Extended Data Fig. 3: Complementary metabolomics data and impact of PHGDH inhibition on immediate functions.

(a) Sorted CD56^Dim NK cells were cultured for 4 hours in the indicated medium. Mass isotopologue distribution after culture with ¹³C-glucose (complete medium: in blue, without pyruvate: in orange) or ¹³C-pyruvate (complete medium: light orange framed) for the indicated metabolites (n = 6 independent donors, values obtained for each individual donor are shown) measured by mass-spectrometry is shown. (b) The concentration of intracellular p-serine in sorted CD56^Dim NK cells determined by MS after a 4-hour culture in the indicated medium is shown (n = 3 independent donors, values obtained for each individual donor are shown). (c) Intracellular serine concentration in in vitro activated NK cells cultured for 4 h in the presence or absence of PKUM was measured using a kit (Sigma) and is shown (Mean +SD, n = 5 independent donors). (d) Sorted CD56^Dim NK cells were pre-incubated in medium +/− pyruvate +/− PKUM for 3 hours and incubated 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. Representative dot-plots (left panel) and the proportion of positive cells (Mean + SEM) for each experiment (right panel, n = 6 independent donors) are shown. (e) The percentage of K562 lysis (mean + SEM, n = 4 independent donors) is shown for the indicated E:T ratio. (f) Total abundance of lactate in supernatant of NK cells cultured for 3 h with or without pyruvate and PKUM were measured by RMN. Absolute quantities (Mean +SD, n = 3 independent donors) are shown. (g) Sorted CD56^Dim NK cells were cultured in the presence or absence of Devimistat for 4 h. Basal ECAR and OCR were measured. The results (Mean +SD) obtained from 3 independent donors are shown. Individual points representing independent donors are shown in all panels. One-way ANOVAs were performed with corrections for multiple testing in panels d, e, and f. Two-tailed T-test were performed comparing the indicated conditions in panel b, c, and g. Exact p-values are shown.