Lymphocyte-mediated activation of cultured endothelial cells (EC). CD4+ T cells inhibit EC class II MHC expression despite secreting IFN-gamma and increasing EC class I MHC and intercellular adhesion molecule-1 expression

Endothelial cells (EC) were cocultured with allogeneic PBL, CD4+ T cells, or CD8+ T cells, and the degrees of EC activation induced examined by determining patterns of endothelial class I and class II MHC and intercellular adhesion molecule-1 (ICAM-1) expression. Coculture with PBL or CD8+ T cells uniformly increases class I MHC and ICAM-1 expression on all EC within a culture, but induces class II MHC expression on only a subpopulation(s) of EC. This heterogeneous EC response to coculture contrasts with the uniform class II expression on all EC induced by IFN-gamma in replicate wells. CD4+ T cells, when compared to equal numbers of unfractionated PBL or CD8+ T cells, are more effective at increasing class I MHC and ICAM-1 but are unable to induce class II MHC expression. The failure of CD4+ T cells to induce EC class II MHC Ag is not due to insufficient activation of the T cells, as PHA-activated CD4+ T cells also do not induce significant class II expression. In addition, conditioned media (CM) from CD4+ T cell/EC contain greater levels of immunoreactive IFN-gamma than do CM from PBL/EC cocultures. Rather, CD4+ T cells appear to actively inhibit the induction of EC class II Ag but not class I or ICAM-1 by IFN-gamma. Inhibition occurs at the time of induction, as CD4+ T cells are not capable of down-regulating previously induced class II Ag. CM from CD4+/EC (but not PBL/EC) cocultures also inhibits IFN-gamma induction of EC class II MHC expression. The inhibitory activity is generated during CD4+ T cell-EC cell contact, and is enhanced by PHA. The inhibitory activity(ies) of the CD4+/EC-CM is as yet unidentified, and is only minimally reversible by cocktails of neutralizing antibodies directed against TNF-alpha, TNF-beta (lymphotoxin), IFN-alpha and IFN-beta. In conclusion, CD4+ and CD8+ T cells are each effective activators of EC, but the patterns of activation produced by these subsets are quite distinct, largely due to generation of a soluble inhibitor(s) of class II MHC induction during coculture of CD4+ T cells with EC.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Cutting edge: induction of the antigen-processing enzyme IFN-gamma-inducible lysosomal thiol reductase in melanoma cells Is STAT1-dependent but CIITA-independent

Presentation and CD4(+) T cell responses to Ag in the context of MHC class II molecules require processing of native proteins into short peptide fragments. Within this pathway, IFN-gamma-inducible lysosomal thiol reductase (GILT) functions to catalyze thiol bond reduction, thus unfolding native protein Ag and facilitating further processing via cellular proteases. In contrast with professional APCs such as B cells, class II-positive human melanomas expressed relatively little to no GILT protein or mRNA. Tumor cell GILT expression was partially restored with IFN-gamma treatment but unlike other genes required for class II Ag presentation, GILT was not regulated by CIITA. Rather, studies revealed STAT1 plays a direct role in IFN-gamma-inducible GILT expression. These results define a molecular mechanism for the uncoupled regulation of MHC class II genes and the processing enzyme GILT in human melanomas.     

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

The kinetics of CD8 T cell IFN-gamma responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-alphabeta and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-gamma response detectable in serum samples and spleen and liver homogenates. Production of IFN-gamma was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-gammadelta T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-gamma induction, and viral peptide stimulation enhanced CD8 T cell IFN-gamma expression in culture. The IFN-gamma response was associated with IL-18 and IFN-alphabeta expression. Furthermore, both factors augmented peptide-driven IFN-gamma production in culture, and mice lacking IL-18 or IFN-alphabeta functions had reduced day 4 IFN-gamma. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.     

IFN-gamma reverses the stop signal allowing migration of antigen-specific T cells into inflammatory sites

In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO(+) B7-independent CD4(+) T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known, and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4(+) CD45RO(+) B7-independent T cells. Alloreactive CD4(+) T cell clones and lines were generated against HLA-DR11, DR13, DR4, and DR1 MHC Ags, and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner, and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration, migrated cells were still able to proliferate, but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-gamma restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate, thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse, but this is overcome in an inflammatory milieu.     

IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1- MHC class I-like/CD1-dependent CD4+ T cells.

NK T cells are an unusual subset of T lymphocytes. They express NK1. 1 Ag, are CD1 restricted, and highly skewed toward Vbeta8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-gamma immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-gamma by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-alphabeta is markedly increased. In contrast, IFN-gamma production remains essentially unchanged. In beta2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L-CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1- cells. This NK1.1-IL-4-producing subset shares three important characteristics with NK T cells: 1) Vbeta8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1- cells, and that these two subsets are biased toward IL-4 production by IL-7.     

Cross presentation of antigen on MHC class II via the draining lymph node after corneal transplantation in mice

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45(+), CD11b(+), CD11c- cells, with a few MHC class II(+) cells, and F4/80(+) cells. However, MHC class II(+) passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c(+) and F4/80(+) APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4-6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4(+) T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.     

