Figure 5.

IL-21 controls expression of IL-10 in Th17/Tc17 primary cells and is required for normal induction of IL-10 by IL-6 or IL-27. (A) Naive CD4⁺ or CD8⁺ T cells from WT or IL-21R KO mice were polarized in vitro in the presence of TGFβ + IL-6 for 4 days and re-stimulated for 6 h with PMA + ionomycin in the presence of Golgi stop for the last 4 h. Intracellular levels of IL-10 and IL-17 were assessed by flow cytometry. The quadrants were determined based on staining with isotype control antibodies. (B) WT CD4⁺ T cells were stimulated under neutral conditions with anti-CD3 + anti-CD28 in the presence or absence of TGFβ and IL-6 or IL-27 for 4 days, at which time culture supernatants were assayed by ELISA for IL-21. (C) CD4⁺ T cells from either WT or IL-21R KO mice were stimulated with anti-CD3 + anti-CD28 + anti-IFNγ + anti-IL-4 in the presence of TGFβ with either IL-6, IL-21, or IL-27 for 4 days and then re-stimulated for 6 h with PMA + ionomycin in the presence of Golgi stop for the last 4 h. Intracellular levels of IL-10 and IL-17 were assessed by flow cytometry. (D) The percentage of IL-10 positive cells in (c) are represented in a bar graph. (E) CD4⁺ and CD8⁺ T cells were pre-activated with anti-CD3 + anti-CD28 for 48 h, washed and rested overnight, and were re-stimulated without or with IL-6, IL-21 or IL-27 for 6 h. RNA was then isolated and relative IL-10 mRNA levels quantified by real-time PCR. (F) Parallel culture supernatants were assayed at 48 h for IL-10 by ELISA. *P<0.01; **P<0.05.