Figure 7
Figure 7. IL-7, IL-15, and TGF-β up-regulate IL-1RI expression on naive CD4+ T cells, leading to enhanced IL-17 production in response to IL-1β and TCR triggering. (A) Flow cytometric analysis of IL-1RI expression on CD4+ T cells in human umbilical cord blood. (B) Flow cytometric analysis of IL-1RI expression on peripheral IL-1RI− naive CD4+ T cells that were incubated for 6 days with the common γ chain cytokine cocktail (IL-2, IL-7, IL-15, IL-21) or IL-17 promoting cytokine cocktail (IL-1β, IL-6, IL-21, IL-23) in the presence or absence of TGF-β as well as with anti-CD3 and anti-CD28 antibody-coated beads. (C) Flow cytometric analysis of the induction of IL-1RI expression on IL-1RI− naive CD4+ T cells that were incubated for 6 days with different combinations of cytokines in the common γ chain cytokine cocktail and TGF-β. The presence and absence of each cytokine are indicated by + and −, respectively. NS indicates not significant; *P < .05, respectively, by paired t test in comparison of cells treated with the common γC cytokine cocktail. (D) Flow cytometric analysis of IL-1RI expression on IL-1RI− naive CD4+ T cells that were treated for 6 days with an inhibitor for AKT (SH-5), JAK1/STAT5 (AG-490), PI3K (LY294002), or MEK1/2 (PD980599) in the presence of IL-7, IL-15, and TGF-β. (E) Flow cytometry of CD45RA and CCR7 expression on IL-1RI− naive CD4+ T cells that were incubated with IL-7, IL-15, and TGF-β as in panel C. (F) ELISA of IL-17 production from sorted IL-1RI− memory CD4+ T cells that were incubated for 6 days with IL-7, IL-15, and TGF-β followed by washing off the cytokines and an additional 7-day stimulation with anti-CD3 and anti-CD28 antibody-coated beads with or without IL-1β in the presence or absence of IL-1R antagonist (IL-1Ra). (G) Flow cytometric analysis of CD31 and IL-1RI expression on naive CD4+ T cells. (H) Flow cytometric analysis of IL-1RI and CD31 expression on IL-1RI− naive CD4+ T cells treated for 6 days with or without IL-7, IL-15, and TGF-β. Data represent 6 (A), 2 (B,D,H) and 4 (E-G) separate experiments (1 experiment per donor) or are from 3 to 5 (C) donors; error bars, mean ± SEM of 3 to 5 experiments (C) or ± SD of triplicates (F). < 15 indicates below the lower limit of detection of IL-17 (15 pg/mL).

IL-7, IL-15, and TGF-β up-regulate IL-1RI expression on naive CD4+ T cells, leading to enhanced IL-17 production in response to IL-1β and TCR triggering. (A) Flow cytometric analysis of IL-1RI expression on CD4+ T cells in human umbilical cord blood. (B) Flow cytometric analysis of IL-1RI expression on peripheral IL-1RI− naive CD4+ T cells that were incubated for 6 days with the common γ chain cytokine cocktail (IL-2, IL-7, IL-15, IL-21) or IL-17 promoting cytokine cocktail (IL-1β, IL-6, IL-21, IL-23) in the presence or absence of TGF-β as well as with anti-CD3 and anti-CD28 antibody-coated beads. (C) Flow cytometric analysis of the induction of IL-1RI expression on IL-1RI− naive CD4+ T cells that were incubated for 6 days with different combinations of cytokines in the common γ chain cytokine cocktail and TGF-β. The presence and absence of each cytokine are indicated by + and −, respectively. NS indicates not significant; *P < .05, respectively, by paired t test in comparison of cells treated with the common γC cytokine cocktail. (D) Flow cytometric analysis of IL-1RI expression on IL-1RI− naive CD4+ T cells that were treated for 6 days with an inhibitor for AKT (SH-5), JAK1/STAT5 (AG-490), PI3K (LY294002), or MEK1/2 (PD980599) in the presence of IL-7, IL-15, and TGF-β. (E) Flow cytometry of CD45RA and CCR7 expression on IL-1RI− naive CD4+ T cells that were incubated with IL-7, IL-15, and TGF-β as in panel C. (F) ELISA of IL-17 production from sorted IL-1RI− memory CD4+ T cells that were incubated for 6 days with IL-7, IL-15, and TGF-β followed by washing off the cytokines and an additional 7-day stimulation with anti-CD3 and anti-CD28 antibody-coated beads with or without IL-1β in the presence or absence of IL-1R antagonist (IL-1Ra). (G) Flow cytometric analysis of CD31 and IL-1RI expression on naive CD4+ T cells. (H) Flow cytometric analysis of IL-1RI and CD31 expression on IL-1RI− naive CD4+ T cells treated for 6 days with or without IL-7, IL-15, and TGF-β. Data represent 6 (A), 2 (B,D,H) and 4 (E-G) separate experiments (1 experiment per donor) or are from 3 to 5 (C) donors; error bars, mean ± SEM of 3 to 5 experiments (C) or ± SD of triplicates (F). < 15 indicates below the lower limit of detection of IL-17 (15 pg/mL).

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