Figure 4. MIF controls IL-2-mediated Treg generation.
(A) Suppression of inducible CD4+ Tregs by anti-IL-2 Ab. Splenic CD4+ cells of MIF+/+ mice were stimulated with anti-CD3/CD28 Ab (CD3/CD28) plus TGF-β (3 ng/ml) in the presence of anti-IL-2 Ab (0.1 µg/ml) or isotype control IgG (IgG). After 72 hours of incubation, the frequency of CD4+CD25+Foxp3+ T cells over CD4+ cells was determined by flow cytometry analysis. Data shows mean±SD. *, P < 0.05 versus isotype control Ab. (B) Restoration of IL-2 production by recombinant MIF. Splenic T cells (2×105) of MIF−/− mice were stimulated with anti-CD3/CD28 Ab in the presence of recombinant MIF (500 ng/ml) for 48 hours. IL-2 levels the culture supernatants were measured by ELISA. *, P < 0.05 versus IL-2 production by splenic cells of MIF+/+ mice treated with anti-CD3/CD28 Ab alone. (C and D) Inhibition of IL-2 production by neutralizing anti-MIF Ab (C) or MIF receptor antagonist (D). Splenic cells (2×105) of MIF+/+ mice were stimulated with anti-CD3/CD28 Ab in the absence or presence of anti-MIF Ab (100 µg/ml) for 48 hours or D1036 (a MIF receptor antagonist: 1, 5, and 50 µM) for 24 hours. Isotype control IgG (100 µg/ml) was used as a control. IL-2 levels in the culture supernatants were measured by ELISA. *, P < 0.05 versus isotype control Ab or anti-CD3/CD28 Ab alone. (E) Effect of anti-MIF Ab on the inducible CD4+Treg production. Splenic CD4+ cells of MIF+/+ mice were cultured in RPMI1640 supplemented with 10% FBS, and stimulated with anti-CD3/CD28 Ab (CD3/CD28) plus TGF-β (3 ng/ml) in the presence of anti-MIF Ab (50 µg/ml) or isotype control IgG (50 µg/ml). After 72 hours of incubation, the frequency of CD4+Tregs was determined by flow cytometry analysis. A representative is shown on the left. Bar graph on the right shows the mean±SD of six independent experiments. *, P < 0.05 versus isotype control Ab. **, P < 0.01 versus anti-MIF Ab alone.