muramidase and hen-egg-lysozyme-peptide-(46-61)

Topics: Animals; Antigens; Histocompatibility Antigens Class II; History, 20th Century; Mice; Muramidase; Peptide Fragments; Protein Binding

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.

Topics: Amino Acid Sequence; Animals; Antibody Formation; Antibody Specificity; Arginine; Cell Division; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class II; Lymphocyte Activation; Mass Spectrometry; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Molecular Sequence Data; Muramidase; Mutagenesis, Site-Directed; Peptide Fragments; Recombinant Proteins; T-Lymphocytes

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.

Topics: Actins; Animals; Antigen Presentation; Antigens; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; beta 2-Microglobulin; CD11c Antigen; Cells, Cultured; Dendritic Cells; Genes, Synthetic; H-2 Antigens; Histocompatibility Antigens Class II; HLA-D Antigens; Humans; Hybridomas; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Ovalbumin; Peptide Fragments; Promoter Regions, Genetic; Recombinant Fusion Proteins

Activation of CD4(+) Th cells requires their cognate interaction with APCs bearing specific relevant MHC class II-peptide complexes. This cognate interaction culminates in the formation of an immunological synapse that contains the various proteins and lipids required for efficient T cell activation. We now show that APC lipid raft membrane microdomains contain specific class II-peptide complexes and serve as platforms that deliver these raft-associated class II molecules to the immunological synapse. APC rafts are required for T cell:APC conjugate formation and T cell activation at low densities of relevant class II-peptide complexes, a requirement that can be overcome at high class II-peptide density. Analysis of confocal microscopy images revealed that over time APC lipid rafts, raft-associated relevant class II-peptide complexes, and even immunologically irrelevant class II molecules accumulate at the immunological synapse. As the immunological synapse matures, relevant class II-peptide complexes are sorted to a central region of the interface, while irrelevant class II molecules are excluded from this site. We propose that T cell activation is facilitated by recruitment of MHC class II-peptide complexes to the immunological synapse by virtue of their constitutive association with lipid raft microdomains.

Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Cell Communication; Dose-Response Relationship, Immunologic; Histocompatibility Antigens Class II; Lymphocyte Activation; Lymphocyte Cooperation; Membrane Microdomains; Mice; Mice, Transgenic; Muramidase; Peptide Fragments; T-Lymphocytes; Tumor Cells, Cultured

We report an inducible system of self Ag expression that examines the relationship between serum protein levels and central T cell tolerance. This transgenic approach is based on tetracycline-regulated expression of a secreted form of hen egg lysozyme, tagged with a murine hemoglobin (Hb) epitope. In the absence of the tetracycline-regulated transactivator, serum levels of the chimeric protein are extremely low (< or = 0.1 ng/ml) and the mice show partial tolerance to both Hb(64-76) and lysozyme epitopes. In the presence of the transactivator, expression increases to 1.5 ng/ml and the mice are completely tolerant. Partial tolerance was further investigated by crossing these mice to strains expressing transgenic TCRs. At the lowest Ag levels, 3.L2tg T cells (specific for Hb(64-76)/I-E(k)) escape the thymus and approximately 10% of CD4(+) splenocytes express the 3.L2 TCR. In contrast, 3A9 T cells (specific for hen egg lysozyme(46-61)/I-A(k)) are completely eliminated by negative selection. These data define a tolerogenic threshold for negative selection of Ag-specific T cells by circulating self proteins that are 100-fold more sensitive than previously demonstrated. They suggest that partial tolerance at extremely low levels of self Ag exposure is the result of a restricted repertoire of responding T cells, rather than a simple reduction in precursor frequency; tolerogenic thresholds are T cell specific.

Topics: Amino Acid Sequence; Animals; Autoantigens; Cell Differentiation; Clone Cells; Epitopes, T-Lymphocyte; Gene Expression Regulation; Hemoglobins; Humans; Immunodominant Epitopes; Mice; Mice, Inbred AKR; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Muramidase; Peptide Fragments; Recombinant Fusion Proteins; Repressor Proteins; Self Tolerance; T-Lymphocyte Subsets; Tetracycline; Thymus Gland; Transgenes; Transplantation Tolerance

DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment(s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived A(k)/CLIP complexes, decreased A(k) surface expression, and enhanced A(k) peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied A(k) conformers. On the other hand, the mutation hardly affects E(k) activities. The appearance of mature compact E(k) dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal A(k)/CLIP and a heterogeneous collection of relatively short peptides bound to E(k) molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Differentiation, B-Lymphocyte; Crosses, Genetic; Female; Gene Deletion; H-2 Antigens; Haplotypes; Histocompatibility Antigens Class II; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Molecular Chaperones; Molecular Sequence Data; Muramidase; Peptide Fragments; RNA Editing; Self Tolerance

