Fig 1: T cells stimulate macrophage IL-27 production by IFNγ signaling and CD40-CD40L interaction(A) CD40L blockade suppresses IL-27 expression in islet macrophages. NOD.Il27p28-eGFP females (6–9 weeks old) were injected with anti-CD40L or control antibodies weekly for 3 weeks. Islet-infiltrating cells were analyzed at 1 week after the last injection. The frequencies of GFP (IL-27p28)+ cells among CD11c+F4/80+ and CD11clowF4/80+ macrophages are pooled from four independent experiments (n = 11–12 per group). ∗p < 0.05 by unpaired t test.(B) IFNγ signaling is important for islet macrophage production of IL-27. Islet-infiltrating cells were analyzed for GFP (IL-27p28) in NOD.Ifngr1+/+ and NOD.Ifngr1+/− female littermates (9–11 weeks old). The frequencies of GFP (IL-27p28)+ cells among CD11c+F4/80+ and CD11clowF4/80+ macrophages are pooled from six independent experiments (n = 10–13 per group). ∗p < 0.05 by unpaired t test.(C) IFNγR and CD40L blockade reduces IL-27p28 production in the coculture of wild-type (WT) CD4 T cells and BMDMs. WT splenic CD4 T cells (1 × 106) were cocultured with WT BMDMs (2 × 105) in the presence or absence of anti-CD3 (2 μg/mL) with the addition of anti-IFNγR and/or anti-CD40L (10 μg/mL) or the corresponding control antibodies for 3 days. Culture media were collected and analyzed for IL-27p28 by ELISA. Summarized results are from four independent experiments as indicated by different symbols. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison.(D) IFNγR blockade reduces IL-27p28 production in the coculture of WT CD8 T cells and BMDMs. WT CD8 T cells and WT BMDMs were cocultured as described in (C). Culture media were collected and analyzed for IL-27p28 by ELISA. Summarized results are from two independent experiments with two biological replicates each time. ∗∗p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison.(E) The ability of macrophages to respond to IFNγ is important for CD4 T cell-induced IL-27p28 production. WT splenic CD4 T cells (1 × 106) were cocultured with WT or Ifngr1−/− BMDMs (2 × 105) in the presence or absence of anti-CD3 (2 μg/mL) for 3 days. Culture media were collected and analyzed for IL-27p28 by ELISA. Results were pooled from two independent experiments with two biological replicates each time. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison.(F) IFNγ and CD40 signaling synergistically enhances macrophage IL-27p28 expression. BMDMs were stimulated with IFNγ (10 ng/mL), anti-CD40 (5 μg/mL), or both for 3 days. Culture media were collected and analyzed for IL-27p28 by ELISA. Results were pooled from three independent experiments with one to two biological replicates each time. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison.(G) IFNγR and CD40L blockade reduces IL-27 production in the coculture of WT CD4 T cells and BMDMs. Culture media from the same cultures shown in (C) were analyzed for IL-27p28/EBI3 heterodimers by ELISA. ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison.(H) Activated human T cells induce IL-27 p28/EBI3 heterodimer production in the PBMC culture. PBMCs (7.5 × 105) were cultured in the presence or absence of anti-CD3 (2 μg/mL) with the addition of anti-IFNγRα and/or anti-CD40L (10 μg/mL) or the corresponding control antibodies for 3 days. Culture media were collected and analyzed for IL-27p28/EBI3 heterodimers by ELISA. Summarized results are from eight subjects as indicated by different symbols. ∗Adjusted p < 0.05 by Wilcoxon signed-rank test.