Processing and presentation of self and foreign antigens by the renal proximal tubule.

The renal proximal tubule (PT) in many ways resembles an APC. The PT is one of the few epithelial cells in the body reported to constitutively express the class II MHC molecules required to present Ag to CD4+ T cells. We questioned whether the PT could function as an APC in vitro and in vivo. Fluorescence cytometry demonstrated that the normal CBA/J PT constitutively expressed low levels of class II MHC and that this expression was markedly augmented by either IFN-gamma or systemic Listeria monocytogenes infection. Functionally, the PT from normal CBA/J mice also stimulated T cell hybridomas when cultured in vitro with Ag, and this ability was markedly up-regulated by both IFN-gamma as well as L. monocytogenes infection. To prove that the PT constitutively processed and presented self Ag in vivo, freshly isolated PT from mice transgenic for human alpha 1-antitrypsin were cultured with the appropriate T cell hybridoma in the absence of exogenous Ag. Strong stimulation of the T cell hybridoma occurred. Our data show that the renal proximal tubule processes and presents foreign Ag both in vitro and in vivo, and that it constitutively processes and presents the self Ag hAAT in vivo. These results have important implications for the understanding of renal interstitial autoimmune diseases as well as the interstitial nephritis that occurs in response to foreign Ag.     

Single-cell analysis of costimulation by B cells, endothelial cells, and fibroblasts demonstrates heterogeneity in responses of CD4(+) memory T cells.

Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.     

Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through "cross-dressing." Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.     

Activation of MHC class I-restricted CD8+ CTL by microbial T cell mitogens. Dependence upon MHC class II expression of the target cells and V beta usage of the responder T cells

The T cell response to microbial T cell mitogens (MTM) such as enterotoxins from Staphylococcus aureus (SE) and the soluble mitogen from Mycoplasma arthritidis, resemble the minor lymphocyte stimulatory locus (Mls) response in several aspects. An important feature of the Mls response is it restriction to CD4+ cells. This study demonstrates that in contrast to Mls, the MTM response includes both CD4+ and CD8+ subsets. Both CD4+ and CD8+ cells expanded in IL-2 after stimulation with SEB showed preferential expression of T cell receptors bearing V beta 8 domains. Mouse and human target cells could be lysed in the presence of MTM both by MTM-stimulated CD8+ lymphocytes and by MHC class I-restricted CTL clones of defined Ag specificity. MTM-induced lysis required the expression of MHC class II, but not class I Ag, on the target cells. Inhibition studies of SEB and Ag-dependent cytolysis by CTL clones underlined the crucial role of CD3 and LFA-1 in both instances, but showed CD8 dependence only for AG-dependent cytolysis. Together these findings suggest important differences between the putative MTM-mediated interaction of TCR with MHC molecules and the classical TCR/MHC interaction involved in MHC-restricted Ag recognition.     

Transient T and B cell activation after neonatal induction of tolerance to MHC class II or Mls alloantigens.

The neonatal injection of semiallogeneic F1 spleen cells into newborn parental mice results in the induction of tolerance to the corresponding alloantigen (alloAg) and chimerism. In these F1 cell-injected mice, we have previously observed that this state of specific tolerance is associated with the development of a transient lupus-like autoimmune syndrome. In this study, we show that neonatal injection of mice with spleen cells differing from the host at major histocompatibility complex (MHC) class I, class II, class (I + II), or minor lymphocyte stimulating (Mls) alloAg induced a state of specific tolerance characterized by the absence of alloreactive CTL and/or Th cell responses in the spleen and the thymus of 6- to 12-week-old injected mice. However, in mice rendered tolerant to MHC class II or class (I + II) alloAg, the presence of high levels of IgG1 antibodies, of circulating immune complexes, of anti-ssDNA autoantibodies, and of tissue lesions were transiently observed. In these mice, an increased Ia Ag expression on lymphoid spleen cells was also detected at 1 wk. The elevated production of IgG1 and the overexpression of Ia Ag were almost completely prevented by treatment with an anti-IL-4 mAb. Such manifestations of B cell activation and autoimmunity were not observed in mice neonatally injected with F1 cells differing from the host only at MHC class I Ag. In mice neonatally tolerized to Mls Ag, a transient increase in IgG2a production and an overexpression of Ia Ag were detected without features of autoimmunity, and were prevented by anti-INF-gamma mAb treatment. In mice rendered tolerant to MHC class II, class (I + II), or Mls alloAg at birth, the manifestations of B cell activation were associated with the presence of in vivo-activated alloreactive CD4+ T cells in the spleen--but not the thymus--of 1-wk-old injected mice. Together, these results suggest that in mice neonatally injected with semiallogeneic F1 cells, the process of tolerance induction is not efficient during the early postnatal period, and could allow the maturation and peripheralization of some alloreactive CD4+ T cells, leading to transient B cell activation and, depending on the alloAg, to autoimmunity.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

