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. Author manuscript; available in PMC: 2017 Nov 7.

Published in final edited form as: Nat Immunol. 2016 Nov 14;18(1):15–25. doi: 10.1038/ni.3619

(a, b) 10⁸ CFU Salmonella Typhimurium or PBS (mock) were injected subcutaneously into footpads of S1pr5^−/− and littermate control mice. Draining LN were harvested 2h later and IFN-γ production was assessed by flow cytometry.

(a) Representative FACS plots of IFN-γ production, gated on NK1.1⁺CD3^– cells.

(b) Percent IFN-γ positive cells among total NK1.1⁺CD3^– cells. Each symbol represents one mouse; lines indicate the mean.

(c) Mixed bone marrow chimeras (mBMCs) were generated as in Fig. 4f using GFP⁺ ( ), CD45.1⁺ (○), or CD45.1⁺CD45.2⁺ (●) mice as WT donors. Mixed chimeras were injected subcutaneously with 10⁸ CFU Salmonella Typhimurium in the footpad. Draining LN were harvested 2 hours later for analysis by flow cytometry. Graph shows the ratio of the [percent IFN-γ⁺ of CD45.2⁺ experimental NK cells] to the [percent IFN-γ⁺ of WT NK cells] in S1pr5^−/− mBMCs and S1pr5^+/+ control mBMCs. Each symbol represents one mouse; lines indicate the mean and SEM.

(d, e) NK cells isolated from LN of S1pr5^−/− or littermate control animals were treated in vitro with IL-15 alone or IL-15, IL-12, and IL-18 (STIM) for 3 hours, and assessed for IFN-γ production by flow cytometry.

(d) Representative FACS plots of IFN-γ production, gated on NK1.1⁺CD3^– cells.

(e) Percent IFN-γ positive cells among total NK1.1⁺CD3^– cells. Each symbol represents the average of duplicate samples from one mouse; lines indicate the mean.

(f, g) 10⁸ CFU Salmonella Typhimurium (ST) or PBS (mock) were injected subcutaneously in footpads of S1pr5^−/− and littermate control mice. Draining LN were harvested 2h later for analysis.

(f) Representative sections from draining LN stained with anti-NK1.1 (NK cells, green), anti- IFN-γ (magenta), anti-Lyve1 (LEC and some sinus-lining macrophages, blue), and anti-B220 (B cells, white). Med indicates medulla, T indicates T zone. Scale bars, 100 μm. Insets show NK1.1 and IFN-γ stains in the boxed areas of medulla and T zone. Scale bars, 20 μm. Representative of 9 (littermate control) or 12 (S1pr5^−/−) images from 3 pairs of mice in 2 experiments.

(g) Percent IFN-γ⁺ among total NK cells within the medulla or T zone of draining LN. Mock-treated mice were controls. For the T zone, sections containing fewer than 10 NK cells within the T zone were excluded from analysis. Each symbol represents one section; lines indicate the mean and SEM.

Graphs compile 2–5 mice per condition, analyzed in 4 experiments (b); 12–14 mice per genotype from 7 sets of donors, analyzed in 9 experiments (c); 3 pairs of mice, analyzed in 2 experiments (e); or 3–12 sections from 3 mice per condition analyzed in 2 experiments (g). *, p<0.05; **, p<0.01; ns, p>0.05, not significant. Unpaired Student’s 2-tailed t-test (b, c, e); Fisher’s LSD test (g).

Inline graphic — (a, b) 10⁸ CFU Salmonella Typhimurium or PBS (mock) were injected subcutaneously into footpads of S1pr5^−/− and littermate control mice. Draining LN were harvested 2h later and IFN-γ production was assessed by flow cytometry.

(a) Representative FACS plots of IFN-γ production, gated on NK1.1⁺CD3^– cells.

(b) Percent IFN-γ positive cells among total NK1.1⁺CD3^– cells. Each symbol represents one mouse; lines indicate the mean.

(c) Mixed bone marrow chimeras (mBMCs) were generated as in Fig. 4f using GFP⁺ ( ), CD45.1⁺ (○), or CD45.1⁺CD45.2⁺ (●) mice as WT donors. Mixed chimeras were injected subcutaneously with 10⁸ CFU Salmonella Typhimurium in the footpad. Draining LN were harvested 2 hours later for analysis by flow cytometry. Graph shows the ratio of the [percent IFN-γ⁺ of CD45.2⁺ experimental NK cells] to the [percent IFN-γ⁺ of WT NK cells] in S1pr5^−/− mBMCs and S1pr5^+/+ control mBMCs. Each symbol represents one mouse; lines indicate the mean and SEM.

(d, e) NK cells isolated from LN of S1pr5^−/− or littermate control animals were treated in vitro with IL-15 alone or IL-15, IL-12, and IL-18 (STIM) for 3 hours, and assessed for IFN-γ production by flow cytometry.

(d) Representative FACS plots of IFN-γ production, gated on NK1.1⁺CD3^– cells.

(e) Percent IFN-γ positive cells among total NK1.1⁺CD3^– cells. Each symbol represents the average of duplicate samples from one mouse; lines indicate the mean.

(f, g) 10⁸ CFU Salmonella Typhimurium (ST) or PBS (mock) were injected subcutaneously in footpads of S1pr5^−/− and littermate control mice. Draining LN were harvested 2h later for analysis.

(f) Representative sections from draining LN stained with anti-NK1.1 (NK cells, green), anti- IFN-γ (magenta), anti-Lyve1 (LEC and some sinus-lining macrophages, blue), and anti-B220 (B cells, white). Med indicates medulla, T indicates T zone. Scale bars, 100 μm. Insets show NK1.1 and IFN-γ stains in the boxed areas of medulla and T zone. Scale bars, 20 μm. Representative of 9 (littermate control) or 12 (S1pr5^−/−) images from 3 pairs of mice in 2 experiments.

(g) Percent IFN-γ⁺ among total NK cells within the medulla or T zone of draining LN. Mock-treated mice were controls. For the T zone, sections containing fewer than 10 NK cells within the T zone were excluded from analysis. Each symbol represents one section; lines indicate the mean and SEM.

Graphs compile 2–5 mice per condition, analyzed in 4 experiments (b); 12–14 mice per genotype from 7 sets of donors, analyzed in 9 experiments (c); 3 pairs of mice, analyzed in 2 experiments (e); or 3–12 sections from 3 mice per condition analyzed in 2 experiments (g). *, p<0.05; **, p<0.01; ns, p>0.05, not significant. Unpaired Student’s 2-tailed t-test (b, c, e); Fisher’s LSD test (g).