High mTOR activity is a hallmark of reactive natural killer cells and amplifies early signaling through activating receptors
- CIRI, Centre International de Recherche en Infectiologie - International Center for Infectiology Research, France
- Inserm, U1111, France
- Ecole Normale Supérieure de Lyon, France
- Université Lyon 1, France
- UMR5308, France
- Karolinska Institutet, Karolinska University Hospital Huddinge, Sweden
- Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, France
- Innate-Pharma, France
- Aix-Marseille Université, CNRS, INSERM, CIML, France
- APHM, Hôpital de la Timone, Service d’Immunologie, France
- University of Bergen, Norway
Figures
Figure 1 with 3 supplements
Basal activity of the mTOR pathway is proportional to the level of NK cell reactivity, and dependent on SHP1.
(A) Heatmap representing the phosphorylation level of the phosphoepitopes indicated on the right in the different subsets of splenic resting NK cells indicated on top and gated as defined in Figure …
Figure 1—figure supplement 1
Bar graph showing the phosphorylation level of the indicated phosphoepitopes in the different subsets of splenic resting C57BL/6 NK cells defined by their expression of CD27 and CD11b.
Mean Fluorescence Intensity was recorded for each phosphoepitope in each subset. Normalized expression was calculated using the CD27+CD11blo subset of C57BL/6 mice as reference (n = 6 mice in three …
Figure 1—figure supplement 2
Flow cytometry density plots presenting the analysis strategy to compare educated versus uneducated NK cells in C57BL/6 mice and the phenotypically equivalent subsets in B2m−/− mice.
Expression of the surface markers CD11b and CD27 by CD3-CD19-NK1.1+ splenocytes defines three maturation subsets (CD11blo, DP and CD27lo), each of these subsets is further divided into four subsets …
Figure 1—figure supplement 3
Phosphorylation level of phospho-epitopes defined in Table 1 was measured by flow-cytometry in splenic NK cells from C57BL/6 or B2m−/− mice following NK1.1 stimulation for the indicated time.
MFI of the indicated phospho-epitope (mean + SD) of 5 mice of each genotype in five independent experiments (t-test, *p<0.05, **p<0.01).
Figure 2 with 2 supplements
Reversion of education is accompanied by loss of the basal activity of the mTOR pathway.
(A) Left: Representative histograms showing the GFP fluorescence levels of Nur77GFP NK cells transferred into C57BL/6 or B2m−/− mice and harvested 1 or 3 days after transfer. Non-transgenic host …
Figure 2—figure supplement 1
Left: Representative histograms showing the GFP fluorescence levels of Nur77GFP NK cells before or after a 4 hr stimulation with anti-NK1.1 or YAC-1 cells.
Non-transgenic C57BL/6 control NK cells are shown. Right: Bar graph showing the GFP levels of the indicated splenic NK cell subsets before or after a 4 hr stimulation with anti-NK1.1 or YAC-1 cells. …
Figure 2—figure supplement 2
Percentage (mean +SD) of IFN-γ+ or CD107a+ cells among splenic host or transferred NK cells of the indicated subset following 4 hr stimulation with (A) coated anti-NKp46 or (B) YAC-1 cells.
The experiment was done 3 days after the transfer (n = 6 mice of each genotype in two independent experiments, two-way ANOVA comparing each subset to its counterpart in C57BL/6 mice, *p<0.05, ***p<0.…
Figure 3 with 1 supplement
mTOR is essential for NK cell reactivity.
(A) Percentage (mean +SD) of CD107a+ cells among splenic CD11blo NK cells of the indicated subset from Ncr1iCre/+ or Ncr1iCre/+ Mtorlox/lox mice following 4 hr stimulation with coated anti-NK1.1 or …
Figure 3—figure supplement 1
Percentage (mean +SD) of IFN-γ+ or CD107a+ cells among splenic NK cells of the indicated subset from C57BL/6 or B2m−/− mice following 4 hr stimulation with coated anti-NKp46 in the presence or absence of 250 nM Torin2 (n = 9–10 mice of each genotype in five independent experiments, 2-way ANOVA comparing each subset to its counterpart in B2m−/− mice, *p<0.05, **p<0.01, ***p<0.001, n.s. non significant).
https://doi.org/10.7554/eLife.26423.012
Figure 4 with 1 supplement
mTOR is a rheostat of NK cell reactivity through NKar.
(A) Bar graph showing the phosphorylation level of S6 (left) and Akt S473 (right) in splenic NK cells following 1 hr treatment with 100 ng/ml IL-15 in the presence or absence of the indicated mTOR …
Figure 4—figure supplement 1
Bar graphs showing (A) the phosphorylation level of STAT5 in splenic NK cells following 1 hr treatment with 100 ng/ml IL-15 or (B) the percentage of live NK cells following a 24 hr culture in the presence or absence of the indicated mTOR inhibitors at the indicated concentration (A) mean of the MFI normalized to the No inhibitor condition or (B) percentage of live cells + SD, n = 9 mice in three independent experiments for pSTAT5 and 4 mice in two independent experiments for Viability, one-way ANOVA comparing the No inhibitor condition with the indicated condition, n.s. non significant).
