Figure 4: Adipocyte MHCII-stimulated IFNγ secretion inhibits Treg differentiation.

(a) IFNγ secretion by CD4⁺ splenic T cells cultured with adipocytes of WT and aMHCII^−/− mice and specific antigen. (b) Treg differentiation in naïve splenic T cell cultures supplemented with recombinant IFNγ or (c,d) 50% conditioned media (CM) from pooled WT or KO cultures (panel A) with IFNγ blocking antibody (αIFN) or non-specific isotype control antibody (IgG). Wild-type VAT (SVF) and spleen lymphocytes were treated with IL-33 or IL-33 plus IFNγ for 3 days. (e) Representative histograms of ST2 expression on adipose Tregs after 4 days culture with or without (NC) IL-33±IFNγ treatment. (f) Graph shows mean fold increase of Treg cells in lymphocytes from SVF or spleen at the end of the culture period. (g) 3H-thymidine incorporation in splenic CD4⁺ effector T cell cultures incubated with adipose (f) or splenic (sp) Tregs±IFNγ. (h) Body weight, fasting plasma glucose, insulin and HOMA-IR for 12 week HFD-fed WT and IFNγR1^−/− (KO) mice (i) Intraperitoneal insulin tolerance test at 12 weeks of HFD. Flow cytometry analysis of epididymal adipose tissue (j) Treg to CD4⁺, (k) ST2⁺ to Treg, (l) and Th1 (Tbet⁺GATA3⁻) to CD4⁺ ratios for WT and KO (Mean±s.e.m.; a,b: N=4 per group, c–g: N=3 per group, h: N=10 (KO), 11 (WT), i–l: N=11(KO), 12 (WT), ^†P<0.05 versus IL-33 treated group, *P<0.05 versus all other groups).