ovalbumin and Lymphopenia

CD8 T cell memory critically contributes to long-term immunity. Both low- and high-affinity TCR signals are able to support the differentiation of memory CD8 T cells. However, it is unclear whether the requirements for memory development change when TCR signal strength is altered. To gain further insight into this question, we used a TCRβ transmembrane domain mutant model that is defective in the generation of memory in response to high-affinity ligands. Surprisingly, lowering TCR signal strength, by stimulation with low-affinity ligands, resulted in normal memory development. Restoration of memory correlated with recovery of TCR-dependent NF-κB signaling. Thus, these data provide novel evidence that the requirements for memory are qualitatively different depending on TCR signal strength.

Topics: Active Transport, Cell Nucleus; Adoptive Transfer; Animals; Antigens; CD8-Positive T-Lymphocytes; Gene Expression Regulation; Immunologic Memory; Immunomagnetic Separation; Ligands; Listeria monocytogenes; Listeriosis; Lymphocyte Count; Lymphopenia; Lymphopoiesis; Mice; Mice, Congenic; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B; Ovalbumin; Peptide Fragments; Point Mutation; Protein Structure, Tertiary; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocyte Subsets

IFN-gamma regulates multiple processes in the immune system. Although its antimicrobial effector functions are well described, less is known about the mechanisms by which IFN-gamma regulates CD8(+) T cell homeostasis. With the help of adoptive T cell transfers, we show in this study that IFN-gammaR signaling in CD8(+) T cells is dispensable for expansion, contraction, and memory differentiation in response to peptide vaccination. In contrast, host IFN-gammaR signaling counterregulates CD8(+) T cell responses and the generation of effector memory T cell processes, which are partially regulated by CD11b(+) cells. Similar to vaccination-induced proliferation, host IFN-gammaR signaling limits the expansion of naive CD8(+) T cells and their differentiation into effector memory-like T cells in lymphopenic mice. In contrast to peptide vaccination, IFN-gammaR signaling in CD8(+) T cells contributes to memory fate decision in response to lymphopenia, an effect that is fully reversed by high-affinity TCR ligands. In conclusion, we show that host IFN-gammaR signaling controls the magnitude of CD8(+) T cell responses and subsequent memory differentiation under lymphopenic and nonlymphopenic conditions. In contrast, IFN-gammaR signaling in CD8(+) T cells does not affect cell numbers under either condition, but it directs memory fate decision in response to weak TCR ligands.

Topics: Adoptive Transfer; Animals; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chickens; Coculture Techniques; Immunization; Immunologic Memory; Interferon gamma Receptor; Interferon-gamma; Ligands; Lipopolysaccharides; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Peptide Fragments; Receptors, Interferon; Signal Transduction

The Toxoplasma gondii population consists of multiple strains, defined by genotype and virulence. Previous studies have established that protective immunity to this organism is mediated by IL-12, which drives T cells to produce IFN-gamma. Paradoxically, although type I and type II strains of T. gondii both induce IL-12 and IFN-gamma in the mouse, type I parasites are lethal, whereas type II strains establish chronic infection. The cellular basis for these strain-dependent differences remains unclear. To better understand these events, the CD8(+) T cell and dendritic cell (DC) responses to transgenic, OVA-expressing type I RH (RH OVA) and type II Prugniuad (Pru OVA) parasites were examined. Pru OVA-infected mice developed a robust DC response at the site of infection and the draining lymph node and generated a population of endogenous OVA-specific CD8(+) T cells. In contrast, RH OVA-infected mice had fewer DCs and OVA-specific CD8(+) T cells. RH OVA-infected mice given preactivated OVA-specific CD8(+) T cells were protected, suggesting that reduced DC-derived signals contributed to the low OVA-specific CD8(+) T cell numbers observed during type I infection. Indeed, DC depletion prior to Pru OVA infection resulted in a failure to generate activated OVA-specific CD8(+) T cells, and IL-12p70 treatment during RH OVA infection modestly increased the number of Ag-specific cells. Together, these data are consistent with a model of immunity to T. gondii in which strain-dependent DC responses shape the generation of Ag-specific CD8(+) T cells and determine the outcome of infection.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Dendritic Cells; Epitopes, T-Lymphocyte; Female; Lymphocyte Activation; Lymphocyte Count; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Toxoplasma; Toxoplasmosis, Animal; Virulence