T-cell receptor-mediated T-cell activation requires cosimulation signal, which can be provided by B7-1 molecule. Our previous study demonstrated that the coexpression of a covalent peptide/major histocompatibility complex class II molecule complex and costimulatory molecule B7-1 by recombinant adenovirus leads to synergy in peptide-specific T-cell activation. However, the viral antigen-specific T-cell activation is not enhanced by B7-1 expressed by the adenovirus. To verify the differential T cell activation by B7-1 and investigate its underlying mechanisms, we constructed an adenovirus coexpressing a covalent complex of hen egg lysozyme peptide/I-Ak (HEL46-61/I-Ak) and B7-1 in the present study. In vivo studies revealed that HEL46-61-specific T-cell response, but not viral antigen-specific T-cell response, was enhanced by B7-1 expression mediated by the adenovirus, suggesting that exogenous B7-1 expression may regulate T-cell response to these two different antigens through distinct mechanisms. Furthermore, our results revealed that antigen-presenting cells were not susceptible to adenovirus infection in vivo. Based on these findings, the possible mechanism of differential B7-1 costimulation on peptide-specific and viral antigen-specific T-cell activation is discussed.

Topics: Adenoviridae; Adenoviridae Infections; Amino Acid Sequence; Animals; Antigen-Presenting Cells; Antigens, Viral; B7-1 Antigen; Blotting, Northern; Cells, Cultured; Gene Transfer Techniques; Lymphocyte Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Muramidase; Peptide Fragments; T-Lymphocytes

It remains unclear which cells present fed antigen and whether their phenotype and/or activation state differs between oral tolerance and priming. Furthermore, it is also controversial whether the presentation occurs locally in the gut and/or systemically. Clarifying these issues will be important for the design and use of oral vaccines, the therapeutic use of oral tolerance and understanding of IBD. Previous studies using activation of T cell receptor transgenic T cells to compare local and systemic distribution of fed antigen have yielded conflicting results. We have therefore employed the C4H3 antibody, specific for HEL peptide (46-61) in the context of I-Ak, to examine antigen distribution in oral tolerance and priming. Our studies indicate that immunologically relevant antigen can be detected in both local and systemic lymphoid tissue soon after feeding antigen in tolerogenic or immunogenic forms. Furthermore, targeting fed antigen to BcR tg B cells via their specific B cell receptor also results in local and systemic presentation and leads to up-regulation of costimulatory molecules on these cells that suggests their role in presentation of the fed antigen.

Topics: Administration, Oral; Animals; Antibodies; Antigen Presentation; Antigens; B-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Dendritic Cells; Histocompatibility Antigens Class II; Immune Tolerance; Immunophenotyping; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Muramidase; Peptide Fragments

We investigated differentiation of CD4 T cells responding to Ag presented by bone marrow-derived dendritic cells (DC) in association with MHC class II (MHC II) molecules. Peptides encapsulated in liposomes opsonized by IgG were taken up by endocytosis. MHC II-peptide-specific T cells responding to this Ag were polarized to a Th1 cytokine profile in a CD40-, CD28-, MyD88-, and IL-12-dependent manner. Th2 responses were obtained from the same transgenic T cell population exposed to the same DC on which MHC-peptide complexes had dispersed for 48 h following uptake of FcR-targeted liposomes. DC that took up the same FcR-targeted liposomes and then were exposed to methyl-beta-cyclodextrin, which chelates cholesterol and dissociates lipid microdomains, also stimulated Th2 differentiation. Incubation of T cells with DC incubated with peptides directly binding to MHC II resulted in Th2 responses, whether or not the DC were coincubated with opsonized liposomes as a maturation stimulus. CD4 Th1 polarization thus appears to depend on MHC II-peptide complex clustering in DC lipid microdomains and the time between peptide loading and T cell encounter.

