Engineering antigen specific T regulatory cells for transplant tolerance (P2148)

Abstract

Abstract T regulatory cells (Tregs) are critical for self-tolerance and are being pursued as a potential cell therapy in transplantation and autoimmunity. While polyclonal Tregs can prevent transplant rejection in mice, allo-antigen specific Tregs are more potent. A chimeric antigen receptor (CAR) may be an effective way to make ag-specific Tregs in suitable numbers for the clinic. CARs are created by combining antibody variable domains with T cell receptor signaling domains. However, CARs for Treg cell therapy must a) function within the distinct signaling network of Tregs and b) recognize a transplant-relevant antigen. CAR function in Tregs was assessed by transducing sorted cells with a highly-expressed, humanized HER2 CAR. Cells were labeled with proliferation dye and assessed for their ability to respond to target antigen. Two CARs specific for HLA-A2 were generated by cloning the variable portions of the anti-A2 antibody from a hybridoma, creating a single chain antibody and ligating it in frame in the humanized CAR. These CARs were assessed for expression and activity by flow cytometry, and cytotoxicity and proliferation assays. Preliminary data suggests that CARs can induce strong, ag-specific proliferation in human Tregs and that an ag-specific HLA-A2 CAR has been generated. Further work includes determining the effect of CAR stimulation on Treg phenotype via cytokine staining and suppression assays, and optimizing the surface expression of the HLA-A2 CAR via phage display.