T-bet(-/-) mice have been shown to have a profound deficiency in the ability to generate invariant NKT (iNKT) cells in the periphery due to a halt in terminal maturation, but despite this deficiency, T-bet(-/-) mice develop spontaneous airway hyperreactivity (AHR) and airway inflammation. Because in some situations the development of AHR requires the presence of iNKT cells, we sought to more clearly understand how AHR develops in T-bet(-/-) mice by examining T-bet(-/-) mice in several distinct mouse models of asthma, including spontaneous, OVA-induced and alpha-galactosylceramide (alpha-GalCer)-induced AHR. Surprisingly, we found that administration of alpha-GalCer, which very specifically activates iNKT cells, greatly increased the AHR response in the T-bet(-/-) mice. Moreover, in T-bet(-/-) mice, spontaneous AHR as well as AHR induced with OVA or alpha-GalCer were all eliminated by blocking CD1d, the restricting element of iNKT cells, using an anti-CD1d-blocking mAb. Although the number of the iNKT cells in T-bet(-/-) mice was reduced compared with that in wild-type mice, the remaining iNKT cells produced primarily IL-4 and IL-13, and only minimal amounts of IFN-gamma. We conclude therefore that the AHR that develops in T-bet(-/-) mice is dependent on the presence of iNKT cells, and that whereas T-bet(-/-) have reduced numbers of iNKT cells, these are sufficient for the development of AHR.

Topics: Animals; Antibodies, Blocking; Antigens, CD1d; Bronchial Hyperreactivity; Disease Models, Animal; Epitopes, T-Lymphocyte; Galactosylceramides; Lymphopenia; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Transgenic; Natural Killer T-Cells; Ovalbumin; T-Box Domain Proteins

Whether memory CD8 T cells can be reactivated in nonlymphoid tissues is unclear. Using mice lacking the spleen, lymph nodes, or both, we show that the secondary T cell response, but not homeostatic maintenance of memory cells, required lymphoid tissue. Whereas primary and secondary CD8 T cell responses to vesicular stomatitis virus infection were lymph node dependent, responses to Listeria monocytogenes infection were driven primarily in the spleen. Memory cell subset reactivation was also regulated by location of the responding population and the pathogen. Thus, CD62Llow effector memory T cells (TEM) cells responded nearly as well as CD62Lhigh central memory T cells (TCM) and TCM cells after L. monocytogenes infection, and both subsets generated equivalent populations of secondary memory cells. In contrast, TCM cells, but not TEM cells, mounted a robust response to vesicular stomatitis virus infection. TCM and TEM cells also required lymphoid tissue to mount recall responses, and the bone marrow did not contribute significantly to the response of either subset. Our findings indicated that characteristics of the infectious agent and the migratory preferences of memory cells dictated the secondary lymphoid tissue requirement for the recall response to infection.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Immunologic Memory; Listeria monocytogenes; Liver; Lung; Lymphoid Tissue; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Specificity; Ovalbumin; Splenectomy; Vesicular stomatitis Indiana virus

CD4(+) T cells produce hematopoietic-related cytokines and are essential for hematopoiesis stimulation during infection and hematologic recovery after bone marrow transplantation. However, it remains unclear if T cells are necessary to maintain normal hematopoiesis. We report here that, in T-cell-deficient mice, terminal differentiation of myeloid progenitors is defective, resulting in very low levels of granulocytes in the periphery. Hematopoiesis is restored after thymus graft or reconstitution with CD4(+) T cells but not CD8(+) T cells. Bone marrow CD4(+) T cells have an activated phenotype and produce cytokines, apparently, in the absence of exogenous stimulation. Transgenic mice carrying T-cell receptor specific for an ovalbumin peptide presented in the context of a specific class II molecule (I-A(d)) (DO11.10 RAG(-/-)) show the same hematopoietic deficiency as athymic mice. Their bone marrow CD4(+) T cells are not activated, suggesting that hematopoiesis maintenance requires the presence of cognate antigen in order to activate bone marrow T-helper cells. In fact, priming of transgenic mice with ovalbumin restores normal hematopoiesis. The data show that the current concept of "normal hematopoiesis" does not reflect a basal bone marrow activity, but it is an antigen-induced state maintained by constant activation of bone marrow CD4(+) T cells.

