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 affected by different cancer cells and different T-cell donors could help identify improved immunotherapeutic strategies. To test the combinatorial interactions among T cell interdonor heterogeneity, cancer cell intertumor heterogeneity (ITH), and multimodal γδ T-cell killing, we performed a systematic single-cell phenoscaping analysis of more than 1,000 γδ T-cell and colorectal cancer patient-derived organoid cultures. Single-cell analysis of posttranslational modification (PTM) signaling, cell cycle, apoptosis, and T-cell immunophenotypes revealed that whereas unmodified γδ T cells have limited antitumor activity, IL15Rα-IL15 fusion protein [stabilized IL15 (stIL15)]-engineered γδ T cells can kill patient-derived organoids 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 anticancer 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. SIGNIFICANCE: Single-cell phenoscaping of more than 1,000 γδ T-cell and patient-derived organoid cultures shows that cancer cells suppress anticancer γδ T-cell cytotoxicity but γδ T cells can use multimodal killing to overcome immunomodulation.