Abstract
Resting natural killer (NK) cells display immediate effector functions after recognizing transformed or infected cells. The environmental nutrients and metabolic requirements to sustain these functions are not fully understood. Here, we show that NK cells rely on the use of extracellular pyruvate to support effector functions, signal transduction and cell viability. Glucose-derived carbons do not generate endogenous pyruvate. Consequently, NK cells import extracellular pyruvate that is reduced to lactate to regenerate glycolytic NAD+ and is oxidized in the tricarboxylic acid (TCA) cycle to produce ATP. This supports serine production through phosphoglycerate dehydrogenase, a pathway required for optimal proliferation following cytokine stimulation but dispensable for effector functions. In addition, like mouse NK cells, human NK cells rely on a citrate–malate configuration of the TCA cycle that is not fed by glutamine. Moreover, supraphysiologic pyruvate concentrations dose-dependently increase the effector functions of NK cells. Overall, this study highlights the role of exogenous pyruvate in NK cell biology, providing knowledge that could be exploited to boost NK cell potential in therapeutic settings.
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Data availability
Data supporting the findings of this study are available on request from the corresponding authors (A.M. or T.W.). Source data are provided with this paper.
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Acknowledgements
We acknowledge the contribution of the SFR Biosciences (Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique (CNRS) UAR3444, Institut National de la Santé et de la Recherche Médicale (INSERM) US8, ENS de Lyon) flow cytometry facility and the AniRA-ImmOs platform. We acknowledge the contributions of the CELPHEDIA Infrastructure (http://www.celphedia.eu/), especially the centre AniRA in Lyon. We thank R. Rossignol (INSERM-Université de Bordeaux, France) for helpful discussions. We acknowledge the contribution of the Etablissement Français du Sang Auvergne-Rhône-Alpes. The T.W. laboratory receives institutional grants from the INSERM, CNRS, Université Claude Bernard Lyon 1 and ENS de Lyon. N.K.C. was an employee of Parfums Christian Dior (thèse CIFRE) at the time of the study. This work was supported by a grant from LVMH-Recherche.
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Contributions
A.M. and T.W. came up with the general approach. N.K.C., A.M. and T.W. designed the experiments with inputs from C.N. and A.-L.B., and N.K.C. performed them with the help of L.P., A.H., P.M., S.B., A.D., M.M., L.F. and A.-L.M. N.L.-L., E.C. and F.B. performed the metabolomics analysis. A.M. wrote the paper with inputs from T.W. and N.K.C. All authors critically read the manuscript.
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Competing interests
N.K.C. was an employee of Parfums Christian Dior at the time of the study. M.M., C.N. and A.L.B. are employees of LVMH-Recherche. The other authors declare no competing interests.
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Nature Metabolism thanks David Finlay, Lydia Lynch and Felix Wensveen for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.
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Extended data
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 CD56Dim 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 CD56Dim 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.
Extended Data Fig. 2 Testing the impact of glutamine, glutaminolysis, AMPK pathway in resting NK cells and the role of glucose in activated NK cells.
(a) Sorted CD56Dim NK cells were cultured in the indicated medium for 4 h. ECAR (a-b) and OCR (c-e) were measured. The results (Mean +SD) obtained from 3 independent donors are shown. ATP/ADP ratio (f) or ATP levels (g) in sorted CD56Dim NK cells cultured in the indicated medium for 4 h was measured and is shown (Mean +SD, n = 3 or 4 independent donors). (h-l) Sorted CD56Dim NK cells were cultured in the presence or absence of pyruvate for 4 h. (h) Absolute AMP concentration measured by mass spectrometry is shown (Mean +SD, n = 3 independent donors). The expression of AMPKα and p-AMPK (i-j) or p-ACC S79 (k-l) was measured by capillary WB. A representative image (i and k) or the result of the quantification (j and l) are shown (Mean +SD, n = 5 independent donors). For p-ACC quantification, sorted NK cells were treated for 1 h with MK8722, an activator of AMPK, to obtain a positive control (l). (m-n) In vitro activated NK cells were cultured in the indicated medium for 4 h. ECAR (m) and OCR (n) were measured. Left panels: One representative experiment (Mean +SD) out of three is shown. Right panels: individual points representing independent experiments are shown. (o) Sorted CD56Dim NK cells were cultured in the indicated medium and the ATP level (Mean +SD, n = 6 independent donors) measured at the indicated time points are shown. One-way ANOVAs were performed with corrections for multiple testing in panels a, b, c, d, e, and f. Two-tailed T-test were performed comparing the indicated conditions in panel g, h, j, and l. Exact p-values are shown.
