OBJECTIVE-We have previously reported a highly diabetogenic CD8 T-cell clone, G9C8, in the nonobese diabetic (NOD) mouse, specific to low-avidity insulin peptide B15-23, and cells responsive to this antigen are among the earliest islet infiltrates. We aimed to study the selection, activation, and development of the diabetogenic capacity of these insulin-reactive T-cells. RESEARCH DESIGN AND METHODS-We generated a T-cell receptor (TCR) transgenic mouse expressing the cloned TCR V␣18/V␤6 receptor of the G9C8 insulin-reactive CD8 T-cell clone. The mice were crossed to TCRC␣ Ϫ/Ϫ mice so that the majority of the T-cells expressed the clonotypic TCR, and the phenotype and function of the cells was investigated.RESULTS-There was good selection of CD8 T-cells with a predominance of CD8 single-positive thymocytes, in spite of thymic insulin expression. Peripheral lymph node T-cells had a naïve phenotype (CD44 lo , CD62L hi ) and proliferated to insulin B15-23 peptide and to insulin. These cells produced interferon-␥ and tumor necrosis factor-␣ in response to insulin peptide and were cytotoxic to insulin peptide-coated targets. In vivo, the TCR transgenic mice developed insulitis but not spontaneous diabetes. However, the mice developed diabetes on immunization, and the activated transgenic T-cells were able to transfer diabetes to immunodeficient NOD.scid mice.CONCLUSIONS-Autoimmune CD8 T-cells responding to a low-affinity insulin B-chain peptide escape from thymic negative selection and require activation in vivo to cause diabetes.

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Objective Apoptotic destruction of insulin-producing pancreatic β-cells is involved in the aetiology of both type 1 and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge whose sustained activation has recently been implicated in pancreatic β-cell apoptosis and in islet cell death post-transplantation. Here, we examine the importance of β-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8+ T cells. Research Design and Methods Clonal MIN6 β-cells or CD1 mouse pancreatic islets were infected with recombinant adenoviruses encoding enhanced green fluorescent protein (eGFP/Null), constitutively-active AMPK (AMPK CA), or dominant-negative AMPK (AMPK DN) and exposed or not to tumour necrosis factor-α (TNF-α) interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Apoptosis was detected by monitoring the cleavage of caspase-3 and DNA fragmentation. The cytotoxic effect of CD8+ purified T cells was examined against pancreatic islets from NOD mice infected with either Null or the AMPK DN-expressing adenoviruses. Results Exposure to cytokines, or expression of AMPK CA, induced apoptosis in clonal MIN6 β-cells and CD1 mouse pancreatic islets. By contrast, over-expression of AMPK DN protected against the proapoptotic effect of these agents, in part by preventing decreases in cellular ATP, and lowered the cytotoxic effect of CD8+ T cells towards NOD mouse islets. Conclusions Inhibition of AMPK activity enhances islet survival in the face of assault by either cytokines or T cells. AMPK may therefore represent an interesting therapeutic target to suppress immune mediated β-cell destruction, and may increase the efficacy of islet allografts in Type 1 diabetes.

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Low-avidity autoreactive CD8 T cells (CTLs) escape from thymic negative selection, and peripheral tolerance mechanisms are essential for their regulation. We report the role of proinsulin (PI) expression on the development and activation of insulin-specific CTLs in the NOD mouse model of type 1 diabetes. We studied insulin B-chain-specific CTL from different T-cell receptor transgenic mice (G9Cα) expressing normal PI1 and PI2 or altered PI expression levels. In the absence of PI2 (Ins2), CTL in pancreatic lymph nodes (PLNs) were more activated, and male G9Cα mice developed T1D. Furthermore, when the insulin-specific CTLs developed in transgenic mice lacking their specific PI epitope, the CTLs demonstrated increased cytotoxicity and proliferation in vitro and in vivo in the PLNs after adoptive transfer into NOD recipients. Dendritic cell-stimulated proliferation of insulin-specific T cells was reduced in the presence of lymph node stromal cells (LNSCs) from NOD mice but not from mice lacking the PI epitope. Our study shows that LNSCs regulate CTL activation and suggests that exposure to PI in the periphery is very important in maintenance of tolerance of autoreactive T cells. This is relevant for human type 1 diabetes and has implications for the use of antigen-specific therapy in tolerance induction.

The lymph node cells of CBA (H-2k), but not BALB/c (H-2d) mice, release interferon (IFN)-gamma into the supernatant when immunized with picryl chloride epicutaneously and then exposed to antigen (haptenized cells) in vitro 4 days later. The failure in IFN-gamma production maps to the major histocompatibility complex (MHC; H-2d) in the congenic BALB/c, BALB/k and BALB/b mice. The evidence that this is an MHC regulation of the class of response to a range of antigens and not a classical Ir gene effect is (a) the difference is seen with several antigens including picryl chloride, "oxazolone" and purified protein derivative of tuberculin and (b) BALB/c mice, which fail to produce IFN-gamma, show excellent contact sensitivity to picryl chloride. It was also found that the crosses between responder and nonresponder strains (CBA x BALB/c)F1 respond to antigen on responder cell but not on nonresponder cells. This influence of MHC on the class of the immune response is a possible basis for some of the associations of MHC with disease.