The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells

Abstract

Interleukin 15 (IL-15) controls both the homeostasis and the peripheral activation of natural killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we found that the metabolic checkpoint kinase mTOR was activated and boosted bioenergetic metabolism after exposure of NK cells to high concentrations of IL-15, whereas low doses of IL-15 triggered only phosphorylation of the transcription factor STAT5. mTOR stimulated the growth and nutrient uptake of NK cells and positively fed back on the receptor for IL-15. This process was essential for sustaining NK cell proliferation during development and the acquisition of cytolytic potential during inflammation or viral infection. The mTORC1 inhibitor rapamycin inhibited NK cell cytotoxicity both in mice and humans; this probably contributes to the immunosuppressive activity of this drug in different clinical settings.

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Figure 1: NK cell metabolism is regulated developmentally and after activation.
Figure 2: mTOR activity decreases as differentiation progresses and is upregulated upon activation.
Figure 3: mTOR activity is mainly under the control of IL-15.
Figure 4: mTOR controls the maturation and homeostasis of NK cells.
Figure 5: mTOR is necessary for the optimal fitness of NK cells, their proliferation in the BM and their maximal response to IL-15.
Figure 6: Defective activation of NK cells in the absence of mTOR.
Figure 7: Acute inhibition of mTOR abrogates inflammation-induced priming.

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Acknowledgements

We thank the Plateau de Biologie Expérimentale de la Souris, and the flow cytometry facility of the SFR Biosciences Gerland and of Institute for Research on Cancer and Aging, Nice (the Cytomed platform funded by the CG06, INSERM and FEDER). Supported by the FINOVI foundation (T.W. laboratory), Agence Nationale de la Recherche (for the T.W. laboratory), the European Research Council (ERC-Stg 281025 for the T.W. laboratory and a THINK Advanced Grant for the E.V. laboratory), Institut National de la Santé et de la Recherche Médicale (T.W. and E.V. laboratories), Centre National de la Recherche Scientifique (T.W. and E.V. laboratories), Université Claude Bernard Lyon1 (T.W. laboratory), Ecole Normale Superieure de Lyon (T.W. laboratory), the “Ligue contre le Cancer” (“équipe labelisée”; E.G. and E.V. laboratories) and Aix-Marseille University to Centre d'Immunologie de Marseille Luminy (E.V. laboratory).

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A.M., J.C.-V., C.V., S.D., S.V., A.F., J.R., K.M. and A.T. did experiments; T.W. designed the study with the help of A.M., J.B., Y.-G.G., E.G. and E.V.; and A.M. and T.W. wrote the manuscript.

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Correspondence to Thierry Walzer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 NK cell metabolism is regulated developmentally and following activation.

(a) Histograms represent FSC and SSC of BM NK (NK1.1+ CD3-CD19-) cell subsets as defined by the expression of CD27 and CD11b, one representative experiment out of 5 is shown. The bar graph shows averaged FSC and SSC MFI of BM and splenic NK cell subsets (n=5 mice in 3 independent experiments). MFI is normalized to the BM CD11blo subset. (b) Splenocytes were cultured in the presence of 100ng/ml IL-15 and analyzed after 24 to 48h. The FSC and SSC of NK cells present in the culture are shown. One representative experiment out of many is shown. (c) The FSC and SSC of NK cells from the spleen of Control or poly(I:C) injected mice are shown. One representative experiment out of many is shown. (d) The FSC and SSC of NK cells from the draining lymph nodes of Control or intranasaly Influenza infected mice at day 3 are shown. One representative experiment out of 3 is shown.

Supplementary Figure 2 mTOR activity decreases as differentiation progresses.

The bar graph shows averaged phosphoproteins MFI of BM and splenic NK cell subsets (n=4 mice in 4 independent experiments). MFI is normalized to the CD11blo subset in the BM.

Supplementary Figure 3 Gating strategies.

(a) Gating strategy employed to analyze BM, Spleen, Liver and Blood NK cells. (b) Gating strategy employed to analyze NK, ILC3 NKR+ and LTi cells in the gut.

Supplementary Figure 4 Characterization of NK-Mtor-/- mice.

(a) Representative histograms show mTOR expression in CD11blo splenic NK cells and in T cells of NK-MtorWT/WT and NK-Mtor-/- mice. Staining obtained using a control isotype is shown. The bar graph shows averaged mTOR MFI among the different NK cell subsets in the BM and Spleen (n=4 mice in 3 independent experiments). Data are normalized to the CD11blo subset of the BM. Doted line represents the limit of detection. (b) The bar graph shows averaged FSC and SSC MFI of CD11blo NK cells from the BM and Spleen of NK-MtorWT/WT and NK-Mtor-/- mice (n=4 mice in 3 independent experiments). (c) Histograms represent phosphorylation levels of different proteins in CD11blo NK cells from the BM of NK-MtorWT/WT and NK-Mtor-/- mice. Staining obtained with an isotype control is shown. The bar graph shows the averaged MFI of different phosphoproteins as indicated (n=4 mice in 4 independent experiments). (d) Representative flow cytometric analysis showing NKp46 and RORγt expression in gut CD45+ CD19- CD3- lymphoid cells from NK-MtorWT/WT and NK-Mtor-/- mice. The bar graphs show percentages of the indicated subsets. Each point represents a single mouse (n=4 mice in 2 independent experiments).