Topics: Animals; Antigen Presentation; beta-Cyclodextrins; CD4-Positive T-Lymphocytes; Centrifugation, Density Gradient; Cyclodextrins; Cytokines; Dendritic Cells; Histocompatibility Antigens Class II; Immunophenotyping; Interleukin-12; Liposomes; Lymphocyte Activation; Membrane Microdomains; Mice; Mice, Inbred CBA; Mice, Transgenic; Muramidase; Peptide Fragments; Receptors, Fc; Th1 Cells; Th2 Cells; Time Factors

The mechanisms that regulate CD4(+) T cells responses in vivo are still poorly understood. We show here that initial Ag stimulation induces in CD4(+) T cells a program of proliferation that can develop, for at least seven cycles of division, in the absence of subsequent Ag or cytokine requirement. Thereafter, proliferation stops but can be reinitiated by novel Ag stimulation. This initial Ag stimulation does not however suffice to induce the differentiation of naive CD4(+) T cells into effector Th1 cells which requires multiple contacts with Ag-loaded APC. Thus, recurrent exposure to both Ag and polarizing cytokines appears to be essential for the differentiation of IFN-gamma-producing cells. Ag and cytokine availability therefore greatly limits the differentiation, but not the initial proliferation, of CD4(+) T cells into IFN-gamma-producing cells.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Cytokines; Flow Cytometry; Kinetics; Lymphocyte Activation; Mice; Mice, Transgenic; Muramidase; Peptide Fragments; Receptors, Antigen, T-Cell; RNA, Messenger; Signal Transduction; T-Box Domain Proteins; Th1 Cells; Transcription Factors; Transcriptional Activation

We have recently reported the lateral and rotational diffusion parameters for I-A(k) molecules expressing various cytoplasmic truncations (Int. Immunol. 12 (2000) 1319). We now describe the membrane dynamics of I-A(k) with various mutations in the presumed contact region between alphabeta-heterodimers in an (alphabeta)2 dimer of dimers structure. Such mutations are known to strongly affect the antigen presentation ability of these molecules (Int. Immunol. 10 (1998) 1237-1249) but cause relatively small changes in the molecular dynamics of I-A(k). Lateral diffusion coefficients of I-A(k) wild-type molecules and mutants obtained via fringe fluorescence photobleaching recovery (FPR) ranged from 1.1 to 2.3x10(-10)cm2/s at room temperature while fractional mobilities averaged 75+/-6%. For all cell types examined, treatment with either hen egg lysozyme 46-61 peptide or db-cAMP reduced the I-A(k) mobile fraction by about 10% relative to untreated cells, suggesting that these treatments may increase lateral confinement of class II in lipid rafts or cytoskeletal interactions of the molecules. Wild-type I-A(k) and mutants capable of normal or partial antigen presentation exhibited, as a group, slightly longer rotational correlation times (RCT) at 4 degrees C than did mutants inactive in antigen presentation, 14+/-4 versus 10+/-1 micros, respectively. Moreover, peptide, cAMP and anti-CD40 mAb treatment all increased rotational correlation times for fully- and partially-functional I-A(k) but not for non-functional molecules. For example, 16 h peptide treatment yielded average RCTs of 28+/-12 and 10+/-1 micros for the groups of functional and non-functional molecules, respectively. Such modulation of the dynamics of functional class II molecules is consistent with these treatments' stabilization of class II or induction of new gene expression. Measurements of fluorescence resonant energy transfer between I-A(k), though complicated by cellular autofluorescence, averaged 6+/-7% over 15 cells or treatments, a result consistent with the presence of a small fraction of I-A(k) as a dimer of dimers species. In summary, our results suggest subtle changes in the molecular motions of class II molecules correlate with a significant impact on class II function. Molecules active in antigen presentation exhibit more restricted motion in the membrane, and thus presumably more extensive intermolecular interactions, than non-functional molecules. Further, treatments, such as db-cAMP a

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; B-Lymphocytes; Bucladesine; CD40 Antigens; Diffusion; Dimerization; Energy Transfer; Fluorescence; Histocompatibility Antigens Class II; Mice; Muramidase; Peptide Fragments; Point Mutation; Rotation; Tumor Cells, Cultured

Major histocompatibility complex class II (MHC II) molecules capture peptides within the endocytic pathway to generate T cell receptor (TCR) ligands. Immature dendritic cells (DCs) sequester intact antigens in lysosomes, processing and converting antigens into peptide-MHC II complexes upon induction of DC maturation. The complexes then accumulate in distinctive, nonlysosomal MHC II+ vesicles that appear to migrate to the cell surface. Although the vesicles exclude soluble lysosomal contents and antigen-processing machinery, many contain MHC I and B7 costimulatory molecules. After arrival at the cell surface, the MHC and costimulatory molecules remain clustered. Thus, transport of peptide-MHC II complexes by DCs not only accomplishes transfer from late endocytic compartments to the plasma membrane, but does so in a manner that selectively concentrates TCR ligands and costimulatory molecules for T cell contact.

