The cytoplasmic domain of CTLA-4 control autoimmunity via inducing regulatory T cells

Abstract Regulatory T cells (Tregs) have critical roles for maintaining immune tolerance and homeostasis. Recently, it is revealed that CTLA-4 is an essential membrane protein for the suppressive function of Treg. However, the molecular mechanism of cytoplasmic domain of CTLA-4 (ctCTLA-4) and its role in Treg function and differentiation is not clearly understood. In this study, we utilized ctCTLA-4 peptide and its various mutant forms conjugated with dNP2, a cell-permeable peptide, to investigate the mechanism of ctCTLA-4 in Treg function and differentiation. We found that the ctCTLA-4 convert naïve T cells to Foxp3-positive cells in T helper 17 cell differentiation condition in vitro, while the lysine-rich motif mutant form could not. The dNP2-ctCTLA-4 also significantly inhibits experimental autoimmune encephalomyelitis (EAE) in vivo, by reducing CNS-infiltrating Th17 cells with significantly increased Treg proportion. As similar with in vitroresults, lysine-rich motif mutant form could not increase Treg proportion and inhibit EAE. To elucidate its molecular mechanisms, we conducted HuProt protein microarray to identify lysine-rich motif dependent binding partner of ctCTLA-4 related with increasing Tregs. With the results, we found that ctCTLA-4 decreased a phosphorylation of MAPK in T cell stimulation conditions resulting enhanced nuclear localization of Smad2/3, a key transcription factor for Foxp3 expression. These results suggest that ctCTLA-4 has a critical molecular mechanism of inducing Tregs to maintain peripheral immune tolerance to control autoimmunity.