The death receptor Fas mediates local bystander killing of antigen-negative variants by antigen-specific CD8 T cells in a heterogeneous tumor

Abstract Antigen-specificity is one of the hallmarks of the adaptive immune system, and CD8 T cells are particularly crucial for eliminating malignant cells with aberrant expression of tumor-associated self-antigens or neoantigens. We established a high-throughput pooled CRISPR/Cas9 screen for functionally assessing cancer cell genes involved in modulating the cytotoxic activity of antigen-specific CD8 T cells. As validation of our approach, we identified many genes known to be altered in cancers refractory to immunotherapy, including MHC component B2m and the checkpoint molecule Cd274. Interestingly, the cell surface death receptor Fas was identified as a strong hit in mediating the killing of antigen-positive lymphoma and was further verified with breast cancer, despite the availability of other cytotoxic effectors such as perforin/granzymes. Using a 2-antigen system, we surprisingly observed that Fas was also mediating the killing of neighboring GFP-negative cancer cells by T cells highly specific for a GFP epitope. This phenomenon was not dependent on MHC Class I expression but was potentiated by interferon gamma, which is known to upregulate Fas expression. Even for T cells that were previously primed, this bystander cytotoxicity required the presence of neighboring antigen-positive cells, suggesting a very hyperlocal and temporally regulated off-target effect that was not dependent on natural killer cells in vivo. We believe this phenomenon to be crucial to the durable remissions seen with highly antigen-specific approaches such as CAR-T cell therapy. Modulating this pathway may be a viable approach for preventing immune escape of rare antigen-loss variants or treating heterogeneous tumors with no clear single target antigen.