Supplementary Figure 4: IL-33 drives proliferation of VAT-T_reg cells.

(a-b) In vitro proliferation of VAT-T_reg cells. Equal number of purified VAT lymphocytes from wild-type (WT) mice were CTV labeled and cultured for 3.5 days with (a) or without (b) plate bound CD3 and soluble CD28 antibodies, cytokines, and with or without IL-2 blocking antibodies as indicated. Bar graph shows relative numbers of T_reg cells at the end of the culture. Figure representative of three experiments. (c) Relative Il33 mRNA expression in the VAT of young (8 weeks) versus old (35 weeks) mice. (d) IL-33 protein expression analyzed by immunoblotting of adipose tissue from young and old mice that were on a normal diet. Il33^–/– mice were used as specificity control. Actin was used as loading control. Representative of 5 experiments. (e-f) ST2 expression on VAT-T_reg cells isolated from WT mice of different ages as indicated. (e) and correlation of age and VAT-T_reg prevalence (f). One way ANOVA for both panels, P<0.0001. (g) Frequency of Foxp3⁺ cells of CD4⁺ T cells in selected lymphoid and non-lymphoid organs from PBS, IL-33 and IL-2/anti-IL-2 Ab complex (IL-2c) treated mice. LPL - Lamina propria lymphocytes of the small intestine. (h) ST2 expression on KLRG1⁺ and KLRG1^- T_reg cells. (i) Flow cytometric analysis of splenic Foxp3⁺ cells showing expression of KLRG1 and ST2 in PBS and IL-33 treated mice (left). Graph showing proportion of KLRG1⁺ cells ± S.D. of total T_reg cells in the spleen in control and IL-33 treated mice (right). (j) Proportion of Foxp3⁺ cells within CD4⁺ T cells in the VAT at different time points post IL-33 injection. One way ANOVA, P=0.0047. Symbols indicate data points for individual mice, values are mean ± S.D. Values in (a-c) are means ± S.D. from 3 experiments. Values in (e-g) are means ± S.D. from 5 individual mice from 2 experiments. *P=0.017; **P<0.008; ***P=0.0001; ****P<0.0001 (unpaired, two tailed t-test).