Topics: Animals; Antigens; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Cells, Cultured; Coculture Techniques; Hematopoiesis; Injections, Intravenous; Lymphocyte Activation; Lymphopenia; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Myelopoiesis; Ovalbumin; Thymus Gland

We have investigated the consequence of lack of IgA on host immunity using a murine model of allergic lung inflammation. Mice with a targeted disruption of the alpha-switch region and 5' H chain gene (IgA(-/-) mice), which lack total IgA, developed significantly reduced pulmonary inflammation with fewer inflammatory cells in lung tissue and bronchoalveolar lavage fluids, as well as reduced levels of total and IgG1 OVA-specific Abs and decreased IL-4 and IL-5 in bronchoalveolar lavage fluids compared with IgA(+/+) controls, following allergen sensitization and challenge. This defect was attributable to fewer B cells in the lungs of IgA(-/-) mice. Polymeric IgR-deficient (pIgR(-/-)) mice, which lack the receptor that transports polymeric IgA across the mucosal epithelium where it is cleaved to form secretory IgA, were used to assess the contribution of secretory IgA vs total IgA in the induction of allergic lung inflammation. pIgR(-/-) and pIgR(+/+) mice had comparable levels of inflammation, demonstrating that IgA bound to secretory component is not necessary for the development of allergic lung inflammation, although this does not necessarily rule out a role for transudated IgA in lung secretions because of "mucosal leakiness" in these mice. The results indicate that Ag-specific B cells are required at mucosal surfaces for induction of inflammation and likely function as major APCs in the lung for soluble protein Ags.

Topics: Administration, Intranasal; Allergens; Animals; B-Lymphocyte Subsets; Cell Migration Inhibition; IgA Deficiency; Immunoglobulin A; Inflammation; Lung; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Respiratory Hypersensitivity; Respiratory Mucosa

The factors that control development of adaptive responses to exogenous Ag remain incompletely understood. An ability to selectively direct immunity toward a specific phenotype would be of clinical benefit in numerous immunological disorders. Administration of chemically modified allergen glutaraldehyde-polymerized OVA (OA-POL) leads to >90% reductions in murine IgE and >500-fold increases in IgG2c responses that develop upon subsequent immunization with native Ag. In the present study, we examine the mechanisms underlying this reorientation of the type 2 dominant response that would normally develop. Lack of endogenous IL-12 or IFN-gamma results in markedly reduced induction of IgG2c responses following OA-POL treatment, but only IFN-gamma(-/-) mice demonstrate reduced capacity to prevent IgE induction. This indicates that while both IL-12 and IFN-gamma are critical promoters of type 1 immunity, only IFN-gamma is required to maximally inhibit development of type 2 immune responses. Compared with OVA-immunized mice, CD69(+) T cells from OA-POL-immunized mice demonstrate elevated IL-12Rbeta(2), IL-18Ralpha, and IL-18Rbeta mRNA levels, as well as increased IFN-gamma production in response to rIL-12 or rIL-18 stimulation. Collectively, these data indicate that preventing induction of type 2 immune responses is critically dependent on altered T cell responsiveness to these cytokines. The finding that targeted, Ag-specific manipulation of IL-12 and IL-18 responsiveness can be used to shape the phenotype of the dominant immune response that develops suggests that specifically targeting IL-12 and IL-18 receptor expression may offer clinical options for clinical prophylaxis or intervention.

