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. 2019 Jun 19;216(9):2024–2037. doi: 10.1084/jem.20182316

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© 2019 Sin et al.

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Figure 5. — iT reg cell induction is augmented in Atf7ip^fl/fl T cells and Atf7ip^fl/fl T cells suppress IL-17A production in trans. (A–C) Naive T cells from CD4-Cre/Atf7ip^+/fl (Atf7ip^+/fl) and CD4-Cre/Atf7ip^fl/fl (Atf7ip^fl/fl) mice were in vitro differentiated in the presence (+IL-2) or the absence (no IL-2) of IL-2 under Th1 (A), Th2 (B), or iT reg cell (C) conditions and analyzed for intracellular cytokine or Foxp3 expression. (D–F) Naive T cells from CD4-Cre/Atf7ip^+/fl (Atf7ip^+/fl) and CD4-Cre/Atf7ip^fl/fl (Atf7ip^fl/fl) mice were in vitro differentiated in the presence (+IL-2) or the absence (no IL-2) of IL-2 under Th1 (D), Th2 (E), or iT reg cell (F) conditions and analyzed for IL-2 expression. (G and H) WT (Thy1.1) naive T cells were mixed at a 50:50 ratio with either CD4-Cre/Atf7ip^+/fl (Thy1.2) or CD4-Cre/Atf7ip^fl/fl (Thy1.2) naive T cells and cultured for 96 h under Th17-inducing conditions with the addition of no IL-2 blocking antibody (No Antibody), 5 µg isotype control antibody (Isotype), or 5 µg S4B6. (G) Flow cytometric analysis of T cells expressing Thy1.1, Thy1.2, and IL-17A. Green box shows that S4B6 is able to rescue the trans defect in IL-17A production. (H) Summary of flow cytometric data. Each data point represents an individual mouse. Each data point in A–F and H represents an individual mouse. Data are representative of two independent experiments (A and D) or one of two experiments (B, C, E, and F) with three mice per group; data in G and H are representative of two experiments with two to four mice per genotype. Error bars (A–F and H) show mean with SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test.