Genome-wide CRISPR screens reveal metabolic and transcriptional regulation of BTN3A and cancer susceptibility to Vγ9Vδ2 T cell targeting

Abstract γδ T cells are potent anti-cancer effectors with potential to target tumors broadly, independent of neoantigens or HLA-background. γδ T cells can sense conserved cell stress signals prevalent in transformed cells, although the mechanisms governing how γδ T cells sense and kill stressed target cells remain poorly characterized. Vγ9Vδ2 T cells – the most abundant subset of human γδ T cells – recognize a protein complex containing butyrophilin 3A1 (BTN3A1), a ubiquitously expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumor cells. Here we performed genome-wide CRISPR screens in target cancer cells to identify pathways that regulate: (1) γδ T cell activity with a functional co-culture killing screen, and (2) BTN3A1 cell surface expression in a phenotypic screen. Multilayered regulation of BTN3A1 expression was uncovered: transcriptional regulation (IRF1, CTBP1, ZNF217, RUNX1), intracellular trafficking, sialylation, oxidative phosphorylation (OXPHOS), purine metabolism, and other metabolic pathways. Consistent with these results, we found upregulated BTN3A1 on cells undergoing an energy crisis due to glucose deprivation, glycolysis inhibition, or OXPHOS inhibition. Furthermore, we discovered that this BTN3A1 upregulation was induced by activation of the AMP-activated protein kinase (AMPK). Also, CRISPR screen-derived gene expression signatures correlated with higher survival in low-grade glioma patients whose tumors had high Vγ9Vδ2 T cell infiltration. Uncovering this AMPK-dependent mechanism of metabolic stress-induced ligand activation deepens our understanding of γδ T cell stress surveillance and suggests new avenues to enhance γδ T cell anti-cancer activity. M.R.M. is a Cancer Research Institute (CRI) Irvington Fellow supported by CRI and was funded by the Human Vaccines Project Michelson Prize for Human Immunology.