics are similar to that of the C57BL/6 major idiotype (NPb), it is referred to as NPa. There may be a second major idiotype associated with at least some of the kappa antibodies.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Binding Sites, Antibody; Guinea Pigs; Immune Sera; Immunoglobulin Heavy Chains; Immunoglobulin Idiotypes; Immunoglobulin Light Chains; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Rabbits

NP-O-Succinimide-induced cutaneous sensitivity (CS) responses can be adoptively transferred by NP-primed lymphoid cells into naive K-, I-, or D-compatible recipients. The distinct fine specificities of I- versus D-restricted T cell clones from various strains of mice suggested that presence of different idiotypic receptors on these T cell subsets. We now demonstrate that NP-O-Su immune lymphoid cells are composed of 2 T cell subsets with distinct antigen recognition patterns, as well as different Lyt 2 phenotypes. Thus, I-restricted cells respond to NP-coupled to a protein carrier but not to NP-coupled cells, whereas D-restricted clones react to NP-cells, but not to NP-protein conjugates.

Topics: Animals; Cross Reactions; Cytotoxicity, Immunologic; H-2 Antigens; Haptens; Hypersensitivity, Delayed; Lymph Nodes; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Succinimides; T-Lymphocytes

Topics: Animals; Antigens; Crosses, Genetic; Female; H-2 Antigens; Hypersensitivity, Delayed; Immunization; Immunization, Passive; Male; Mice; Mice, Inbred Strains; Nitrophenols; Phenylacetates; T-Lymphocytes

The ability of suppressor cells induced by the intravenous administration of 4-hydro-3-nitrophenyl acetyl (NP)-modified syngeneic cells to reduce an idiotypic B cell response was studied in both an in vivo and an in vitro system. Idiotype-positive B cells were assayed by the ability of guinea pig anti-idiotypic antiserum to specifically inhibit idiotype-positive plaque formation. It was found that up to 57% of the PFC response in vivo and 100% of the PFC response in vitro was inhibitable with antiidiotypic antiserum. The expression of these idiotype-positive B cells could be suppressed by the transfer of spleen cells form mice treated 7 d previously with NP coupled syngeneic cels. T cells are both required and sufficient for the transfer of idiotype specific suppression. The induction of these idiotype-specific T suppressor cells directly with antigen suggests that recognition of unique determinants on cell surfaces is important for regulation of lymphoid cell interactions. The role of idiotype-specific suppressor cells in the network of lymphoid interactions is discussed.

Topics: Animals; Antibody Specificity; Antibody-Producing Cells; Female; Ficoll; Guinea Pigs; Haptens; Hemolytic Plaque Technique; Immune Sera; Immunoglobulin Idiotypes; Immunosuppression Therapy; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Nude; Nitrophenols; Peptides; Phenylacetates; Rabbits; T-Lymphocytes

Topics: Animals; Antibodies, Monoclonal; Antibody Diversity; Antibody Specificity; Antigen-Antibody Reactions; Electrophoresis, Polyacrylamide Gel; Haptens; Hybridomas; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; Thermodynamics

4-Hydroxy-3-nitrophenyl (NP) derivatized syngeneic spleen cells injected intravenously stimulate maturation of an antigen-binding, idiotype-bearing induction-phase suppressor cell population, as well as an idiotype-binding anti-idiotype-bearing effector-phase suppressor cell population. Both cell types are present simultaneously in the spleen cell population 7-d after their induction. Furthermore, the cell population with antigen-binding properties can, in the presence of NP-derivatized syngeneic cells, induce a population of effector suppressor cells. The precursors of the effector suppressor population are not sensitive to concentrations of cyclophosphamide which prevented the generation of induction phase suppressor cells. These data provide direct evidence in support of the theory of network regulation of immune suppression. X

Topics: Animals; Antibodies, Anti-Idiotypic; Cyclophosphamide; Epitopes; Haptens; Hypersensitivity, Delayed; Immunization; Immunoglobulin Idiotypes; Mice; Nitrophenols; Phenylacetates; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Transplantation, Homologous

4-Hydroxy-3-nitrophenyl acetyl (NP)-derivatized syngeneic spleen cells administered intravenously induced a population of suppressor T cells that could suppress mice previously primed to NP. The effect was demonstrable when the suppressor cells were transferred to NP-primed mice on the day of challenge for delayed-type hypersensitivity (DTH) responses. In contrast to the suppressor T cell population, which abrogates 5-iodo derivative of NP (NIP)-specific DTH responses when administered before antigen priming, the effector-phase suppressors did not efficiently suppress NIP-specific DTH responses, and were not lysed by treatment with antiidiotype plus complement. Adoptive transfer experiments between major histocompatibility complex and allotype congenic strains of mice allowed demonstration of both Igh-V and I-A restrictions in the transfer of this cell population. The implications of these data in terms of network theories and proposed cellular models for negative immunoregulation were discussed.

