CAR T cells, CAR NK cells and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines

Chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy against cancer. Although there is a growing interest in other cell types, a comparison of CAR immune effector cells in challenging solid tumor contexts is lacking. Here, we compare mouse and human NKG2D-CAR expressing T cells, NK cells and macrophages against glioblastoma, the most aggressive primary brain tumor. In vitro we show that T cell cancer killing is CAR-dependent, whereas intrinsic cytotoxicity overrules CAR-dependence for NK cells and CAR macrophages reduce glioma cells in co-culture assays. In orthotopic immunocompetent glioma mouse models, systemically administered CAR T cells demonstrate superior accumulation in the tumor and each immune cell type induces distinct changes in the tumor microenvironment. An otherwise low therapeutic efficacy is significantly enhanced by co-expression of pro-inflammatory cytokines in all CAR immune effector cells, underscoring the necessity for multifaceted cell engineering strategies to overcome the immunosuppressive solid tumor microenvironment. Overall design: Mice bearing Gl261 syngeneic tumors were injected with different CAR cells and analyzed using single-cell RNA-Sequencing. Biological replicates were multiplexed with barcoded lipids. Library preparation was conducted using 10X 3' mRNA-Sequencing reagents.