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).