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; Antigens, CD; B-Lymphocytes; B7-2 Antigen; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Cells, Cultured; Dendritic Cells; Endocytosis; Endosomes; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Kinetics; Ligands; Lipopolysaccharides; Lysosomes; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Muramidase; Peptide Fragments; Receptors, Antigen, T-Cell; Thiazoles; Thiazolidines

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; B-Lymphocytes; CD40 Ligand; CD8 Antigens; Cells, Cultured; Dendritic Cells; Epitopes, T-Lymphocyte; Female; Injections, Subcutaneous; Lipopolysaccharides; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Muramidase; Peptide Fragments; T-Lymphocytes; Up-Regulation

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.

Topics: Animals; Antigen Presentation; Brucella abortus; Cell Membrane; Dimerization; Dose-Response Relationship, Immunologic; Down-Regulation; Epitopes, T-Lymphocyte; Female; Flow Cytometry; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Immunosuppressive Agents; Injections, Intraperitoneal; Lipopolysaccharides; Lymphocyte Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Muramidase; Peptide Fragments; Sodium Dodecyl Sulfate; T-Lymphocytes

MHC class II-restricted tumor Ags presented by class II(+) tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II(+) tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and whether class II(+) tumor cells directly present Ag in vivo. To address these questions, a model Ag, hen egg lysozyme, was targeted to various subcellular compartments of mouse sarcoma cells, and the resulting cells were tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear Ag in vivo. In in vitro studies, Ags localized to all tested compartments (nuclei, cytoplasm, mitochondria, and endoplasmic reticulum) are presented in the absence invariant chain and H-2M. Coexpression of invariant chain and H-2M inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II(+) tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear Ags. Because class II(+) tumor cells are effective APC in vivo and probably present novel tumor Ag epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4(+) T cells.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; Cell Compartmentation; Cell Nucleus; Cytosol; Histocompatibility Antigens Class II; HLA-D Antigens; Humans; Hybridomas; Immunodominant Epitopes; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Mitochondria; Molecular Sequence Data; Muramidase; Neoplasm Transplantation; Peptide Fragments; Sarcoma, Experimental; Spleen; Transfection; Tumor Cells, Cultured

Mice expressing hen egg-white lysozyme (HEL) as a transgene are unresponsive to immunization with the HEL protein. Profound tolerance was found even in situations where the amounts of l-A(k)-peptide complexes was 100 or less per APC. Among the few T cells that escaped tolerance, we did not observe differential responses to the different HEL epitopes, perhaps because of the very high sensitivity of the negative selection process. The same HEL transgenic mice that did not respond to HEL responded to immunization with the 46-61 peptide of HEL. These peptide-specific T cells that escaped negative selection belonged to a set that reacted with a particular conformer of the HEL peptide-l-A(k) (type B). The presence of type B reactive T cells should be considered in autoimmunity.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Chickens; Clonal Deletion; Epitopes; Histocompatibility Antigens Class II; Immune Tolerance; Immunization; Lymphocyte Count; Mice; Mice, Transgenic; Molecular Sequence Data; Muramidase; Peptide Fragments; T-Lymphocyte Subsets

We have examined the role of Fas and Bcl-2 in T cell survival and responses to antigen in vivo using T cells that express a transgenic antigen receptor specific for hen egg lysozyme (HEL) and that either lack functional Fas or Fas ligand (FasL) or overexpress Bcl-2 as a transgene. HEL-specific, Bcl-2-transgenic T cells showed prolonged responses to immunization with cognate peptide but were eliminated rapidly when exposed to HEL expressed systemically as a self antigen. In contrast, Fas- and FasL-defective T cells did not display exaggerated responses to immunization with HEL peptide, but did show increased expansion and survival in response to systemic self antigen and were able to activate anti-HEL (self) antibody-forming cells. Thus, Bcl-2 and Fas play different roles in the regulation of T cell responses to antigen in vivo and in self tolerance.

