Phenoscaping reveals multimodal γδ T cell cytotoxicity as a strategy to overcome cancer cell-mediated immunomodulation.

γδ T cells can kill cancer cells via antibody-independent cytotoxicity (AIC) and antibody-dependent cellular cytotoxicity (ADCC). A better understanding of how these cytotoxic mechanisms are impacted by different cancer cells and different T cell donors could help identify improved immunotherapeutic strategies. To test the combinatorial interactions between T cell inter-donor heterogeneity (IDH), cancer cell inter-tumor heterogeneity (ITH), and multimodal γδ T cell killing, we performed a systematic single-cell phenoscaping analysis of >1,000 cultures of γδ T cells and colorectal cancer (CRC) patient-derived organoids (PDO). Phenoscaping analysis of post-translational modification (PTM) signaling, cell-cycle, apoptosis, and T cell immunophenotypes revealed that while unmodified γδ T cells have limited anti-tumor activity, IL-15Rα-IL-15 fusion protein (stIL15)-engineered γδ T cells can kill PDOs via AIC without exogenous cytokine support. However, when stIL15 γδ T cells only killed via AIC, cancer cells reciprocally rewired γδ T cell PTM signal networks in an ITH-specific manner to suppress anti-cancer cytotoxicity. stIL15 γδ T cells could overcome this cancer cell immunomodulation by also engaging B7-H3-targeted ADCC independent of B7-H3 checkpoint activity. Combined AIC and ADCC rescued γδ T cell PTM signaling flux and enabled γδ T cells to kill chemorefactory revival colon cancer stem cells. Together, these results demonstrate that multimodal γδ T cell cytoxicity mechanisms can overcome ITH-specific immunomodulation to kill chemorefractory cancer cells.