Summary We describe the generation of ovalbumin (OVA)-speci®c, MHC class II-restricted ab T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate a-and b-chains of the TCR. Unexpectedly, T cells bearing the transgenic ab TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modi®cation of the approach which enabled expression of the TCR b-chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen-dependent proliferation. These results show that successful generation of MHC class II-restricted, OVA-speci®c abTCR transgenic mice was dependent upon combining cDNA-and genomic DNA-based constructs for expression of the respective a-and b-chains of the TCR.

SummaryOvalbumin (OVA)-specific CD8 + T cells from the T cell receptor-transgenic line OT-I (OT-I cells) were injected into unirradiated transgenic 1KIP-mOVA mice, which express a membranebound form of OVA (mOVA) in the pancreatic islet [3 cells and the renal proximal tubular cells. OT-I cells accumulated in the draining lymph nodes (LN) of the kidneys and pancreas and in no other LN. They displayed an activated phenotype and a proportion entered cell cycle. Unilateral nephrectomy 7-13 d before inoculation of OT-I ceils into 1KIP-mOVA mice allowed the injected T cells to home only to the regional LN of the remaining kidney (and pancreas), but when the operation was performed 4 h before injecting the T ceils, homing to the LN of the excised kidney was evident. When the bone marrow of RIP-mOVA mice was replaced with one of a major histocompatibility haplotype incapable of presenting OVA to OT-I cells, no homing or activation was detectable. Therefore, OT-I cells were activated by OVA presented by short-lived antigen-presenting cells of bone marrow origin present in the draining LN of OVA-expressing tissue. These results provide the first evidence that tissue-associated "self" antigens can be presented in the context of class I via an exogenous processing pathway. This offers a constitutive mechanism whereby T cells can be primed to antigens that are present in nonlymphoid tissues, which are not normally surveyed by recirculating naive T cells.

Self-antigens expressed in extrathymic tissues such as the pancreas can be transported to draining lymph nodes and presented in a class I–restricted manner by bone marrow-derived antigen-presenting cells. Such cross-presentation of self-antigens leads to CD8+ T cell tolerance induction via deletion. In this report, we investigate the influence of CD4+ T cell help on this process. Small numbers of autoreactive OVA-specific CD8+ T cells were unable to cause diabetes when adoptively transferred into mice expressing ovalbumin in the pancreatic β cells. Coinjection of OVA-specific CD4+ helper T cells, however, led to diabetes in a large proportion of mice (68%), suggesting that provision of help favored induction of autoimmunity. Analysis of the fate of CD8+ T cells indicated that CD4+ T cell help impaired their deletion. These data indicate that control of such help is critical for the maintenance of CD8+ T cell tolerance induced by cross-presentation.

ABSTRACT

An antigen administered orally can induce immunological tolerance to a subsequent challenge with the same antigen. Evidence has been provided for the efficacy of this approach in the treatment of human autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. However, oral administration of autoantigen in mice was found to induce a cytotoxic T lymphocyte response that could lead to the onset of autoimmune diabetes. Thus, feeding autoantigen can cause autoimmunity, which suggests that caution should be used when applying this approach to the treatment of human autoimmune diseases.

A class I histocompatibility gene, H-2Kb, linked to the rat insulin promoter, is overexpressed in the pancreatic beta cells of transgenic mice. The mice, whether syngeneic or allogeneic to the transgene, develop insulin dependent diabetes without detectable T cell infiltration, suggesting a direct, non-immune role for the transgenic class I molecules in the disease process.

A pathogenic role for self-reactive cells against the stress protein Hsp6O has been proposed as one of the events leading to autoimmune destruction of pancreatic is cells in the diabetes of nonobese diabetic (NOD) mice. To examine this hypothesis, we generated transgenic NOD mice carrying a murine Hsp6O transgene driven by the H-2Ea class II promoter. This would be expected to direct expression of the transgene to antigen-presenting cells including those in the thymus and so induce immunological tolerance by deletion. Detailed analysis of Hsp6O expression revealed that the en-dogenous gene is itself expressed strongly in thymic medullary epithelium (and weakly in cortex) yet fails to induce tolerance. Transgenic mice with retargeted Hsp6O showed overexpres-sion of the gene in thymic cortical epithelium and in bone marrow-derived cells. Analysis of spontaneous T-cell responses to a panel of self and heterologous Hsp6O antigens showed that tolerance to the protein had not been induced, although responses to an immunodominant 437-460 epitope implicated in disease were suppressed, probably indicating an epitope shift. This correlated with changes in disease susceptibility: insulitis in transgenic mice was substantially reduced so that pathology rarely progressed beyond periislet infiltration. This was reflected in a substantial reduction in hyper-glycemia and disease. These data indicate that T cells specific for some epitopes of murine Hsp6O are likely to be involved in the islet-cell destruction that occurs in NOD mice.

In type 1 diabetes, cytokine action on β cells potentially contributes to β cell destruction by direct cytotoxicity, inducing Fas expression, and up-regulating class I MHC and chemokine expression to increase immune recognition. To simultaneously block β cell responsiveness to multiple cytokines, we overexpressed suppressor of cytokine signaling-1 (SOCS-1). This completely prevented progression to diabetes in CD8+ TCR transgenic nonobese diabetic (NOD) 8.3 mice without affecting pancreas infiltration and partially prevented diabetes in nontransgenic NOD mice. SOCS-1 appeared to protect at least in part by inhibiting TNF- and IFN-γ-induced Fas expression on β cells. Fas expression was up-regulated on β cells in vivo in prediabetic NOD8.3 mice, and this was inhibited by SOCS-1. Additionally, IFN-γ-induced class I MHC up-regulation and TNF- and IFN-γ-induced IL-15 expression by β cells were inhibited by SOCS-1, which correlated with suppressed 8.3 T cell proliferation in vitro. Despite this, 8.3 T cell priming in vivo appeared unaffected. Therefore, blocking β cell responses to cytokines impairs recognition by CD8+ T cells and blocks multiple mechanisms of β cell destruction, but does not prevent T cell priming and recruitment to the islets. Our findings suggest that increasing SOCS-1 expression may be useful as a strategy to block CD8+ T cell-mediated type 1 diabetes as well as to more generally prevent cytokine-dependent tissue destruction in inflammatory diseases.