Topics: Allergens; Animals; B-Lymphocytes; Cells, Cultured; Cytokines; Down-Regulation; Epitopes, T-Lymphocyte; Immunity, Active; Immunity, Cellular; Immunoglobulin E; Interleukin-12; Interleukin-18; Lymphopenia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Ovalbumin; Rats; Rats, Sprague-Dawley; Th2 Cells; Up-Regulation

Human and mouse Ig alpha molecules share only 58% amino acid sequence identity in their extracellular regions. However, mice immunized with a recombinant Fc fusion protein containing the extracellular portion of human Ig alpha produced significant amounts of IgG capable of binding to Ig alpha on mouse B cells. The induced auto/cross-reactive Abs could down-regulate B cell levels and the consequent humoral immune responses against an irrelevant Ag in treated mice. Analogous immunization with an Fc fusion protein containing the extracellular portion of human Ig beta gave a much weaker response to mouse Ig beta, although human and mouse Ig beta, like their Ig alpha counterparts, share 56% sequence identity in their extracellular regions. Protein sequence analyses indicated that a potential immunogenic segment, located at the C-terminal loop of the extracellular domain, has an amino acid sequence that is identical between human and mouse Ig alpha. A mAb A01, which could bind to both human and mouse Ig alpha, was found to be specific to a peptide encompassing this immunogenic segment. These findings suggest that specific auto/cross-reactivity against self Ig alpha can be induced by a molecular mimicry presented by a foreign Ig alpha.

Topics: Amino Acid Sequence; Animals; Antibodies, Bispecific; Antigen-Antibody Reactions; Antigens, CD; Autoantibodies; B-Lymphocytes; Binding Sites, Antibody; CD79 Antigens; Cell Death; Cell Line; Down-Regulation; Humans; Immune Sera; Immunization; Immunoglobulin Fc Fragments; Injections, Intraperitoneal; Lymphopenia; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Ovalbumin; Receptors, Antigen, B-Cell; Recombinant Fusion Proteins

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.

Topics: Administration, Intranasal; Agammaglobulinemia; Allergens; Ambrosia; Animals; Antibody Specificity; B-Lymphocytes; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokines; Epitopes; Female; Immunization; Immunoglobulin E; Immunoglobulin G; Lymphopenia; Macrophages, Alveolar; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Ovalbumin; Receptors, IgG

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.

Topics: Allergens; Animals; Bronchial Hyperreactivity; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Down-Regulation; Epitopes, T-Lymphocyte; Goblet Cells; Influenza A virus; Interferon-gamma; Interleukin-5; Lung; Lymphocyte Count; Lymphopenia; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nippostrongylus; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; Strongylida Infections; Th2 Cells; Up-Regulation

NF-kappaB activity in mammalian cells is regulated through the IkappaB kinase (IKK) complex, consisting of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma). Targeted deletion of Ikkbeta results in early embryonic lethality, thus complicating the examination of IKKbeta function in adult tissues. Here we describe the role of IKKbeta in B lymphocytes made possible by generation of a mouse strain that expresses a conditional Ikkbeta allele. We find that the loss of IKKbeta results in a dramatic reduction in all peripheral B cell subsets due to associated defects in cell survival. IKKbeta-deficient B cells are also impaired in mitogenic responses to LPS, anti-CD40, and anti-IgM, indicating a general defect in the ability to activate the canonical NF-kappaB signaling pathway. These findings are consistent with a failure to mount effective Ab responses to T cell-dependent and independent Ags. Thus, IKKbeta provides a requisite role in B cell activation and maintenance and thus is a key determinant of humoral immunity.