Extended Data Fig. 3 Complementary metabolomics data and impact of PHGDH inhibition on immediate functions.
(a) Sorted CD56Dim 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 CD56Dim 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 CD56Dim 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 CD56Dim 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.
Extended Data Fig. 4 Impact of TCA cycle inhibition on bioenergetic parameters and effector functions.
Sorted CD56Dim NK cells were cultured for 4 hours in complete medium in the presence or absence of Devimistat or BMS 303141. (a) NAD/NADH ratio, (b) ATP level or (c) the proportion of cells expressing CD107a, IFN-γ and MIP1-β measured by flow cytometry after K562 stimulation are shown (Mean +SD, n = 3 independent donors). One-way ANOVAs were performed with corrections for multiple testing in all panels. Exact p-values are shown.
Extended Data Fig. 5 Test of alternative TCA fuels and final working model.
(a) Sorted CD56Dim NK cells were cultured for 3 hours in medium containing or not pyruvate and β-Hydroxybutyrate (BHB). The proportion of cells expressing CD107a, IFN-γ and MIP1-β measured by flow cytometry after stimulation with K562 is shown (Mean +SD, n = 4 independent donors). Sorted CD56Dim NK cells were cultured for 4 hours in the indicated medium. (b) The Mean Fluorescence Intensity (MFI) for p-STAT5, p-S6 and p-AKT S473 was measured by flow cytometry and normalized to the complete medium (CM) condition for each experiment. The normalized MFI (Mean +SD) in independent donors (n = 4) is shown. (c) ATP level and NAD/NADH ratio were measured and normalized to the complete medium condition and are shown (Mean +SD, n = 3 independent donors for NAD/NADH ratio and 6 independent donors for ATP levels). (d) Sorted CD56Dim NK cells were cultured for 4 hours in the indicated medium. ATP level and NAD/NADH ratio are shown (upper panel). The proportion of cells expressing CD107a, IFN-γ and MIP1-β measured by flow cytometry after K562 stimulation is shown (lower panel). Heatmaps depicting the results are shown (n = 3 independent donors for ATP and NAD/NADH ratio and n = 1 for functional parameters). (e) Model summarizing the principal findings of this study. See text for details. Blue and orange arrows represent metabolic pathways fed by glucose or pyruvate respectively. One-way ANOVAs were performed with corrections for multiple testing in panels a, b, and c. Exact p-values are shown.
Supplementary information
Supplementary Table 1
IsoCor MS data related to C13 metabolomics analysis.
Supplementary Table 2
Raw data related to C12 quantification 1.
Supplementary Table 3
Raw data related to C12 quantification 2.
Source data
Source Data Fig. 3
Unprocessed capillary western blots for Fig. 3g.
Source Data Extended Data Fig. 2
Uncropped blots for Extended Data Fig. 2i,k.
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Kern Coquillat, N., Picq, L., Hamond, A. et al. Pivotal role of exogenous pyruvate in human natural killer cell metabolism. Nat Metab 7, 336–347 (2025). https://doi.org/10.1038/s42255-024-01188-4
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DOI: https://doi.org/10.1038/s42255-024-01188-4
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