Figure 1.

MHC class II impedes the induction of nonregulatory CD4⁺ T cells into iDNT cells. (A, B) Freshly sorted CD4⁺CD127^hiCD25^- T cells were cocultured with mature bone marrow dendritic cells (CD86^-MHC-II⁺ DCs) at a ratio of 1×10⁵ T cells to 2.5×10⁴ DCs for up to 7 days in 96-well round-bottom plates in complete RPMI 1640. Different concentrations of IL-2 (0, 50 ng/mL, 100 ng/mL or 200 ng/mL) were added to the culture system. TCRαβ⁺CD3⁺CD4^-CD8^- iDNT cells were analyzed to evaluate the influence of IL-2 on induction. (A) The TCRαβ⁺CD3⁺CD4^-CD8^- cell population is gated. Representative flow cytometry profiles of different concentrations of IL-2 (0, 50 ng/mL, 100 ng/mL or 200 ng/mL). (B) The bar graphs are representative of three independent experiments. (C) Representative data of the use of anti-CD86 and anti-MHC-II antibodies to divide bone marrow DCs into two subgroups: mature DCs (CD86⁺MHC-II⁺) and immature DCs (CD86^-MHC-II⁺, CD86⁺MHC-II^- or CD86^-MHC-II^-). (D, E) Immature DCs (CD86^-MHC-II⁺) or mature DCs (CD86⁺MHC-II⁺) were cocultured with freshly sorted nonregulatory CD4⁺ T cells. IL-2 (50 ng/mL) was added to the culture system. iDNT cells were analyzed to evaluate the influence of CD86 on induction. (D) Representative flow cytometry profiles of these two mixed lymphocyte reactions. (E) The bar graphs are representative of three independent experiments. (F, G) Immature DCs (CD86⁺MHC-II^-), mature DCs (CD86⁺MHC-II⁺) or mature DCs with anti-MHC-II antibody (20 μg/mL) were cocultured with freshly sorted nonregulatory CD4⁺ T cells. IL-2 (50 ng/mL) was added to the culture system. iDNT cells were analyzed to evaluate the influence of MHC class II on induction. (F) Representative flow cytometry profiles of these three mixed lymphocyte reactions. (G) The bar graphs are representative of three independent experiments. Student’s t-test was used to compare two independent variables (ns, not significant, **p < 0.01, and ***p < 0.001).