Processing in the endoplasmic reticulum generates an epitope on the insulin A chain that stimulates diabetogenic CD8 T cell responses.

RIP-B7.1 mice express the costimulator molecule B7.1 (CD80) on pancreatic beta cells and are a well-established model for studying de novo induction of diabetogenic CD8 T cells. Immunization of RIP-B7.1 mice with preproinsulin (ppins)-encoding plasmid DNA efficiently induces experimental autoimmune diabetes (EAD). EAD is associated with an influx of CD8 T cells specific for the K(b)/A(12-21) epitope into the pancreatic islets and the subsequent destruction of beta cells. In this study, we used this model to investigate how ppins-derived Ags are expressed and processed to prime diabetogenic, K(b)/A(12-21)-specific CD8 T cells. Targeting the K(b)/A(12-21) epitope, the insulin A chain, or the ppins to the endoplasmic reticulum (ER) (but not to the cytosol and/or nucleus) efficiently elicited K(b)/A(12-21)-specific CD8 T cell responses. The K(b)/A(12-21) epitope represents the COOH terminus of the ppins molecule and, hence, did not require COOH-terminal processing before binding its restriction element in the ER. However, K(b)/A(12-21)-specific CD8 T cells were also induced by COOH-terminally extended ppins-specific polypeptides expressed in the ER, indicating that the epitope position at the COOH terminus is less important for its diabetogenicity than is targeting the Ag to the ER. The K(b)/A(12-21) epitope had a low avidity for K(b) molecules. When epitopes of unrelated Ags were coprimed at the same site of Ag delivery, "strong" K(b)-restricted (but not D(b)-restricted) CD8 T cell responses led to the suppression of K(b)/A(12-21)-specific CD8 T cell priming and reduced EAD. Thus, direct expression and processing of the "weak" K(b)/A(12-21) epitope in the ER favor priming of autoreactive CD8 T cells.

A missing PD-L1/PD-1 coinhibition regulates diabetes induction by preproinsulin-specific CD8 T-cells in an epitope-specific manner.

Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1(-/-) and PD-1(-/-) mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K(b)/A(12-21)-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA(12-21) DNA (encoding ppins without the COOH-terminal A(12-21) epitope) elicited K(b)/B(22-29)-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA(12-21) (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K(b)/B(22-29)-specific CD8 T-cells. The A(12-21) epitope binds K(b) molecules with a very low avidity as compared with B(22-29). Interestingly, immunization of coinhibition-deficient PD-L1(-/-) or PD-1(-/-) mice with pCI/ppins induced K(b)/A(12-21)-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA(12-21) did neither recruit K(b)/B(22-29)-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA(12-21)/(K(b)/B(22-29))-mediated EAD was efficiently restored in RIP-B7.1(+)/PD-L1(-/-) mice, differing from PD-L1(-/-) mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K(b)/A(12-21))-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA(12-21) co-immunized PD-L1(-/-) mice, facilitated the expansion of ppinsΔA(12-21)/(K(b)/B(22-29))-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K(b)/B(22-29)- (but not the low-affinity K(b)/A(12-21))-epitope thus require stimulatory help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.

Deficiency in B7-H1 (PD-L1)/PD-1 coinhibition triggers pancreatic beta-cell destruction by insulin-specific, murine CD8 T-cells.

OBJECTIVE: RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic beta-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic beta-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells. RESEARCH DESIGN AND METHODS: DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1(-/-), PD-1(-/-) or bone marrow chimeric mice. RESULTS: Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1(-/-) or PD-1(-/-) mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic beta-cells or of PD-1 in autoreactive CD8 T-cells induced EAD. CONCLUSIONS: An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic beta-cells can trigger pancreatic beta-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the beta-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.