Figure 8.

Increased VAT-T_R accumulation in the absence of ICOS signaling is associated with elevated CCR3 expression. (A) ST2 expression in splenic T_Rs (n = 3–5 per group from eight independent experiments). (B) ST2 expression in CD45.1⁺ and CD45.2⁺ donor splenic T_Rs in WT:YF and WT:KO chimeric mice. Lines connect CD45.1⁺ and CD45.2⁺ donor splenic T_Rs within the same chimera (n = 3–5 chimeric mice per group from three independent experiments). (C) Expression of CCR3 in VAT-T_Rs in mice ≤8 wk and >8 wk of age (n = 3–5 per group from seven independent experiments). (D) Expression of CCR2 and CCR3 by VAT-T_Rs (n = 3–5 per group from two independent experiments). DP, CCR2⁺CCR3⁺; DN, CCR2⁻CCR3⁻. (E) Expression of indicated CCR3 ligands in total VAT normalized to Tbp as measured by qPCR (n.d. indicates not detected; n = 5 per group). (F) Schematic of in vitro culture experiments examining the impact of ICOS signaling on CCR3 expression (left). Graphs indicating fold change in T_R frequency of CD4⁺ cells and %CCR3⁺ of T_Rs between individual culture samples stimulated (stim) with or without αICOS for 2 d (middle). Representative flow cytometry plots with frequency of CCR3⁺ T_Rs after 2 d in specified culture conditions (right; n = 1–3 per group from three independent experiments). (G) Left: Representative flow cytometry plots indicating VAT-T_R frequency with or without CCL11/24 blockade. Graphs summarize T_R frequencies in indicated tissues after 2 wk (n = 3–4 per group). Mice were age matched within independent experiments and collectively; pooled data are from experiments using male mice aged 8–16 wk unless otherwise indicated. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (A and C); two-tailed, paired Student’s t test for expression in donor cells within the same chimeric mouse (B); and two-tailed Student’s t test (F and G). All data are presented as mean values ± SD.