Topics: Animals; H-2 Antigens; Haptens; Hypersensitivity, Delayed; Immune Tolerance; Immunity, Cellular; Immunoglobulin Allotypes; Immunoglobulin Idiotypes; Major Histocompatibility Complex; Mice; Nitrophenols; Phenylacetates; Receptors, Immunologic; T-Lymphocytes; T-Lymphocytes, Regulatory

The chain composition and fine specificity of the anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) response was examined at the level of individual clones in BALB/c (Ig-1a allotype) and B10.D2 (Ig-1b) mice. The primary anti-NP serum response of B10.D2 is distinguished from that of BALB/c by its restricted heterogeneity and unusual heteroclitic fine specificity, which appear to be associated with antibodies bearing a unique allotype-linked idiotype(s) (NP-b) paired with lambda light (L) chains. At the clonal precursor level, the 10.d2 anti-NP primary response was found to be characterized by less diversity than that of BALB/c and is dominated by several clonotypes with the heteroclitic fine specificity. However, BALB/c also possess abundant heteroclitic precursors (53%), which are expressed but apparently obscured in its more heterogeneous serum response. Heteroclitic fine specificity in both strains is occasionally associated with clones bearing kappa as well as lambda L chains.

Topics: Animals; Antibody Specificity; B-Lymphocytes; Clone Cells; Cross Reactions; Haptens; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Mice; Mice, Inbred BALB C; Nitrophenols; Phenylacetates; Time Factors

The primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody response is known to have a heteroclitic fine specificity, i.e., anti-NP antibodies bind (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP) with greater affinity than NP itself. Past studies of NP-specific DTH responses and NP-specific T cell-mediated suppression have demonstrated sharing of fine specificity patterns and idiotypic structure between receptors on NP-specific T cells and anti-NP antibodies. We now analyze the fine specificity of NP-specific cutaneous sensitivity (CS) reactions to NP-O-succinimide (NP-O-Su) and NIP-O-succinimide (NIP-O-Su). The specificity of these responses is shown to be controlled by genes in the Igh gene complex. Cross-reactive CS responses induced by NP-O-Su elicited by NIP-O-Su were observed in strains of mice possessing the Igh-1b allotype but not in strains bearing the Igh-1c or Igh-1j allotypes. The CS reactivity could be adoptively transferred to naive recipients, and the ability of transfer CS reactivity was T cell dependent. In contrast to the genetic requirement for I-A region homology to adoptively transfer DTH reactions, compatibility at either the H-2K, H-21, or H-2D regions was sufficient to transfer NP-specific CS reactivity to naive recipients. Furthermore, in contrast to DTH responses, cyclophosphamide pretreatment was not required to induce CS responsiveness. Thus, the specificity of NP-O-Su-induced CS responses is controlled by both H-2- and Igh-linked genes.

Topics: Animals; Antibody Formation; Cross Reactions; Cyclophosphamide; Genes; Haptens; Hypersensitivity, Delayed; Immunization, Passive; Kinetics; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Nitrophenols; Phenylacetates; Succinimides; T-Lymphocytes

We have previously shown that cross-reactive sensitivity (CS) responses induced by 4-hydroxy-3-nitrophenyl acetyl-O-succinimide (NP-O-Su) and elicited by its 5-iodo analogue, 4-hydroxy-5-iodo-3-nitrophenyl acetyl-O-succinimide were observed in strains of mice possessing the Igh-1b allotype, but not in strains bearing allotypes Igh-1c or Igh-1j. These CS responses are mediated by T cells and can be transferred to naive recipients that are homologous at either the H-2K, H-2I, or H-2D regions of the major histocompatibility complex. We now extend our analysis of cross-reactive 4-hydroxy-3-nitrophenyl-acetyl (NP)-induced CS responses to inbred strains of mice expressing additional Igh-1 allotypes. In contrast to NP-induced delayed-type hypersensitivity responses, which only display 4-hydroxy-5-iodo-3-nitrophenyl acetyl (NIP) cross-reactivity in Igh-1b-bearing mice, cross-reactive CS responses can also be elicited in NP-primed mice carrying the Igh-1d, Igh-1e, or Igh-1f allotypes. Moreover, cross-reactive NP-induced CS responses could be transferred by NP-O-Su-primed lymph node cells from the AKR (Igh-1d) strain, into naive recipients homologous at the H-2D region, but only non-cross-reactive NP responses could be transferred into strains homologous at the H-2I region. Furthermore, the lack of cross-reactivity in the Igh-1j-bearing C3H strain was not the result of an inability of these mice to recognize NP in association with H-2K/D products, because NP-O-Su-primed cells from C3H donors transferred NP-specific CS responses into both H-2D and H02I homologous recipients. The results are discussed with respect to the nature of the T cell receptors that control NP responses.

