Extended Data Fig. 1: Heterogeneity of Treg cells in male and female VAT.

a, Gating strategy to identify and isolate CD4⁺ T cells in the VAT. b, Single cell RNA sequencing (scRNAseq) heatmap showing expression of signature transcripts for VAT Treg cell clusters 1-5 (yellow denotes upregulated genes). c, Heat map showing expression of selected genes in clusters 1, 2 and 3-6 (red - high, blue - low expression). d, Bar graph showing percentages of cell clusters in the Treg cell compartment in male and female mice identified by scRNAseq. e, Flow cytometric analysis of 25-week-old male and female Cxcr3^CIBER mice showing ST2 and CXCR3 expression in VAT Treg cells. f, Volcano plot shows differential expression of genes between ST2⁺ and CXCR3⁺ VAT Treg cells (FDR 0.2). g, Graphs show frequencies of ST2⁺ (left) and CXCR3⁺ (right) VAT Treg cells in different adipose tissue depots of male (VAT – visceral adipose tissue, sAT – subcutaneous adipose tissue, pAT – perinephral adipose tissue) (n = 10 VAT, n = 6 sAT, n = 5 pAT) and female (n = 9 VAT, n = 8 sAT and pAT each). Symbols represent individual mice; horizontal lines indicate means. Data are representative (e) or pooled (g) from at least two independent experiments. Error bars indicate the standard deviation. Statistical analyses were performed using two-way ANOVA (g).