Topics: Animals; Antigens, CD19; Antigens, T-Independent; B-Lymphocyte Subsets; Cell Differentiation; Cell Division; Cell Survival; Cells, Cultured; Haptens; I-kappa B Kinase; Immunoglobulin G; Immunoglobulin M; Integrases; Lymph Nodes; Lymphoid Tissue; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mitogens; Ovalbumin; Protein Serine-Threonine Kinases; Spleen; Trinitrobenzenes; Viral Proteins

Eosinophilic inflammation plays a key role in tissue damage that characterizes asthma. Eosinophils are produced in bone marrow and recent observations in both mice and humans suggest that allergen exposure results in increased output of eosinophils from hemopoietic tissue in individuals with asthma. However, specific mechanisms that alter eosinophilopoiesis in this disease are poorly understood. The current study used a well-characterized murine animal model of asthma to evaluate alterations of eosinophil and eosinophil progenitor cells (CFU-eo) in mice during initial sensitization to allergen and to determine whether observed changes in either cell population were regulated by T lymphocytes. Following the first intranasal installation of OVA, we observed sequential temporal elevation of eosinophils in bone marrow, blood, and lung. In immunocompetent BALB/c mice, elevation of bone marrow eosinophils was accompanied by transient depletion of CFU-eo in that tissue. CFU-eo rebounded to elevated numbers before returning to normal baseline values following intranasal OVA exposure. In T cell-deficient BALB/c nude (BALB/c(nu/nu)) mice, CFU-eo were markedly elevated following allergen sensitization, in the absence of bone marrow or peripheral blood eosinophilia. These data suggest that eosinophilia of asthma results from alterations in two distinct hemopoietic regulatory mechanisms. Elevation of eosinophil progenitor cells in the bone marrow is T cell independent and likely results from altered bone marrow stromal cell function. Differentiation of eosinophil progenitor cells and phenotypic eosinophilia is T cell dependent and does not occur in athymic nude mice exposed to intranasal allergen.

Topics: Administration, Intranasal; Allergens; Animals; Asthma; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Colony-Forming Units Assay; Desensitization, Immunologic; Disease Models, Animal; Eosinophils; Female; Immunoglobulin E; Injections, Intraperitoneal; Interleukin-5; Leukopoiesis; Lung; Lymphopenia; Mice; Mice, Inbred BALB C; Mice, Nude; Ovalbumin; T-Lymphocyte Subsets

Allergen provocation of allergic asthma patients is often characterized by an initial period of bronchoconstriction, or early phase reaction (EPR), that leads to maximal airway narrowing within 15-30 min, followed by a recovery period returning airway function to baseline within 1-2 h. In this study, we used a defined OVA provocation model and mice deficient for specific leukocyte populations to investigate the cellular/molecular origins of the EPR. OVA-sensitized/challenged wild-type (C57BL/6J) mice displayed an EPR following OVA provocation. However, this response was absent in gene knockout animals deficient of either B or T cells. Moreover, transfer of OVA-specific IgG, but not IgE, before the OVA provocation, was capable of inducing the EPR in both strains of lymphocyte-deficient mice. Interestingly, an EPR was also observed in sensitized/challenged mast cell-deficient mice following an OVA provocation. These data show that the EPR in the mouse is an immunologically based pathophysiological response that requires allergen-specific IgG but occurs independent of mast cell activities. Thus, in the mouse the initial period of bronchoconstriction following allergen exposure may involve neither mast cells nor IgE-mediated events.

Topics: Administration, Inhalation; Aerosols; Allergens; Animals; Antibody Specificity; B-Lymphocytes; Bronchoconstriction; Crosses, Genetic; Immunoglobulin E; Immunoglobulin G; Injections, Intraperitoneal; Lymphopenia; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; T-Lymphocytes; Time Factors

Naïve T cells proliferate and differentiate into memory cells after antigenic stimulation or in a lymphopenic environment. We showed here transient increases in memory phenotype CD8+ T cell numbers in the lymphopenic environment of spleens of very young mice. The magnitude of the increase correlated with Bcl-6 expression in the T cells. Bcl-6 controlled the generation and maintenance of antigen-specific memory phenotype CD8+ T cells in the spleens of immunized mice. These data suggest that Bcl-6, which is essential for memory B cell development in germinal centers, is a key molecule for the establishment not only of memory T cells but also of the peripheral T cell compartment in infancy.

