Figure 2
Figure 2. STAT3-dependent IL-21 production regulates hematopoiesis. (A) Naive CD4+ T cells from wild-type mice were cultured under conditions that promote Th1, Th2, Th9 or Th17 differentiation. Cells were stained with antibodies for phospho-STAT5 and phospho-STAT3 on culture initiation, and after 2 or 4 days of differentiation. Results are the average ± SEM of 3-5 cultures for percent positive cells, based on quadrants set from staining at day 0, or mean fluorescence intensity (MFI) of the entire population. (B-C) Naive CD4+ T cells from wild-type, Stat3CD4−/− or Rorcgfp/gfp mice were cultured under the conditions indicated and after 5 days of culture, cells were restimulated with anti-CD3 for 24 hours before cell-free supernatants were analyzed for IL-21 concentration using ELISA. UD indicates undetectable. (D) Naive CD4+ T cells from wild-type or Stat3CD4−/− mice were cultured under Th17 differentiation conditions and transduced with control or active STAT3-expressing retroviruses. After 5 days of culture, cells were restimulated with anti-CD3 for 24 hours before cell-free supernatants were analyzed for IL-21 concentration using ELISA. UD indicates undetectable. (E-F) Bone marrow (top) or spleen (middle) cells were analyzed for hematopoietic progenitor cell numbers and bone marrow cells for cycling status (bottom). (E) C57BL/6 mice were injected intraperitoneally at 0 and 72 hours with PBS, anti–IL-21, or anti–IL-22 (100 μg/injection; Abs from R&D Systems). Mice were euthanized 24 hours after the second injection for analysis of HPC. Results are the average ± SEM of 3 individually assessed mice for each treatment group. (F) Wild-type and Stat3CD4−/− mice were injected every 12 hours for 48 hours with PBS or Stat3CD4−/− mice were injected with IL-21 (2 μg in 100 μL of PBS twice per day for 2 days; cytokines from R&D Systems or Peprotech), and mice were euthanized for analysis of HPC numbers and cycling 12 hours after the last injection. Results are the average ± SEM of 4 individually assessed mice and are representative of 2 separate experiments. SEM of the percentage of progenitors in S-phase was < 3%. Asterisks indicate significant difference from wild-type or control mice: *P < .05; **P < .005. Statistics were performed using the Student t test.

STAT3-dependent IL-21 production regulates hematopoiesis. (A) Naive CD4+ T cells from wild-type mice were cultured under conditions that promote Th1, Th2, Th9 or Th17 differentiation. Cells were stained with antibodies for phospho-STAT5 and phospho-STAT3 on culture initiation, and after 2 or 4 days of differentiation. Results are the average ± SEM of 3-5 cultures for percent positive cells, based on quadrants set from staining at day 0, or mean fluorescence intensity (MFI) of the entire population. (B-C) Naive CD4+ T cells from wild-type, Stat3CD4−/− or Rorcgfp/gfp mice were cultured under the conditions indicated and after 5 days of culture, cells were restimulated with anti-CD3 for 24 hours before cell-free supernatants were analyzed for IL-21 concentration using ELISA. UD indicates undetectable. (D) Naive CD4+ T cells from wild-type or Stat3CD4−/− mice were cultured under Th17 differentiation conditions and transduced with control or active STAT3-expressing retroviruses. After 5 days of culture, cells were restimulated with anti-CD3 for 24 hours before cell-free supernatants were analyzed for IL-21 concentration using ELISA. UD indicates undetectable. (E-F) Bone marrow (top) or spleen (middle) cells were analyzed for hematopoietic progenitor cell numbers and bone marrow cells for cycling status (bottom). (E) C57BL/6 mice were injected intraperitoneally at 0 and 72 hours with PBS, anti–IL-21, or anti–IL-22 (100 μg/injection; Abs from R&D Systems). Mice were euthanized 24 hours after the second injection for analysis of HPC. Results are the average ± SEM of 3 individually assessed mice for each treatment group. (F) Wild-type and Stat3CD4−/− mice were injected every 12 hours for 48 hours with PBS or Stat3CD4−/− mice were injected with IL-21 (2 μg in 100 μL of PBS twice per day for 2 days; cytokines from R&D Systems or Peprotech), and mice were euthanized for analysis of HPC numbers and cycling 12 hours after the last injection. Results are the average ± SEM of 4 individually assessed mice and are representative of 2 separate experiments. SEM of the percentage of progenitors in S-phase was < 3%. Asterisks indicate significant difference from wild-type or control mice: *P < .05; **P < .005. Statistics were performed using the Student t test.

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