Fig 1.
Influence of NOS2 deficiency on γδ T cells in vivo.
(A) Percentages of CD4+ and CD8+ αβ T cells from pLNs of WT (n = 12) and Nos2KO (n = 12) mice. (B-D) Representative dot plot of peripheral γδ T cells defined as CD3ε+TCRγδ+ cells (B—left). Percentages and absolute numbers of γδ T cells from pLNs (B), thymus (C) and skin (D) of WT (n = 12, except for E n = 5) and Nos2KO (n = 12, except for E n = 5) mice. (E) Proportion of dead γδ T cells from pLNs of WT (n = 10) and Nos2KO (n = 9) mice analyzed ex vivo by annexin V staining. Data are pooled from one (D), two (B, C) or three (A, E) experiments. Bars are mean or mean ± SEM and each point represents one mouse. ** p<0.01, *** p<0.001 (Mann-Whitney’s test).
Fig 2.
Effect of NOS2 deficiency on IL-2 production by pLNs γδ T cells.
(A-B) Representative microscopy images showing a γδ T cell positive (A) or negative (B) for NOS2 derived respectively from pLNs of WT and Nos2KO mice and stained with antibodies to TCR γδ (red), NOS2 (green) and counterstained with DAPI (blue). The arrow indicates a NOS2+ γδ T cell. Bars 10 μM. 40 X objective. The experiment was performed from WT (n = 3) and Nos2KO (n = 2) mice. (C-D) IL-2 production by γδ T cells after 4h PMA/ionomycin stimulation of pLNs from WT (n = 12) and Nos2KO (n = 12) mice. (C) Representative dot plots of IL-2 staining among γδ T cells in WT and Nos2KO mice. (D) Percentages of IL-2+ cells among γδ T cells are shown in left and geometric mean of fluorescence intensity (MFI) in right. Data are pooled from two experiments. Point represents individual mouse, bars are mean and box and whiskers are min to max values and median. ** p<0.01, *** p<0.001 (Mann-Whitney’s test).
Fig 3.
Effect of autocrine NOS2-derived NO on γδ T cell proliferation.
γδ T cells sorted from pLNs of WT or Nos2KO mice and labeled with CFSE were cultured for 2 days in presence of CD3- and CD28-specific antibodies, 0,5 mM L-NIL and 10 mM L-NMMA when indicated. (A) Representative histograms of CFSE dilution. Numbers above line on CFSE plots indicate percent of proliferating cells (left). Percentages of γδ T cell proliferation undergoing division (right). (B) Number of γδ T cells by division. (C) IL-2 levels quantified in supernatants by ELISA after 24h and 48h of γδ T cell cultures. (D) Proportion of living γδ T cells after 48h culture. Data are from five independent experiments with 12 WT, 5 Nos2KO, 3 L-NIL and 6 L-NMMA replicates. Point represents individual replicate, bars mean ± SEM (except in B right mean) * p<0.05 ** p<0.01, *** p<0.001 (Mann-Whitney’s test).
Fig 4.
Glycolytic metabolism assessed in competent or NOS2 deficient γδ T cells.
Sorted γδ T cells from pLNs of WT or Nos2KO mice were expanded in vitro for 4 days in presence of CD3- and CD28- specific antibodies, 15 μg/mL IL-7 and 15U/mL IL-2. Glycolytic metabolism was analyzed using a Seahorse XF-24 analyzer either directly (A), or after 18 h of resting followed by an additional 4h of stimulation with media containing 5 mM L-NMMA when indicated (B). ECAR was assessed after glucose (gluc) addition and in response to metabolic inhibitors oligomycin (oligo) and 2-Deoxy-D-glucose (2DG). Shown are time courses (A left), normalized time courses as % of baseline (B left) and calculations of rate of glycolysis (A, B right panels). Data are from one experiment with 3 (Nos2KO) and 4 (WT) replicates (A) and are pooled from three independent experiments with 7 (WT), 5 (Nos2KO) and 5 (L-NMMA) replicates (B). Mean ± SEM are shown. ** p < 0.01 (Mann-Whitney’s test).
Fig 5.
Proliferation and glycolysis of NOS2-deficient γδ T cells in presence of IL-2.
(A) γδ T cells sorted from pLNs of WT and Nos2KO mice and labeled with CFSE were cultured for 2 days in presence of CD3 and CD28-specific antibodies and 15U/mL IL-2. Numbers above line on CFSE plots indicate percent of proliferating cells. Percentages of γδ T cell proliferation are shown below. Data are representative of two experiments with 4 WT and 5 Nos2KO replicates. (B) Sorted γδ T cells from pLNs were expanded in vitro for 4 days in presence of CD3 and CD28-specific antibodies, 15 μg/mL IL-7 and 15U/mL IL-2. ECAR was analyzed after 18h of resting followed by an additional 4h of stimulation with 15U/mL IL-2. Media contain 5 mM L-NMMA when indicated. Shown are normalized time courses as % of baseline and calculations of rate of glycolysis Data are pooled from three independent experiments with 7 (WT), 5 (Nos2KO) and 5 (L-NMMA) replicates Mean ± SEM are shown. (Mann-Whitney’s test).