CRISPR screens decode cancer cell pathways that trigger γδ T cell detection

Nature. 2023 Sep;621(7977):188-195. doi: 10.1038/s41586-023-06482-x. Epub 2023 Aug 30.


γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1-5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells4-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ 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 both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (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 Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.

MeSH terms

  • AMP-Activated Protein Kinases / genetics
  • AMP-Activated Protein Kinases / metabolism
  • CRISPR-Cas Systems*
  • Cell Line
  • Cell Membrane / metabolism
  • Gene Editing*
  • Humans
  • Neoplasms* / genetics
  • Neoplasms* / immunology
  • Neoplasms* / metabolism
  • Receptors, Antigen, T-Cell, gamma-delta* / immunology
  • Receptors, Antigen, T-Cell, gamma-delta* / metabolism
  • T-Lymphocytes* / immunology
  • T-Lymphocytes* / metabolism


  • AMP-Activated Protein Kinases
  • BTN2A1 protein, human
  • BTN3A1 protein, human
  • Receptors, Antigen, T-Cell, gamma-delta