Sensitizing Resistant Cancer Cells to T cell-secreted Cytokines

Tumor heterogeneity is a major barrier to cancer therapy, including immunotherapy. Activated T cells can efficiently kill tumor cells following recognition of MHC class I (MHC-I) bound peptides, but this selection pressure favors outgrowth of MHC-I deficient tumor cells. We performed a genome-scale screen to discover alternative pathways for T cell-mediated killing of MHC-I deficient tumor cells. Autophagy and TNF signaling emerged as top pathways, and inactivation of Rnf31 (TNF signaling) and Atg5 (autophagy) sensitized MHC-I deficient tumor cells to apoptosis by T cell-derived cytokines. Mechanistic studies demonstrated that inhibition of autophagy amplified pro-apoptotic effects of cytokines in tumor cells. Antigens from apoptotic MHC-I deficient tumor cells were efficiently cross-presented by dendritic cells, resulting in heightened tumor infiltration by IFNg and TNFa-producing T cells. Tumors with a substantial population of MHC-I deficient cancer cells could be controlled by T cells when both pathways were targeted using genetic or pharmacological approaches. B16 ctrl-KO, B16 Rnf31-KO, B16 Atg5-KO, B16 Rnf31/Atg5-dKO tumor cells (5x105/well) were plated in 6-well plates and treated with and without TNFa (2 ng/ml, Peprotech) for 24h. Three biological replicates were used to extract total RNA using the RNeasy Plus Micro Kit (Qiagen #74034) according to the manufacturer’s protocol.