(C) Linear regression plots showing the correlation between pSTAT5 and the percentage of IFN-γ+ or CD107a+ NK cells following 4 hr stimulation with coated anti-NK1.1 in the presence of 100 ng/ml …
Figure 5
mTOR is essential for calcium response and integrin activation following NKar engagement.
(A) Left: Representative histogram overlay showing the Ca2+ flux intensity in splenic CD11blo NK cells from Ncr1iCre/+ or Ncr1iCre/+ Mtorlox/lox mice. NK cells were activated following incubation …
Figure 6
Metabolic parameters of reactive and hyporesponsive NK cells.
(A) Bar graphs showing FSC and SSC values of splenic NK cell subsets from C57BL/6 or B2m−/− mice (mean +SD, n = 6 mice of each genotype in three independent experiments, t-test, ***p<0.001). MFI …
Figure 7 with 2 supplements
Cytokine stimulation overcomes NK cell education by inducing high mTOR activity that restores NKar signaling.
(A) Percentage (mean +SD) of IFN-γ or CD107a positive cells among splenic NK cells from C57BL/6 or B2m−/− mice following 4 hr stimulation with coated anti-NK1.1 or anti-NKp46 in the presence or …
Figure 7—figure supplement 1
Percentage (mean +SD) of IFN-γ or CD107a positive cells among splenic NK cells from C57BL/6 or B2m−/− mice following 4 hr stimulation with coated anti-NK1.1 or anti-NKp46 in the presence or absence of 10 ng/ml IL-15 and 250 nM Torin2 as indicated (n = 4 mice of each genotype in two independent experiments, one-way ANOVA comparing each condition to the C57BL/6 condition, *p<0.05, **p<0.01, ***p<0.001, n.s. non significant).
https://doi.org/10.7554/eLife.26423.018
Figure 7—figure supplement 2
Left: Representative histogram overlay showing the Ca2+ flux intensity in splenic NK cells from C57BL/6 or B2m−/− mice with or without IL-15 (100 ng/ml) and Torin2 (500 nM).
NK cells were activated following incubation with biotinylated anti-NK1.1 (Arrow) followed by cross-linking with streptavidin (Arrowhead). Right: Bar graph showing the Ratio Peak/basal normalized to …
Author response image 1
Percentage (mean + SD) of IFNγ+ cells among splenic CD11blo NK cells of the indicated subset from Ncr1iCre/+ or Ncr1iCre/+ Mtorlox/lox mice following 4-hour stimulation with coated anti-NK1.1 or anti-NKp46 (n=5 mice of each genotype in 3 independent experiments, t-tests comparing each subset in both genotype, **p<0.01, n.s. non significant).
Author response image 2
Bar graphs showing MFI of Glut1 staining, MitoSox staining, Bodipy incorporation and Lipid peroxidation staining of splenic NK cell subsets from C57BL/6 or β2m-/- mice (mean +SD, n=10 mice of each genotype in 3 independent experiments, except Glut1: 4 mice in 2 independent experiments, no statistical differences were detected between C57BL/6 and β2m-/-, t-test).
MFI were normalized to the CD11blo subset of C57BL/6 mice.
Tables
Table 1
List of the antibodies used in this study and the phosphoepitopes they recognize.
https://doi.org/10.7554/eLife.26423.007| Phosphoepitope | Clone (Supplier) |
|---|---|
| pCD3ζ (Y142) | K25-407.69 (BD) |
| pLck (Y505) | 4/LCK-Y505 (BD) |
| pSyk (Y342) | I120-722 (BD) |
| pSLP76 (Y128) | J141-668.36.58 (BD) |
| pItk (Y180) | N35-86 (BD) |
| pPLCg2 (Y759) | K86-689.37 (BD) |
| pWIP (S478) | K32-824 (BD) |
| p-p38 (T180/Y182) | 36/p38 (pT180/pY182) (BD) |
| pERK1/2 (T203/Y205) | 20A (BD) |
| p-c-Cbl (Y698) | 47/c-Cbl (BD) |
| pJNK (T183/Y185) | N9-66 (BD) |
| pNFkB p65 (S468) | #3039 (CST) |
| pNFkB p65 (S529) | K10-895.12.50 (CST) |
| pNFkB p65 (S536) | 93H1 (CST) |
| pAkt (T308) | C31E5E (CST) |
| pAkt (S473) | M89-61 (BD) |
| pS6 (S235/236) | D57.2.2E (CST) |
| p4EBP1 (T36/45) | 236B4 (CST) |
| p-mTOR (S2448) | D9C2 (CST) |
| p-mTOR (S2481) | #2974 (CST) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.26423.020
