Report from the 1st International NOD Mouse T-Cell Workshop and the follow-up mini-workshop.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.

Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice.

B cells can serve dual roles in modulating T cell immunity through their potent capacity to present Ag and induce regulatory tolerance. Although B cells are necessary components for the initiation of spontaneous T cell autoimmunity to beta cell Ags in nonobese diabetic (NOD) mice, the role of activated B cells in the autoimmune process is poorly understood. In this study, we show that LPS-activated B cells, but not control B cells, express Fas ligand and secrete TGF-beta. Coincubation of diabetogenic T cells with activated B cells in vitro leads to the apoptosis of both T and B lymphocytes. Transfusion of activated B cells, but not control B cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta cell autoantigens. Furthermore, this treatment induced mononuclear cell apoptosis predominantly in the spleen and temporarily impaired the activity of APCs. Cotransfer of activated B cells with diabetogenic splenic T cells prevented the adoptive transfer of type I diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of NOD mice treated with control B cells developed T1DM within 27 wk, <20% of the NOD mice treated with activated B cells became hyperglycemic up to 1 year of age. Our data suggest that activated B cells can down-regulate pathogenic Th1 immunity through triggering the apoptosis of Th1 cells and/or inhibition of APC activity by the secretion of TGF-beta. These findings provide new insights into T-B cell interactions and may aid in the design of new therapies for human T1DM.

Antigen-based immunotherapy for autoimmune disease: from animal models to humans?

Insights into tolerance and autoimmune processes have led to novel immunotherapeutics for inhibiting autoimmune disease in animal models. However, recent studies question the immune basis of some of these therapeutic strategies and raise concerns about their efficacy and safety. Here, we discuss the feasibility of extending the success of antigen-based immunotherapeutics for T-cell-mediated autoimmune diseases from animal models to humans.

Infectious Th1 and Th2 autoimmunity in diabetes-prone mice.

In the non-obese diabetic (NOD) mouse, a Th1-biased autoimmune response arises spontaneously against glutamic acid decarboxylase, concurrent with the onset of insulitis. Subsequently, Th1-type autoreactivity spreads intra- and intermolecularly to other beta-cell autoantigens (beta CAAs), suggesting that a spontaneous Th1 cascade underlies disease progression. Induction of Th2 immunity to a single beta CAA results in the spreading of Th2-type T-cell and humoral responses to other beta CAAs in an infectious manner. Thus, both Th1 and Th2 autoimmunity can evolve in amplificatory cascades defined by site-specific, but not antigen-specific, positive feedback circuits. Despite the continued presence of Th1 autoimmunity, the induction of Th2 spreading is associated with active tolerance to beta CAAs and reduced disease incidence. With disease progression there is an attenuation of beta CAA-inducible Th2 spreading, presumably because of a reduced availability of uncommitted beta CAA-reactive precursor T cells. We discuss the implications of these findings for the rational design of antigen-based immunotherapeutics.

Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy.

CD45 is a family of transmembrane protein tyrosine phosphatases exclusively expressed by hematopoietic cells and critically involved in the regulation of T cell activation signals. We now demonstrate that three 100-microg doses of anti-CD45RB mAb MB23G2 can induce long-term engraftment of islets into major histcompatibility complex-disparate chemically diabetic mice. Long-term graft survivors (>120 days) were tolerant to new islet allografts from the original donor strain. MB23G2 induced a temporary decrease in number circulating leukocytes but had no effect on leukocyte number in other lymphoid compartments. Histologic examination of allografts from treated and untreated recipients revealed a similar peri-islet infiltration on day 6. Eleven days after transplant, the peri-islet infiltrate in treated animals persisted, but in marked contrast to untreated control animals, there was no insulitis and islet integrity was preserved. The peri-islet infiltrate from treated animals showed a mild increase in CD4 cells, a decrease in CD8 cells, and decreased intensity of CD45RB expression. Treatment of naive animals with anti-CD45RB (MB23G2) resulted in a shift in CD45 isoform expression on T cells with a loss of higher molecular weight isoforms and increased expression of lower molecular weight (CD45R0) isoform. This shift in CD45 isoform expression from CD45RBHi to CD45RBLo was associated with an increase in the intragraft expression of transcripts for interleukin (IL) 4 and IL-10, consistent with the expected activity of this distinct immunoregulatory T cell subset. Antibody-mediated targeting of CD45 may induce tolerance through novel mechanisms and have direct applicability to clinical transplantation in humans.

GAD-reactive CD4+ Th1 cells induce diabetes in NOD/SCID mice.

Although glutamic acid decarboxylase (GAD) has been implicated in IDDM, there is no direct evidence showing GAD-reactive T cells are diabetogenic in vivo. To address this issue, 3-wk-old NOD mice received two injections of purified rat brain GAD; one mouse rapidly developed diabetes 3 wk later. Splenocytes from this mouse showed a proliferative response to purified GAD, and were used to generate a CD4+ T cell line, designated 5A, that expresses TCRs encoding Vbeta2 and Vbeta12. 5A T cells exhibit a MHC restricted proliferative response to purified GAD, as well as GAD65 peptide 524-543. After antigen-specific stimulation, 5A T cells secrete IFNgamma and TNFalpha/beta, but not IL-4. They are also cytotoxic against NOD-derived hybridoma cells (expressing I-Ag7) that were transfected with rat GAD65, but not nontransfected hybridoma cells. Adoptive transfer of 5A cells into NOD/SCID mice produced insulitis in all mice. Diabetes occurred in 83% of the mice. We conclude that GAD injection in young NOD mice may, in some cases, provoke diabetes due to the activation of diabetogenic T cells reactive to GAD65 peptides. Our data provide direct evidence that GAD65 autoimmunity may be a critical event in the pathogenesis of IDDM.

Inhibition of diabetes by an insulin-reactive CD4 T-cell clone in the nonobese diabetic mouse.

A cloned Th1 cell line was isolated from pancreatic lymph nodes of NOD mice that carries a T-cell receptor encoding Vbeta14 and proliferates in response to NOD islets, islet supernatant, and crystalline bovine and rat insulin, specifically to a B-chain peptide bound to IA(g7). The response to islet supernatant was reduced by 75% by anti-insulin antibody treatment. The insulin-reactive clone reduced insulitis and totally blocked the development of spontaneous diabetes in NOD mice (n = 8) as well as the adoptive transfer of diabetes into irradiated NOD mice following the injection of splenocytes from diabetic mice (n = 13). Trafficking of the adoptively transferred cells was assessed by labeling the clone or diabetic splenocytes with a fluorescent marker (DiI). The labeled clone was detected in the islet periphery, whereas labeled splenocytes alone invaded the islets by 3 days. In contrast, the protective clone dramatically delayed and reduced the number of labeled diabetic splenocytes infiltrating the islet, although their appearance in the spleen was unaffected. In vitro, the clone as well as supernatant derived from the clone blocked the proliferation of diabetic NOD splenocytes to islets. This inhibitory effect was diminished by anti-transforming growth factor-beta. In conclusion, an insulin-specific Th1 cell was isolated from NOD mice that traffics to the islet and prevents the spontaneous development and the adoptive transfer of diabetes. It appears to act locally by releasing transforming growth factor-beta and/or other factors that inhibit homing to and/or proliferation of diabetic splenocytes within the islet. These findings may provide insights into and suggest mechanisms for the protective effects of insulin therapy against diabetes.