Delineating mechanisms underlying the enhanced stability and functionality of CD2-KO Tregs and chimeric antigen receptor (CAR) Tregs and their application in xenotransplantation

PROJECT SUMMARY Regulatory T cell (Treg) therapy has been widely investigated for control of immune responses in different models of autoimmunity and transplantation. Despite promising results in using Tregs and chimeric antigen receptor (CAR) Tregs for tolerance induction, there are still major concerns regarding the plasticity of Tregs and the effect of T cell-depleting conditioning regimens on these cells that need to be addressed before their clinical application. We have discovered that CD2-KO Tregs and CAR Tregs are not only resistant to a conditioning regimen that includes anti-CD2 antibody (siplizumab), but also have increased FOXP3 expression and Treg functionality in vitro and in vivo. CD2 is a costimulatory molecule that is present in all T cells, including Tregs. Potential direct or indirect link between CD2 signaling and FOXP3 is not known. Our preliminary data show that CD2-KO Tregs have increased mTORc1 activity, glycolysis and mitochondrial respiration. We hypothesize that metabolically- active CD2-KO Tregs generate metabolites that through histone modification or post-translational modifications increase FOXP3 levels. In Aim 1, we will address these hypotheses. We will study CD2+ and CD2-KO Tregs at the genome, epigenome, transcriptome and protein levels and evaluate their metabolism and signaling pathways. In Aim 2, we will explore the translational aspects of this discovery in xenotransplantation setting. We have developed and validated a CAR against a universal pig antigen (class-I swine leukocyte antigen (SLA)) and have shown that SLA CAR Tregs harbor higher suppressive capacity in suppressing anti-pig immune responses in vitro as compared to polyclonal Tregs. We hypothesize that CD2-KO SLA CAR Tregs resist conversion to effector T cells and protect transplanted pig organs in an antigen-specific manner. We will evaluate the resistance of CD2-KO SLA CAR Tregs to conversion in inflammatory conditions and also the efficacy of CD2- KO SLA CAR Tregs in preventing the rejection of pig islets in a humanized mouse model of xeno-islet transplantation. Taken together, this work can potentially lead to the development of safer and more efficient Treg-based immunotherapies. Mechanistic studies could uncover molecules and/or signaling pathways involved in determining the fate/function of Tregs, targeting of which could alter the immune balance toward tolerance.