Topics: Adoptive Transfer; Animals; Apoptosis; Autoantibodies; Enzyme-Linked Immunosorbent Assay; Fas Ligand Protein; fas Receptor; Homeostasis; Immune Tolerance; Ligands; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Mutant Strains; Mice, Transgenic; Muramidase; Peptide Fragments; Phenotype; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, T-Cell; T-Lymphocytes; T-Lymphocytes, Helper-Inducer

Transgenic 3.L2 T cells are stimulated by Hb(64-76)/I-Ek and are positively selected on I-Ek plus self-peptides. To this pool of self-peptides we have added a single, well-defined 3.L2 TCR antagonist (A72) in vivo. We find that mice expressing both the 3.L2 TCR and A72 have a minimal loss of T cells expressing the clonotypic TCR in the thymus and spleen. Importantly, the proliferative response of 3.L2 x A72 splenocytes is significantly reduced compared with splenocytes from 3.L2 mice. This reduced response can be attributed to peripheral antagonism. Thus we have identified a new class of self-ligands whose predominant effect is constitutive peripheral antagonism rather than negative selection. The net effect of these ligands is to avoid potential self-reactivity while maintaining as large a repertoire as possible.

Topics: Animals; Antigen-Presenting Cells; Chickens; Clone Cells; Hemoglobins; Histocompatibility Antigens Class II; Immune Tolerance; Ligands; Lymphocyte Activation; Lymphocyte Count; Mice; Mice, Inbred AKR; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Peptide Fragments; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Spleen; T-Lymphocyte Subsets; Thymus Gland; Transgenes

Solid-phase immunoassays such as enzyme-linked immunosorbent assays require one of the assay components to be immobilized. Most frequently this is achieved by passive adsorption of the antigen or antibody to a hydrophobic polymer surface composed of, e.g., polystyrene. Alternatively the biomolecule can be bound indirectly via passively adsorbed carrier proteins or directly via functional groups on the solid phase using cross-linking agents. Here we describe a new technique--hydrocoating--for covalent immobilization of biomolecules, such as peptides, in highly hydrophilic surroundings. Peptides were immobilized on microtiter plates via covalent bonds to an activated hydrophilic polymer. Soluble dextran was activated using 2,2,2-triflouroethanesulphonyl chloride (tresyl chloride) leading to activation of hydroxyl groups on the dextran polymer. This activated dextran molecule was immobilized on a surface containing amino groups leaving a sufficient number of active groups for secondary binding of other biomolecules. Peptides, that were either undetectable or poorly recognized when adsorbed on polystyrene, were readily recognized when immobilized by the hydrocoating technique. Furthermore, peptides immobilized by this method were recognized 5-10-fold better compared to the same peptides immobilized covalently on a surface containing secondary amino groups. The technique appears to provide an alternative to passive adsorption of biomolecules on solid phases and may be useful in the future development of immunoassays.

Topics: Amino Acid Sequence; Animals; Biotin; Cross-Linking Reagents; Dextrans; Enzyme-Linked Immunosorbent Assay; Ligands; Mice; Microchemistry; Molecular Sequence Data; Muramidase; Peptide Fragments; Peptide T; Peptides

More than 90% of the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APC) have in their binding site a peptide derived from an extracellular protein ingested by the APC or from a protein of the APC itself. These self-peptides can be eluted from affinity-purified MHC class II molecules by acid elution, and have been studied with a variety of techniques. We show here that the self-peptides eluted from the mouse MHC class II molecules Ad, Ed and Ek bind specifically to MHC class II molecules of the allelic type from which they were derived. The pH optimum for binding is around 5.0, i.e. the same optimum at which synthetic peptides representing sequences of foreign antigens bind to MHC class II molecules. This suggests that the physiological compartment where MHC class II molecules bind self-peptides may be very late in the endocytic pathway. The chemical properties of the eluted and labelled MHC class II peptides were studied by isoelectric focusing. This method was able to separate the peptides very efficiently, and enabled a rapid comparison of peptides eluted from different MHC molecules. The 125I-labelled peptides displayed a broad range of isoelectric points with values predominantly below neutral. This suggests that such peptides bind to MHC in a predominantly non-charged state.

Topics: Amino Acid Sequence; Animals; Electrophoresis, Polyacrylamide Gel; Histocompatibility Antigens Class II; Hydrogen-Ion Concentration; Isoelectric Focusing; Mice; Mice, Inbred Strains; Molecular Sequence Data; Muramidase; Peptide Fragments

