CAR T cell-induced interferon gamma enhances MHC class I expression and sensitizes neuroblastoma to TCR-engineered T cell therapy

Abstract

T cell receptor (TCR)-engineered T cells target antigens presented on major histocompatibility complex (MHC) class I molecules. MHC class I expression is often low in childhood tumors such as neuroblastoma but can be stimulated by interferon gamma (IFNγ). Chimeric antigen receptor (CAR) T cell effector function, including IFNγ production, is MHC class I-independent. We aimed to enhance neuroblastoma immunotherapy by combining CAR- and TCR-engineered T cells. Our strategy exploits CAR T cell-derived effector molecules, including IFNγ, to increase tumor cell MHC class I expression and thereby enhance subsequent TCR T cell activity. IFNγ-mediated induction and persistence of MHC class I expression were analyzed in neuroblastoma cell lines by flow cytometry. Primary human T cells were engineered to express either a high-affinity TCR targeting the cancer testis antigen NY-ESO-1 or an L1CAM-targeted CAR. Cytotoxic activity of CAR and TCR T cells, applied alone or sequentially, was quantified against LAN-1 neuroblastoma cells using IncuCyte-based real-time killing assays. To model heterogeneous antigen expression, mixed tumor populations containing L1CAM-positive and CRISPR/Cas9-generated L1CAM-knockout cells were analyzed. In vivo MHC class I upregulation was assessed in a human tumor xenograft model following treatment with human L1CAM-CAR T cells and in a syngeneic tumor model following treatment with GD2-CAR T cells. NY-ESO-1 TCR T cells alone showed limited cytotoxicity against LAN-1 cells in vitro , whereas L1CAM-CAR T cell pretreatment increased MHC class I expression and enhanced subsequent TCR-mediated killing. In tumor cells with heterogeneous CAR antigen expression, CAR T cell recognition of antigen-positive cells induced HLA-A*02 upregulation across the tumor population, enabling subsequent NY-ESO-1 TCR T cell recognition of cells not directly recognized by CAR T cells. Exploratory in vivo analyses further showed increased tumor MHC class I expression following CAR T cell treatment, providing preliminary evidence of CAR T cell-mediated inflammatory priming in vivo . Sequential CAR- and TCR-engineered T cell therapy may help overcome limitations imposed by low MHC class I and heterogeneous antigen expression in neuroblastoma, supporting further investigation of this combinatorial strategy.