Extended Data Fig. 1: Experimental set-up, gating strategy and impact of glutamine or pyruvate deprivation.

(a) Model depicting the experimental set-up. (b) Sorted CD56^Dim NK cells were pre-incubated in the presence or absence of extracellular glutamine for 3 hours and stimulated for 4 hours with K562 cells expressing the nanoluc. The proportion of cells expressing CD107a, IFN-γ and MIP1-β was measured by flow cytometry. The proportion of positive cells (Mean +/-SD, n = 3 independent donors) is shown. (c) Sorted CD56^Dim NK cells were pre-incubated in the presence or absence of extracellular pyruvate for 3 hours and stimulated with IL-12/IL-18 cells for 4 hours. The proportion of cells expressing IFN-γ and MIP1-β was measured by flow cytometry. The proportion of positive cells (Mean +/-SD, n = 3 independent donors) is shown. (d) Sorted CD56Dim NK cells were cultured for 4 hours in the presence or absence of pyruvate with 1 ng/ml IL-15. Annexin V was labelled according to manufacturer’s instructions (BD kit). PI was added extemporaneously, and cells were analysed by flow cytometry. The percentage of cells (Mean +/-SD; 2 independent donors) in the indicated cell populations is shown. Two-tailed T-test were performed comparing the indicated conditions in panel b, and c. Exact p-values are shown.