Topics: Animals; Animals, Newborn; Antigens; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Division; DNA-Binding Proteins; Immunologic Memory; Lymphocyte Activation; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-6; Transcription Factors

Peripheral immune tolerance following i.v. administration of Ag has been shown to occur in the absence of B cells. Because different mechanisms have been identified for i.v. vs low dose oral tolerance and B cells are a predominant component of the gut-associated lymphoid tissue (GALT) they may play a role in tolerance induction following oral Ag. To examine the role of B cells in oral tolerance we fed low doses of OVA or myelin oligodendrocyte glycoprotein to B cell-deficient ( microMT) and wild-type C57BL/6 mice. Results showed that the GALT of naive wild-type and microMT mice was characterized by major differences in the cytokine microenvironment. Feeding low doses of 0.5 mg OVA or 250 microg myelin oligodendrocyte glycoprotein resulted in up-regulation of IL-4, IL-10, and TGF-beta in the GALT of wild-type but not microMT mice. Upon stimulation of popliteal node cells, in vitro induction of regulatory cytokines TGF-beta and IL-10 was observed in wild-type but not microMT mice. Greater protection against experimental autoimmune encephalomyelitis was found in wild-type mice. Oral tolerance in microMT and wild-type mice was found to proceed by different mechanisms. Anergy was observed from 0.5 mg to 250 ng in microMT mice but not in wild-type mice. Increased Ag was detected in the lymph of microMT mice. No cytokine-mediated suppression was found following lower doses from 100 ng to 500 pg in either group. These results demonstrate the importance of the B cell for the induction of cytokine-mediated suppression associated with low doses of Ag.

Topics: Administration, Oral; Animals; B-Lymphocytes; Cells, Cultured; Clonal Anergy; Cytokines; Dose-Response Relationship, Immunologic; Down-Regulation; Immune Tolerance; Immunoglobulin mu-Chains; Immunohistochemistry; Immunosuppressive Agents; Interleukin-2; Intestinal Mucosa; Lymph; Lymph Nodes; Lymphoid Tissue; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Species Specificity; Thoracic Duct

Although induction of T cell responses to fed Ag (oral tolerance) is thought to happen within the organized lymphoid tissue of the gut, we found that mice lacking Peyer's patches, B cells, and the specialized Ag-handling M cells had no defect in the induction of T cell responses to fed Ag, whether assayed in vitro by T cell proliferation or cytokine production, or in vivo by delayed-type hypersensitivity or bystander suppression against mycobacterial Ags in CFA. Feeding of Ag had a major influence on dendritic cells from fed wild-type or muMT mice, such that these APCs were able to elicit a different class of response from naive T cells in vitro. These results suggest that systemic immune responses to soluble oral Ags do not require an organized gut-associated lymphoid tissue but are most likely induced by gut-conditioned dendritic cells that function both to initiate the gut-oriented response and to impart the characteristic features that discriminate it from responses induced parenterally.

Topics: Administration, Oral; Animals; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Dendritic Cells; Diet; Dose-Response Relationship, Immunologic; Hypersensitivity, Delayed; Immune Tolerance; Immunoglobulin mu-Chains; Injections, Intradermal; Interphase; Intestinal Mucosa; Lymphopenia; Mice; Mice, Inbred A; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; T-Lymphocytes, Helper-Inducer

Ag-specific proliferation of CD4+ T cells is regulated, in part, by costimulatory signals through CD28. The proliferative response during primary activation is an important determinant of the ability of the T cell to respond to Ag re-encounter. Proliferation of mature CD4+ T cells during lymphopenia (homeostatic proliferation) requires interaction with endogenous peptide MHC. However, the role of costimulation during homeostatic proliferation is unclear, as is the ability of homeostatic proliferation to regulate secondary T cell responses. Using a TCR transgenic system and serial adoptive transfers we find that homeostatic proliferation of CD4+ T cells occurs for at least 5 wk after adoptive transfer into recombination-activating gene (RAG)-/- recipients. Two discrete populations of proliferating T cells can be resolved, one that is highly proliferative and dependent on CD28 signaling, and the other that contains cells undergoing low levels of CD28-independent proliferation. Importantly, naive CD4+ T cells that have undergone homeostatic proliferation acquire both phenotypic and functional characteristics of true memory cells. These studies indicate that functional memory T cells can be generated by encounters with endogenous Ags only. This mechanism of T cell regeneration is possibly active during lymphopenia due to viral infections, such as HIV, transplantation, or cancer therapy, and may explain selected autoimmune diseases.