Supplementary Figure 5 mTOR controls NK cell maturation.

(a) The bar graphs show averaged repartition of BM and Spleen NK cells from NK-MtorWT/WT and NK-Mtor-/- mice in each subset as defined by CD27 and CD11b expression (n=4 mice in 3 independent experiments. (b) Histograms show Ly49H expression level of NK cells from NK-MtorWT/WT and NK-Mtor-/- mice in each subset. The bar graphs show averaged percentage of Ly49H expressing cells among BM and Spleen NK cell of the different subsets (n=4 mice in 3 independent experiments). (c) MFI of the indicated marker was measured on CD11b- BM NK cells from NK-MtorWT/WT and NK-Mtor-/- mice. The bar graph shows the average of the ratio of MFIs NK-Mtor-/- over NK-MtorWT/WT NK cells for each marker (n=3 mice in 3 independent experiments). The red line indicates a ratio of 1.

Supplementary Figure 6 mTOR is necessary for NK cell proliferation in the BM and maximal expression of IL-15R.

(a) CD45.1 MtorWT/WT or CD45.2 Mtorlox/lox splenocytes were treated in vitro with TATCre as described in the Methods section and transferred into CD45.1/CD45.2 hosts. 3 days later, the CD45.2 Mtorlox/lox / CD45.1 MtorWT/WT ratio in the spleen was determined. The average of 3 independent experiments is shown (n=6 mice). (b) Flow cytometric analyzes show Ki67 staining of CD11blo subset BM NK cells from NK-MtorWT/WT and NK-Mtor-/- mice. One representative experiment is shown. The bar graph shows average percentages of Ki67 positive cells (n=6 mice in 3 independent experiments). (c) The bar graph shows averaged MFI of CD122, CD132 and pSTAT5 in CD11blo BM NK cells from NK-MtorWT/WT and NK-Mtor-/- mice (n=4 mice in 4 independent experiments). (d) CD122 MFI of TATCre treated NK cells of the indicated genotype was measured 3 days after transfer and is shown (n=5 mice in 3 independent experiments).

Supplementary Figure 7 Defective NK cell activation in the absence of mTOR.

(a-d) NK-MtorWT/WT and NK-Mtor-/- mice were injected or not with poly(I:C) and sacrificed 18h later. Spleens were harvested and cell suspension analyzed by flow cytometry. The CD11blo NK cell subset was gated and the results are presented. (a) Histograms represent phosphorylation levels of different proteins as indicated. One representative experiment is shown (n=4). (b) Histograms represent expression of CD71, CD98 and CD69 or the level of 2-NBDG staining (n=4). The numbers indicate the geometric mean fluorescence intensity. (c) Histograms show intracellular expression of GzmB and GzmA. One representative experiment is shown (n=4). (d) Splenocytes were cultured for 1h on plates coated with the indicated antagonistic antibodies or with the indicated cytokine. Cells were subsequently stained and analyzed by flow cytometry. Flow cytometric analyzes from one representative experiment are shown for the control, NKp46 and IL-12/18 stimulation conditions. The bar graphs show the averaged percentages of CD107a and IFNγ positive cells of 3 experiments. (e) As in Fig. 6e for KLRG1 and intracellular CCL3. To assess degranulation, cells were incubated for 4h in vitro with an anti-CD107a antibody before staining. The proportion of positive NK cells for each of these markers (average +/- SD and individual mouse, statistical comparisons have been made between WT and mTOR deficient NK cells for each time point) is shown (4 independent experiments, n>6).

Supplementary Figure 8 Acute inhibition of mTOR abrogates inflammation-induced priming.

(a) Rapamycin treated or non-treated mice were injected with poly(I:C) and sacrificed 18h later. Splenocytes were analyzed by flow cytometry. The graphs show the averaged MFI for pAkt S473 in gated NK cells. Each dot represents a single mouse, bars indicate average and SD (n=9 mice in 4 independent experiments for poly(I:C) and 18 mice in 4 independent experiments for Rapa+poly(I:C)). The MFI was normalized to control non-poly(I:C) injected mice. (b) The graph shows averaged MFI of CD69 in NK cells from control or poly(I:C) injected animals, treated or not with Rapamycin (n=4 mice in 4 independent experiments). (c) Human PBMCs were cultured for 36h as indicated. Cells were then stained and analyzed by flow cytometry. The bar graphs show the averaged MFI for Perforin1 in gated NK cells (n=9 individual donors in 3 independent experiments).

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Marçais, A., Cherfils-Vicini, J., Viant, C. et al. The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells. Nat Immunol 15, 749–757 (2014). https://doi.org/10.1038/ni.2936

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