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. 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.