Hen egg lysozyme (HEL)-specific T hybridomas were used to study the presentation of HEL as a self component in tolerant, HEL-transgenic mice expressing different serum levels of HEL (AL3, 3-7 nM: ML5, 1-3 nM). Constitutive presentation of the HEL46-61 determinant was detectable in antigen presenting cell (APC)-enriched preparations of spleen, thymus, and lymph node from AL3 transgenic mice. The most likely cell responsible for constitutive presentation of HEL antigen was a non-B cell. In addition, constitutive presentation of HEL46-61 was clearly evident in thymic rosettes (complexes of macrophages or dendritic cells and lymphoid cells) derived from AL3 HEL-transgenic mice, but not from ML5 HEL-transgenic mice. In contrast to the HEL46-61 determinant, constitutive presentation of the HEL25-43 and HEL112-129 determinants was not detectable on APC-enriched preparations from either of the HEL-transgenic lines tested. Thus, although T cell tolerance was evident in transgenic mice expressing a range of serum HEL concentrations, constitutive presentation of processed HEL antigen was not detectable on APCs from mice expressing less than 3-7 nM. This threshold of detectable antigen presentation is consistent with the notion that the concentration of nominal antigen needed for tolerizing T cells is less than that required for activation of mature T cells.

Topics: Animals; Antigen-Presenting Cells; Chickens; Histocompatibility Antigens Class II; Immune Tolerance; Immunodominant Epitopes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muramidase; Peptide Fragments; T-Lymphocytes

Hen's egg white lysozyme (HEL) is one of the minor allergen in hen's egg white. HEL is commonly used to treat disease of respiratory tract, because it have the effect to dissolve mucopolysaccharide and anti-inflammatory action. We examined specific IgE antibody titers (IgE-HEL) in patients with egg allergy and allergic patients to other antigen than egg. Results indicated that 16.37 +/- 29.56 (PRU/ml) (mean +/- SD) of IgE-HEL was found in 30 out of the 39 allergic patients to egg, and 23 (66.7%) out of the 39 patients studied showed RAST scores of more than 2. On the other hand, 1.08 +/- 0.92 (PRU/ml) of IgE-HEL in 12 out of the 44 allergic patients to other antigen than egg, and 5 (11.4%) out of the 44 patients studied showed RAST scores of more than 2. Moreover, we treated a patient who developed anaphylaxis after taking HEL. 1.0 (PRU/ml) of HEL-IgE was found in this patient. These results suggest that we should be careful in treating allergic patients with HEL.

Topics: Adolescent; Age Factors; Anaphylaxis; Antibody Specificity; Child; Child, Preschool; Eggs; Female; Food Hypersensitivity; Humans; Immunoglobulin E; Infant; Male; Muramidase; Peptide Fragments; Radioallergosorbent Test

During antigen presentation, a close association between CD4 and the T cell receptor (TCR) occurs as a result of interacting with the same major histocompatibility complex class II molecule. The potential consequences of such an intimate interaction on TCR specificity was addressed using CD4 loss variants of four different murine T cell hybridomas specific for the immunodominant hen egg lysozyme (HEL) peptide 46-61. While all the CD4+ and CD4- variants tested possessed comparable surface expression of TCR, CD3, CD2 and LFA-1, and responded similarly to immobilized anti-TCR and anti-CD3 monoclonal antibodies, they differed dramatically in their responses to either the naturally processed HEL antigen, synthetic peptide 46-61 or staphylococcal enterotoxin superantigens. While one hybridoma was comparatively unaffected by the loss of CD4, another lost its responsiveness to antigen and peptide completely while retaining reactivity to SE. In contrast, two other hybridomas still responded to antigen but lost reactivity to synthetic peptide and SE. These data could not be readily explained on the basis of affinity or signal transduction requirements alone, and thus suggest that the intimate association of CD4 with the TCR may result in a subtle modulation of its fine specificity for some but not all T cells.

Topics: Animals; Antibodies, Monoclonal; CD4 Antigens; Enterotoxins; Histocompatibility Antigens Class II; Hybridomas; Muramidase; Peptide Fragments; Receptors, Antigen, T-Cell; Staphylococcus; T-Lymphocytes

This report describes a detailed mutational analysis of a major histocompatibility complex class II molecule--the alpha chain of the Ak complex. Each residue from 50-79 was replaced by an alanine, and the effects on recognition of Ak by panels of antibodies and T cells determined. The results provide the strongest existing experimental evidence that the antigen binding site on a class II molecule can be modelled on the crystal structure of a class I molecule. The data have also permitted the delineation of residues that actually contact antigenic peptides.

Topics: Alanine; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding, Competitive; Histocompatibility Antigens Class II; Hybridomas; Mice; Mice, Inbred Strains; Models, Molecular; Molecular Sequence Data; Muramidase; Mutagenesis, Site-Directed; Peptide Fragments; Protein Conformation; Reproducibility of Results; Ribonucleases; T-Lymphocytes; X-Ray Diffraction