T-dependent destruction of thyroid isografts exposed to IFN-gamma

Several autoimmune diseases are accompanied by tissue-specific expression of class II molecules of the MHC, and it has been suggested that this elicits a T cell response against tissue-specific Ag to which the individual is not tolerant. However, recent transgenic studies have indicated that non-lymphoid expression of class II genes in the pancreas, liver, and kidney is either innocuous or induces peripheral tolerance. To test this hypothesis in another organ-specific autoimmune disease, we attempted to induce autoimmune thyroiditis in normal mice with class II+ thyroid tissue. Normal thyroid lobes were cultured with and without IFN-gamma and then transplanted to adult isogeneic recipients. The thyroid that had been induced to express class II genes by IFN-gamma was destroyed in normal mice, whereas the control cultured thyroid and the native cervical gland survived. Both types of transplants remained intact and functional in congenic nu/nu recipients, indicating that neither exposure to IFN-gamma nor expression of class II genes compromised the thyroid. Thus, in some tissues, exposure to IFN-gamma and/or the induction of class II expression can lead to T-dependent autoimmune disease.     

T cells induce extended class II MHC compartments in dendritic cells in a Toll-like receptor-dependent manner

Interaction of Ag-loaded dendritic cells with Ag-specific CD4 T cells induces the formation of long tubular class II MHC-positive compartments that polarize toward the T cell. We show involvement of a Toll-like receptor-mediated signal in this unusual form of intracellular class II MHC trafficking. First, wild-type dendritic cells loaded with LPS-free Ag failed to show formation of class II-positive tubules upon Ag-specific T cell engagement, but did so upon supplementation of the Ag with low concentrations of LPS. Second, Ag-loaded myeloid differentiation factor 88 -deficient dendritic cells failed to form these tubules upon interaction with T cells, regardless of the presence of LPS. Finally, inclusion of a cell-permeable peptide that blocks TNFR-associated factor 6 function, downstream of myeloid differentiation factor 88, blocked T cell-dependent tubulation. A Toll-like receptor-dependent signal is thus required to allow Ag-loaded dendritic cells to respond to T cell contact by formation of extended endosomal compartments. This activation does not result in massive translocation of class II MHC molecules to the cell surface.     

Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18

Production of IFN-gamma by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-gamma in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-gamma in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.     

Dual roles for IFN-gamma,but not for IL-4, in spontaneous autoimmune thyroiditis in NOD.H-2h4 mice

Spontaneous autoimmune thyroiditis (SAT) is an organ-specific autoimmune disease characterized by chronic inflammation of the thyroid by T and B lymphocytes. To investigate the roles of Th1 and Th2 cytokines in the pathogenesis of SAT, IFN-gamma(-/-) and IL-4(-/-) NOD.H-2h4 mice were generated. IL-4(-/-) mice developed lymphocytic SAT (L-SAT) comparable to that of wild-type (WT) mice. They produced little anti-mouse thyroglobulin (MTg) IgG1, but had levels of anti-MTg IgG2b comparable to WT mice. Compared with WT mice, IFN-gamma(-/-) mice produced significantly less anti-MTg IgG1 and IgG2b. Absence of IFN-gamma resulted in abnormal proliferation of thyroid epithelial cells with minimal lymphocyte infiltration. Thyroids of IFN-gamma(-/-) mice had markedly reduced B lymphocyte chemoattractant expression, B cell and plasma cell infiltration, and decreased MHC class II expression on thyrocytes compared with WT mice. Adoptive transfer of WT splenocytes to IFN-gamma(-/-) mice restored the capacity to develop typical L-SAT, enhanced anti-MTg IgG1 and IgG2b production, up-regulated MHC class II expression on thyrocytes and decreased thyrocyte proliferation. These results suggest that IFN-gamma plays a dual role in the development of SAT. IFN-gamma is required for development of L-SAT, and it also functions to inhibit thyroid epithelial cell proliferation.     

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.     

Efficient in vivo presentation of Listeria monocytogenes- derived CD4 and CD8 T cell epitopes in the absence of IFN-gamma

IFN-gamma is an essential component of the early Listeria monocytogenes-specific immune response, and is also an important regulator of Ag processing and presentation. Ag presentation is required for the induction and also the effector function of antimicrobial T cells. To evaluate the effect of IFN-gamma on bacterial Ag presentation in vivo, macrophages and dendritic cells were separated from L. monocytogenes-infected tissues and analyzed with peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based ex vivo Ag presentation assay. The comparison of professional APCs isolated from infected IFN-gamma-deficient and wild-type mice revealed different peptide presentation patterns of L. monocytogenes-derived CD8 T cell epitopes, while the presentation pattern of CD4 T cell epitopes remained unchanged. The further in vitro analysis of the generation of CD8 T cell epitopes revealed a peptide-specific effect of IFN-gamma on MHC class I-restricted Ag presentation. These results show that despite this modulation of the Ag presentation pattern of CD8 T cell epitopes, IFN-gamma is not generally required for the MHC class I- and MHC class II-restricted presentation of L. monocytogenes-derived antigenic peptides by professional APCs in vivo.