γδ T Cell Phenoscaping Reveals Multimodal Cytotoxicity Overcomes Cellular Therapy Immunomodulation

γδ T cells can kill cancer cells via antibody-independent cytotoxicity (AIC) and antibody-dependent cellular cytotoxicity (ADCC), but how these cytotoxic mechanisms are regulated by different cancer cells (inter-tumour heterogeneity (ITH)) and different T cell donors (inter-donor heterogeneity (IDH)) is unknown. To test the combinatorial interactions between T cell IDH, cancer cell ITH, and multimodal γδ T cell killing we performed a systematic single-cell phenoscaping analysis of >1,000 Vγ9Vδ2 T cell and colorectal cancer (CRC) patient-derived organoid (PDO) cultures. Phenoscaping analysis of post-translational modification (PTM) signalling, cell-cycle, apoptosis, and T cell immunophenotypes revealed that while unmodified γδ T cells have limited anti-tumour 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 kill via AIC, cancer cells reciprocally re-wire γδ T cell PTM signal networks in an ITH-specific manner. stIL15 γδ T cells can overcome this cancer cell immunomodulation by also engaging B7-H3-targeted ADCC independent of B7-H3 checkpoint activity. Combined AIC and ADCC rescue γδ T cell PTM signalling flux and enables γδ T cells to kill chemorefactory revival colonic stem cells (revCSCs). These results demonstrate that multimodal γδ T cell cytotoxicity mechanisms can overcome ITH-specific immunomodulation to kill chemorefractory cancer cells.This repo contains the processed scRNA-seq Scanpy AnnData object generated from the study.