Topics: Adoptive Transfer; Animals; CD28 Antigens; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Fluoresceins; Fluorescent Dyes; Genes, RAG-1; Homeostasis; Immunologic Memory; Immunophenotyping; Kinetics; Lymphocyte Activation; Lymphopenia; Mice; Mice, Knockout; Ovalbumin; Succinimides; T-Lymphocyte Subsets

We demonstrated previously that CD81(-/-) mice have an impaired Th2 response. To determine whether this impairment affected allergen-induced airway hyperreactivity (AHR), CD81(-/-) BALB/c mice and CD81(+/+) littermates were sensitized i.p. and challenged intranasally with OVA. Although wild type developed severe AHR, CD81(-/-) mice showed normal airway reactivity and reduced airway inflammation. Nevertheless, OVA-specific T cell proliferation was similar in both groups of mice. Analysis of cytokines secreted by the responding CD81(-/-) T cells, particularly those derived from peribronchial draining lymph nodes, revealed a dramatic reduction in IL-4, IL-5, and IL-13 synthesis. The decrease in cytokine production was not due to an intrinsic T cell deficiency because naive CD81(-/-) T cells responded to polyclonal Th1 and Th2 stimulation with normal proliferation and cytokine production. Moreover, there was an increase in T cells and a decrease in B cells in peribronchial lymph nodes and in spleens of immunized CD81(-/-) mice compared with wild-type animals. Interestingly, OVA-specific Ig levels, including IgE, were similar in CD81(-/-) and CD81(+/+) mice. Thus, CD81 plays a role in the development of AHR not by influencing Ag-specific IgE production but by regulating local cytokine production.

Topics: Administration, Intranasal; Allergens; Animals; Antigens, CD; B-Lymphocytes; Bronchial Hyperreactivity; Cytokines; Down-Regulation; Eosinophilia; Epitopes, T-Lymphocyte; Immunoglobulin E; Immunologic Deficiency Syndromes; Inflammation; Injections, Intraperitoneal; Interphase; Lung; Lymph Nodes; Lymphocyte Activation; Lymphocyte Count; Lymphopenia; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Mucins; Ovalbumin; Species Specificity; Spleen; Tetraspanin 28; Th1 Cells; Th2 Cells

Cytokines are central regulatory elements in peripheral lymphocyte differentiation, but their role in T cell ontogeny is poorly defined. In the present study, we evaluated the role of IL-12 in thymocyte selection more directly by determining its role in two models of in vivo negative selection. In initial studies we demonstrated that abundant intrathymic IL-12 synthesis occurs during OVA peptide-induced negative selection of thymocytes in neonatal OVA-TCR transgenic mice, and such synthesis is associated with increased IL-12R beta2-chain expression as well as STAT4 intracellular signaling. In further studies, we showed that this form of negative selection was occurring at the alphabetaTCRlowCD4lowCD8low stage and was prevented by the coadministration of anti-IL-12. In addition, the IL-12-dependent thymocyte depletion was occurring through an intrathymic apoptosis mechanism, also prevented by administration of anti-IL-12. Finally, we showed that IL-12 p40-/- mice displayed aberrant negative selection of double positive CD4+CD8+ thymocytes when injected with anti-CD3 mAb. These studies suggest that intact intrathymic IL-12 production is necessary for the negative selection of thymocytes occurring in relation to a high "self" Ag load, possible through its ability to induce the thymocyte maturation and cytokine production necessary for such selection.