Topics: Animals; H-2 Antigens; Haptens; Hypersensitivity, Delayed; Immunity, Cellular; Immunization, Passive; Immunoglobulin Allotypes; Mice; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell; Skin Tests; T-Lymphocytes

Earlier studies have demonstrated that antibodies of the primary response (days 14 to 30) to (4-hydroxy-3-nitrophenyl)acetyl (NP) of C57BL, LP, and 101 mice share idiotype NPb and are heteroclitic (the affinity for certain chemical analogues of NP is higher than the affinity for NP), whereas BALB/c antibodies share idiotype NPa and are homoclitic. Both idiotypes are inherited as allotype-linked traits and have the lambda-chain as the predominant light chain. This study was extended to earlier anti-NP antibodies (7 days) of eight mouse strains (42 individuals) and to anti-NP hybridoma proteins originating from day 6 spleen cells. Only one of the early 42 antisera was found to be homoclitic, and no genetic polymorphism was detectable in fine specificity. In contrast, idiotypes NPa and NPb still followed the genetic rules that had been established earlier. lambda-Chain-bearing BALB/c hybridoma proteins from day 6 of the primary response were more heteroclitic than kappa-chain bearing ones (Krel greater than 20), but two out of three kappa proteins were also heteroclitic (Krel approximately 2). Our results caution against the use of the heteroclitic fine specificity as a typing method. In contrast, they reinforce the value of anti-idiotypic antibodies for this purpose.

Topics: Animals; Antibody Specificity; Immune Sera; Immunoglobulin Idiotypes; Mice; Mice, Inbred A; Mice, Inbred AKR; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Nitrophenols; Phenylacetates; Radioimmunoassay; Time Factors

We have investigated whether cytolytic cells specific for hapten-coupled syngeneic cells have the same public idiotype as serum antibodies against the same hapten, NP-aminocaproyl (NP-cap). Anti-NP-cap antibodies of C57BL mice possess a public idiotype that can be detected by antisera against the whole V region of the antibody molecule or against the VH domain. Neither reagent had an effect on the cytolytic response either when they were used to sensitize the effector cells for complement killing or when used at high concentrations in the culture medium during the secondary stimulation in vitro.

Topics: Animals; Cytotoxicity, Immunologic; Immunity, Cellular; Immunoglobulin Idiotypes; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates; T-Lymphocytes, Cytotoxic

The ability of NP-coupled syngeneic spleen cells to induce antigen-specific T-suppressor cells capable of binding to NP-BSA-coated Petri dishes and mediating transfer of specific suppressive activity to NP was demonstrated. Furthermore, in strains of mice bearing the Ig-1b allotype, including SJL, and in (non-Ig-1b x Ig-1b)F1 hybrids, the NP-specific suppressor cells also interferes with expression of immunity after priming with NIP-BGG. Anti-NPb anti-idiotype antiserum plus complement treatment effectively abrogated the ability to transfer suppression. Formal genetic mapping of the fine specificity of cross-reactivity with Ig-1 allotypic congenic mice implies that expression of this trait is linked to the Ig-1b heavy chain linkage group. The sensitivity of NP-suppressor cells of appropriate strains to anti-idiotype treatment was also consistent with the formal mapping data. These experiments suggest that there are shared V-region structures on antibody and T cells that are crucial in the suppression pathway for the same antigen.

Topics: Animals; Epitopes; gamma-Globulins; Haptens; Hypersensitivity, Delayed; Immunization; Immunoglobulin Heavy Chains; Immunosuppression Therapy; Mice; Mice, Inbred Strains; Nitrophenols; Phenylacetates; Receptors, Antigen, T-Cell; Serum Albumin, Bovine; T-Lymphocytes, Regulatory