Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic β cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in β cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4+ diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This β cell ER stress increased activity of the calcium (Ca2+)-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, β cell ER stress induced by chemical and physiological triggers leads to β cell immunogenicity through Ca2+-dependent PTM. These findings elucidate a mechanism of how β cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing β cell recognition by autoreactive T cells.

As a result of less than optimal outcomes the use of islet allografts as a standard insulin replacement therapy is limited to adults with a history of extreme glucose dysregulation and hypoglycemia unawareness. In this study, we examined the use of prophylactic immunotherapy to prevent islet allograft rejection in the absence of antirejection drugs. Our protocol to achieve allograft acceptance used a negative vaccination strategy that is comprised of apoptotic donor cells delivered in Incomplete Freund’s Adjuvant (IFA) 1 week prior to islet transplantation. The goal of this new protocol is to elicit hyporesponsiveness to alloantigen prior to islet transplantation. First, we examined our protocol without islet allograft transplants and determined that the negative vaccination was not globally immunosuppressive or immunostimulatory. Islet allograft experiments using fully MHC-mismatched islet donors and recipients demonstrated that the negative vaccination strategy induced long-term islet allograft acceptance. Upon rechallenge with alloantigen, the negative vaccination protocol successfully achieved hyporesponsiveness. In addition, the microenvironment at the site of the tolerant allograft revealed a decrease in proinflammatory mediators (IFN-γ, TNF-α) and an increase in the anti-inflammatory mediator IL-10, as well as increased expression of the master regulator of T-regulatory cells, FOXP3. Our data suggest that pretreating allograft recipients with apoptotic donor alloantigen delivered in IFA induced long-term islet allograft acceptance and glycemic control by introducing alloantigen to the recipient immune system in a nonimmunostimulatory manner prior to transplant.

Type 1 diabetes (T1D) is an autoimmune disease, largely mediated by autoreactive T cells, in which islet β cells are destroyed. The events that break peripheral tolerance in individuals genetically predisposed to autoimmunity are poorly understood. Many physiological and environmental triggers associated with T1D, such as viral infection, chemicals, reactive oxygen species, and dynamic glucose sensing, result in endoplasmic reticulum (ER) stress. ER stress increases the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress generates neo-antigens that activate autoreactive T cells and worsen T1D. Indeed, chemical (Thapsigargin) induction of ER stress in murine islets or insulinomas increased IFNγ secretion from diabetogenic BDC2.5 T cells (28-1500 fold). Chelation of stress-induced calcium (Ca2+) flux reduced this response (27-64%), supporting a role for Ca2+-dependent enzymes such as tissue transglutaminase 2 (Tgase). Tgase activity is higher (18 fold) in β cells under stress, and reduced Tgase expression decreased the BDC2.5 response to β cells under stress (58%). Finally, nonantigenic insulinomas were exposed to normal physiological conditions in vivo. At harvest, these cells had increased ER stress (300 fold), increased Tgase activity (44 fold), and elicited higher BDC2.5 IFNγ responses (22 fold) compared to cultured cells. Thus, β cell ER stress causes formation of neo-antigens that activate autoreactive T cells.