Construction of CAR-iTreg (Chimeric antigen receptor-inducible Treg) for inducing tolerance in transplantation

Abstract Tolerance induction is ultimate goal in the field of transplantation. Cell-based therapy with regulatory T cells (Treg) has been effective and evolving, Chimeric antigen receptor (CAR) T cells to target specific antigen are new promising cell-based therapy. Engineering of CAR-Treg have many advantages that can be used in transplantation. Here, we designed development of CAR-iTreg (Chimeric antigen receptor-inducible Treg), which is engineered to become inducible Treg in vivo. DNA for single chain fragment variable (scFv) was obtained from MR1 hybridoma to target CD40L as the antigen. Cytoplasmic signaling part was constituted with CD3z and CD28 for T cell activation, and with TGFβR1 and IL-2Rβ for Treg induction. The CAR DNA construct was inserted into pLVX-IRES-ZsGreen1 expression plasmid vector. The expression vector and packaging vectors were used to produce lentiviral vector in HEK293FT cells. Harvested lentivirus has been tested in Jurkat and EL4 cells, and the expression of ZsGreen fluorescence and the CAR could be confirmed. This CAR-containing lentivirus can be used to transfect naïve CD4+ T cells and be transferred to syngeneic mice. These CAR-T cells target CD40L expressed on activated T cell and convert into inducible Treg cells in the graft environment. Thus, this uniquely engineered CAR construct allows tolerance induction in transplantation model. Furthermore, this CAR-iTreg strategy with other scFv has potential application in autoimmune diseases.