Gamma-delta T cells are potent anti-cancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or HLA background. Gamma delta T cells can sense conserved cell stress signals prevalent in transformed cells, although the mechanisms behind the targeting of stressed target cells remain poorly characterized.

Vg9Vd2 T cells – the most abundant subset of human gamma delta T cells – recognize a protein complex containing BTN2A1 and BTN3A1, a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells.

Combined genome-wide CRISPR screens in target cancer cells identified pathways that regulate gamma delta T cell killing and BTN3A cell surface expression. The screens revealed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of gamma delta T cells through transcription, post-translational modifications, and membrane trafficking.

In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of BTN3A, as well as BTN2A1, during metabolic crises was dependent on the energy sensor AMPK.

Finally, small molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vg9Vd2 TCR-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation suggests new avenues to enhance gamma-delta T cell anti-cancer activity.