229-LB: Generation of Human CAR-Treg Cells for Inducing Immunological Tolerance against Diabetogenic T Cells in Human Pancreatic Islets

Abstract

We have developed pancreatic beta-cell, antigen-specific, Chimeric Antigen Receptor (CAR) T regulatory cells (Tregs) and explored their therapeutic potential against type 1 diabetes (T1D)/Latent Autoimmune Diabetes of Adult (LADA) in human pancreatic tissues ex-vivo. Two human Glutamic Acid Decarboxylase 65 kD isoform (GAD65) B-cell paratopes known to bind to two immunodominant regions in the N-terminal (CAR-N) and Middle (CAR-M) regions of human GAD65 were selected for assembly onto T cell receptors of Tregs. We first show that GAD65-CAR Tregs had a superior proliferation index (3 times than naïve Tregs) and were able to suppress diabetogenic, T cells in-vitro and this suppression was significantly superior to naïve Tregs at 2:1 (Tresp vs Treg) ratio (p<0.004). Further, in ex vivo co-culture experimentation with pancreatic islets, confocal images demonstrated homing of GAD65-CAR Tregs to pancreatic islets as early as 24 hrs. With live immunofluorescence imaging and flow cytometry we demonstrated that GAD65-CAR Tregs from T1D/LADA patient were able to suppress diabetogenic cytotoxic T lymphocytes (CTLs) (p<0.04) in the immune cell microenvironment in presence of heterologous pancreatic islet ex vivo. These findings verify that GAD65-CAR Tregs recognize endogenous GAD65 on isolated islets in an MHC-independent manner, are proliferative robustly and suppress autologous CTLs. Thus, GAD65-CAR Tregs can induce tolerance against T1D diabetogenic T cells and the use of GAD65-CAR Tregs can prove a promising strategy for inducing tolerance in islet transplantation to replace the immunosuppressive drugs in a safe potent manner. Finally, homing of the GAD65-CAR Tregs to the islets, provided evidence for antigen-specific re-directionality. The robust proliferation and superior suppression of diabetogenic T cells by GAD65-CAR Tregs validate the functionality of GAD65-CAR Tregs in inducing tolerance in T1D patients and host vs graft islet rejections. Disclosure S. Imam: None. S. Rafiqi: None. J. C. Jaume: None. Funding University of Toledo