Topics: Animals; Animals, Newborn; Antigens; Apoptosis; CD4 Antigens; CD8 Antigens; Cell Differentiation; DNA-Binding Proteins; Female; Immune Sera; Injections, Intraperitoneal; Interleukin-12; Lymphocyte Depletion; Lymphopenia; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Peptide Fragments; Phosphorylation; Receptors, Antigen, T-Cell; Receptors, Interleukin; Receptors, Interleukin-12; STAT4 Transcription Factor; T-Lymphocyte Subsets; T-Lymphocytes; Thymus Gland; Trans-Activators

NK cells have been shown to play a role in the modulation of B cell differentiation and Ab production. Using a novel murine model of NK cell deficiency, we analyzed the in vivo role of NK cells in the regulation of Ag-specific Ab production. After immunization with OVA or keyhole limpet hemocyanin in CFA, NK cell-deficient (NK-T+) mice developed an efficient Th1 response and produced significant levels of IFN-gamma but displayed markedly reduced or absent Ag-specific IgG2a production. There were no differences in the levels of Ag-specific IgG, IgG1, and IgG2b between NK-T+ and NK+T+ mice. Furthermore, NK cell-reconstituted, NK+T+ (tgepsilon26Y) mice produced significant amounts of Ag-specific IgG2a after immunization with OVA. These results indicate that NK cells are involved in the induction of Ag-specific IgG2a production in vivo. Moreover, they also demonstrate that the lack of Ag-specific IgG2a Ab production in NK-T+ mice is not associated with the impaired Th1 response and IFN-gamma production.

Topics: Animals; Cell Differentiation; Epitopes; Freund's Adjuvant; Hemocyanins; Immunoglobulin G; Injections, Subcutaneous; Interferon-gamma; Killer Cells, Natural; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Mollusca; Ovalbumin; Th1 Cells

Polysaccharide vaccines to encapsulated bacteria such as Neisseria meningitidis and Streptococcus pneumoniae are weakly immunogenic due to their T-independent (TI) nature. Even when converted to T-dependent forms through conjugation to foreign proteins, polysaccharides induce responses that are deficient in many respects, such as induction of murine IgG2a Ab, the isotype that mediates optimal complement fixation and opsonization. We now show that IL-12 treatment of mice induces significantly increased levels of IgG2a Ab to the model TI-2 Ag, DNP-Ficoll, and to vaccines composed of polysaccharides from pneumococci and meningococci. Use of immunodeficient mice lacking T cells and/or NK cells demonstrated that such cells were not responsible for the observed Ab enhancement. Furthermore, the use of IFN-gamma knockout mice showed that stimulation of TI-2 Ab responses by IL-12 was only partially dependent on IFN-gamma. The ability of IL-12 to dramatically enhance TI Ab responses suggests that IL-12 will be useful as a powerful vaccine adjuvant to induce protective immune responses against encapsulated pathogens.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antibody Formation; Antigens, T-Independent; Bacterial Vaccines; Dinitrophenols; Ficoll; Haptens; Immunoglobulin G; Interferon-alpha; Interferon-gamma; Interleukin-12; Killer Cells, Natural; Lymphopenia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Mice, Transgenic; Neisseria meningitidis; Ovalbumin; Pneumococcal Vaccines; Polysaccharides, Bacterial; Streptococcus pneumoniae; T-Lymphocytes

Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.

Topics: Animals; Antibodies, Blocking; B-Lymphocytes; Blotting, Northern; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Movement; Chemokine CCL11; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5; Chemokines, CC; Cytokines; Eosinophilia; Female; Immunocompromised Host; Immunohistochemistry; Intercellular Adhesion Molecule-1; L-Selectin; Lung; Lymphopenia; Macrophage Inflammatory Proteins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Ovalbumin; P-Selectin; Respiratory Hypersensitivity; RNA, Messenger; T-Lymphocytes; Vascular Cell Adhesion Molecule-1

Topics: Animals; Animals, Newborn; Antibody Formation; Antilymphocyte Serum; Ferritins; Fetus; gamma-Globulins; Gestational Age; Immunosuppression Therapy; Lymph Nodes; Lymphocyte Depletion; Lymphopenia; Mice; Ovalbumin; Rabbits; Sheep; Skin Transplantation; Spleen; T-Lymphocytes; Thymectomy; Thymus Gland; Transplantation Immunology; Transplantation, Heterologous; Transplantation, Homologous