키메라 항원 수용체 (CAR)-γδ T 세포의 생산 및 평가

Abstract

Background: Chimeric antigen receptor (CAR)-T cell therapy has been successful in clinics, but complications and side-effects hamper wide application. Gamma delta (γδ) T cells can recognize antigens without MHC restriction, reduceing the risk of graft-versus-host disease (GvHD). Thus, I set up the experiments to characterize expanded γδ T cells and produce CAR-γδ T cells. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers. Cell surface markers of γδ T cells were characterized using flow cytometry. γδ T cells were cultured for 2 weeks in three different conditions. Lentiviral vector expressing CD19-CAR (FMC63-CD8 hinge) was transduced at day 10 in culture. Cytotoxicity was analyzed using 7-amino actinomycin D (7-AAD) and Annexin Ⅴ (AV) in OCI-Ly7 cells, a human B cell lymphoma cell line. NSG mice were injected intraperitoneally (i.p.) with SU- DHL6 cells labeled with Carboxyfluorescein succinimidyl ester (CFSE) followed by injection of CD19-CAR-transduced γδ T cells. After 48 hours, the peritoneal lavage was harvested and analyzed by flow cytometry to evaluate in vivo efficacy. Results: Vδ1 γδ T cells were expanded in the presence of IL-2+IL-15, while Vδ2 γδ T significantly proliferated in the presence of IL-2+ZOL+IL-15. γδ T cells expressed the activating receptors (NKG2D, CD16, and DNAM-1), cell death receptors (TRAIL and FasL) and, immune checkpoint receptors (PD-1 and BTLA) molecules sustainably. CAR-γδ T cells exhibited higher cytotoxicity than that of γδ T cells without CAR in vitro. Mice injected with ex vivo expanded CAR- γδ T cells showed lower numbers of SU-DHL6 cells than mice injected with γδ T cells without CAR. Conclusion: This study shows ex vivo expanded γδ T cells expressed cell surface markers robustly, and γδ T cells transduced with CAR exhibited advanced tumor cell killing ability in vitro and in vivo.