Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes

Abstract

Type 1 diabetes is a T cell-mediated autoimmune disease, and insulin is an important target of the autoimmune response associated with beta cell destruction. The mechanism of destruction is still unknown. Here, we provide evidence for CD8 T cell autoreactivity associated with recurrent autoimmunity and loss of beta cell function in type 1 diabetic islet transplant recipients. We first identified an insulin B chain peptide (insB10-18) with extraordinary binding affinity to HLA-A2(*0201) that is expressed by the majority of type 1 diabetes patients. We next demonstrated that this peptide is naturally processed by both constitutive and immuno proteasomes and translocated to the endoplasmic reticulum by the peptide transporter TAP1 to allow binding to HLA-A2 in the endoplasmic reticulum and cell surface presentation. Peripheral blood mononuclear cells from a healthy donor were primed in vitro with this peptide, and CD8 T cells were isolated that specifically recognize target cells expressing the insulin B chain peptide. HLA-A2(insB10-18) tetramer staining revealed a strong association between detection of autoreactive CD8 T cells and recurrent autoimmunity after islet transplantation and graft failure in type 1 diabetic patients. We demonstrate that CD8 T cell autoreactivity is associated with beta cell destruction in type 1 diabetes in humans.

Fig. 1.

Proteasome digestion of the insulin B chain. In vitro proteasome-mediated digestions of the 30-mer insulin B chain peptide containing potential HLA-A*0201–restricted epitopes. 20S proteasomes isolated from an Epstein–Barr virus-transformed B cell line (immuno proteasome) and an HeLa cell line (constitutive proteasome) were coincubated with 30-mer insulin B chain peptides at 37°C for 20 h. Digestion mixtures were analyzed by MS as described in Materials and Methods. The intensity is expressed as the percentage of total summed mass-peak intensities of digested 30-mer. Predicted epitope used for CTL induction is in bold.

Fig. 2.

Cytotoxic cytokine release by insulin B_10-18-specific CD8⁺ T cells after stimulation with insulin B_10-18. A total of 50,000 CD8⁺ T cells, either untreated (open bars) or treated (filled bars) with insulin B_10-18, were cultured on anti-INF-γ-coated, anti-granzyme B-coated, or IL-10 antibody-coated culture plates. INF-γ, anti-granzyme B, and IL-10 release was measured by ELISpot analysis. The bars show the number of cells producing IFN-γ, granzyme B, or IL-10. Data are expressed as means ± SD. The cut-off line represents the mean plus 3 × SD of specific CTLs frequencies in HLA-A2-positive nondiabetic subjects. *, P < 0.0005 compared with the untreated CTLs.

Fig. 3.

Insulin B_10-18-specific CD8 frequencies in peripheral blood samples of islet allograft recipients who have persistent islet graft function versus recurrent autoimmunity within 1 year after islet transplantation. (A) Representative examples of tetramer stainings of HLA-A2-negative subjects (Left), patients with recurrent type 1 diabetes after islet transplantation (Center), and patients with successfully restored β cell function without islet autoimmunity (Right). (B) Insulin B_10-18-specific CD8 frequencies in the circulation of type 1 diabetic islet transplant recipients with recurrent autoimmunity and islet allograft loss (with or without concurrent alloreactivity against the allograft), patients with persistent β cell function, and patients who lost islet function in combination with induction of allograft rejection without evidence of autoimmunity. HLA-A2^insB10-18 tetramer staining revealed a positive correlation between detection of autoreactive CD8⁺ T cells and development of recurrent autoimmunity after islet transplantation and subsequent graft failure in type 1 diabetic patients.