Figure 2.

MINK1 suppress Th17 cell differentiation through a cell-intrinsic mechanism. (A, left) Splenocytes from Mink1^−/− and WT mice were stimulated with PMA + ionomycin for 5 h, and the CD44⁺CD4⁺ population was gated and then analyzed for IL-17A⁺, IFN-γ⁺, and Foxp3⁺ cells as indicated. Numbers in quadrants indicate the percentages of cells in each throughout. (Right) Summary of IL-17A⁺, IFN-γ⁺, and Foxp3⁺ cells from the CD44⁺CD4⁺ population of Mink1^−/− and WT mice. (B, left) FACS analysis of LP CD4⁺ T cells in the small intestine for IL-17A, Foxp3, and IFN-γ expression. (Right) Summary of CD4⁺IL-17A⁺, CD4⁺Foxp3⁺ T, and CD4⁺IFN-γ⁺ cells in Mink1^−/− and WT small intestine. (C) Splenic γδT cells derived from Mink1^−/− or WT animals were stimulated with PMA + ionomycin for 5 h and stained for IL-17A production. (D) Rag1^−/− mice were reconstituted with mixed bone marrow cells from B6.SJL (CD45.1⁺) and Mink1^−/− (CD45.2⁺) mice or from B6.SJL (CD45.1⁺) and WT (CD45.2⁺) mice at a 1:1 ratio. Total splenocytes in the recipient mice were analyzed 8 wk later for IL-17A⁺CD4⁺ and Foxp3⁺CD4⁺ T cells. The data shown were gated on CD4⁺CD45.1⁺ or CD4⁺CD45.2⁺ (Mink1^−/− or WT) populations. (E, top) Total splenocytes in the recipient mice as in Fig. 2 D were analyzed for CD62L and CD44 expression. (Bottom) Percentages of naive (CD4⁺CD62L⁺) and memory (CD4⁺CD44⁺) T cells in the spleen of host mice. The numbers in the flow cytometry graphs show the percentages of the gated populations. Error bars show mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. n = 3–6 in each group; Student’s t test. Data are representative of three (A and B) or two (C–E) experiments.