Figure 6
Figure 6. Toso deficiency results in defective TNFα signaling and protects from TNFα-mediated liver damage. (A) Toso expression in peripheral lymphoid organs. Leukocytes from lymph node (LN), spleen, peripheral blood, naive CD8+ T cells, CD3/CD28 activated CD8+ T cells and NKT cells (DX5+CD3+) were stained with anti–mouse Toso antibody. Flow cytometric histograms of Toso−/− cells (gray filled) and Toso+/+ cells (red) are shown in overlays. Data are representative of 2 experiments using 4 mice per group. (B) Toso deficiency results in decreased TNFα-induced coactivation of CD8+ T cells. Purified naive CD8+ T cells from spleen of Toso+/+ or Toso−/− mice were incubated with 2 ng/mL of IL-2 (top panel) or IL-2 + 20 ng/mL of TNFα (bottom panel) for 12 hours and analyzed for CD69 surface expression. Flow cytometric histograms of Toso−/− cells (gray filled) and Toso+/+ cells (red) are shown in overlays. Data shown are representative of 3 experiments using 6 mice per group. (C) Toso deficiency results in increased TNFα-induced apoptosis in CD8+ T cells. CD8+ T cells were stimulated for 24 hours via CD3/CD28. Subsequently, activated CD8+ T blasts were stimulated with 20 ng/mL of TNFα for another 24 hours or left unstimulated. Cellular apoptosis of Toso+/+ (black bars) or Toso−/− (white bars) CD8+ T cells were assessed by staining with annexinV and 7-AAD. Results shown are mean percentages ± SEM of apoptotic cells (n = 6/group). Data shown are representative of 3 experiments. Statistical analysis: Student t test (**P < .01). (D) Toso deficiency protects from TNFα-induced liver injury. Toso+/+ and Toso−/− mice were injected intraperitoneally with GalN followed an IV administration of TNFα. Liver damage was analyzed by measuring alanine-aminotransferase (ALT) activity in the serum of Toso+/+ (black) and Toso−/− (white) mice at 4 and 6 hours after TNFα administration. Results shown are mean values ± SEM (n = 5/group). Statistical analysis: Student t test (**P < .01). Data shown are representative of 3 experiments. (E) Survival of wt C57BL/6 and Toso-deficient mice. Toso+/+ and Toso−/− mice were injected intraperitoneally with GalN followed an intravenous administration of TNFα. Results shown are from 7 mice per genotype (n = 7, P = .027, Mantel Cox test). Data shown are representative of 3 experiments.

Toso deficiency results in defective TNFα signaling and protects from TNFα-mediated liver damage. (A) Toso expression in peripheral lymphoid organs. Leukocytes from lymph node (LN), spleen, peripheral blood, naive CD8+ T cells, CD3/CD28 activated CD8+ T cells and NKT cells (DX5+CD3+) were stained with anti–mouse Toso antibody. Flow cytometric histograms of Toso−/− cells (gray filled) and Toso+/+ cells (red) are shown in overlays. Data are representative of 2 experiments using 4 mice per group. (B) Toso deficiency results in decreased TNFα-induced coactivation of CD8+ T cells. Purified naive CD8+ T cells from spleen of Toso+/+ or Toso−/− mice were incubated with 2 ng/mL of IL-2 (top panel) or IL-2 + 20 ng/mL of TNFα (bottom panel) for 12 hours and analyzed for CD69 surface expression. Flow cytometric histograms of Toso−/− cells (gray filled) and Toso+/+ cells (red) are shown in overlays. Data shown are representative of 3 experiments using 6 mice per group. (C) Toso deficiency results in increased TNFα-induced apoptosis in CD8+ T cells. CD8+ T cells were stimulated for 24 hours via CD3/CD28. Subsequently, activated CD8+ T blasts were stimulated with 20 ng/mL of TNFα for another 24 hours or left unstimulated. Cellular apoptosis of Toso+/+ (black bars) or Toso−/− (white bars) CD8+ T cells were assessed by staining with annexinV and 7-AAD. Results shown are mean percentages ± SEM of apoptotic cells (n = 6/group). Data shown are representative of 3 experiments. Statistical analysis: Student t test (**P < .01). (D) Toso deficiency protects from TNFα-induced liver injury. Toso+/+ and Toso−/− mice were injected intraperitoneally with GalN followed an IV administration of TNFα. Liver damage was analyzed by measuring alanine-aminotransferase (ALT) activity in the serum of Toso+/+ (black) and Toso−/− (white) mice at 4 and 6 hours after TNFα administration. Results shown are mean values ± SEM (n = 5/group). Statistical analysis: Student t test (**P < .01). Data shown are representative of 3 experiments. (E) Survival of wt C57BL/6 and Toso-deficient mice. Toso+/+ and Toso−/− mice were injected intraperitoneally with GalN followed an intravenous administration of TNFα. Results shown are from 7 mice per genotype (n = 7, P = .027, Mantel Cox test). Data shown are representative of 3 experiments.

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