Regulatory T cells targeting a pathogenic MHC class II: Insulin peptide epitope postpone spontaneous autoimmune diabetes
In spontaneous type 1 diabetes (T1D) non-obese diabetic (NOD) mice, the insulin B chain peptide 9-23 (B:9-23) can bind to the MHC class II molecule (IAg7) in register 3 (R3), creating a bimolecular IAg7/InsulinB:9-23 register 3 conformational epitope (InsB:R3). Previously, we showed that the InsB:R3-specific chimeric antigen receptor (CAR), constructed using an InsB:R3-monoclonal antibody, could guide CAR-expressing CD8 T cells to migrate to the islets and pancreatic lymph nodes. Regulatory T cells (Tregs) specific for an islet antigen can broadly suppress various pathogenic immune cells in the islets and effectively halt the progression of islet destruction. Therefore, we hypothesized that InsB:R3 specific Tregs would suppress autoimmune reactivity in islets and efficiently protect against T1D. To test our hypothesis, we produced InsB:R3-Tregs and tested their disease-protective effects in spontaneous T1D NOD CD28-/- mice. InsB:R3-CAR expressing Tregs secrete IL-10 dominated cytokines upon engagement with InsB:R3 antigens. A single infusion of InsB:R3 Tregs delayed the onset of T1D in 95% of treated mice, with 35% maintaining euglycemia for two healthy lifespans, while whereas control Tregs did not. Our data demonstrate that Tregs specific for MHC class II: Insulin peptide epitope (MHCII/Insulin) protect mice against T1D more efficiently than polyclonal Tregs lacking islet antigen specificity, suggesting that the MHC II/insulin-specific Treg approach is a promising immune therapy for safely preventing T1D. Overall design: The pancreas of mice treated with InsB:R3-Tregs, Ctl-Tregs, or saline buffer (Non-T), three mice per group, was digested, and islets were handpicked on day 8 post-transfer. Using independent parametric significance testing between two groups and assuming a large effect size (Cohen's d) equal to 4, equal variances, and power of 0.80, significant results (can be achieved with three samples per group. Three diabetic NOD.CD28-/- mice (DM) diagnosed within one week were included for comparison. Fresh islets from each group were pooled, and RNAs were extracted using an RNA Miniprep kit (Zymo Research, R1054). High-quality RNA samples that passed the quality control test were subjected to RNA-Seq analysis (Novogene Corporation, Inc.).