Figure 5.

CXCR3⁺ Treg cells are superior suppressors of Th1 responses. Foxp3-GFP reporter mice were infected with LCMV (days 10–14) or left naïve and CD4⁺Foxp3⁺ Treg cells expressing the indicated markers were sorted from pooled spleen and LNs. (A,B) CD4⁺Foxp3⁺CXCR3⁺ (solid line) or CD4⁺Foxp3⁺CXCR3⁻ (gray line) Treg cells from LCMV-infected mice or CD4⁺Foxp3⁺ (dotted line) Treg cells from naïve mice were titrated onto (A) CD4⁺Foxp3⁻CD62L^high or (B) CD4⁺Foxp3⁻CD44^high effector T cells (Teff) from LCMV-infected mice (days 10–14) stimulated with anti-CD3 in the presence of irradiated antigen presenting cells for 72 h. ³H-thymidine was added for the last 18–22 h to measure proliferation (left) (mean ± SD, technical replicates, n = 3, representative experiment of >4 independent experiments). Cytokine levels in the supernatants were measured by cytometric bead array (right) (mean ± SD, technical replicates, n = 4, summary data from two independent experiments). (C–F) Suppression assays were performed as in (A,B) using total CD4⁺Foxp3-GFP⁻ effector T cells isolated from LCMV-infected mice in the presence of the indicated Treg subset (I–IV or naïve) at a 1:8 (D–F) or 1:4 (C) ratio. [(C–F), left] Suppression of proliferation (mean ± SD, technical replicates, CD85k and Lag-3 n = 3; Tim-3 and TIGIT n = 6, representative experiment of ≥3 independent experiments, one-way ANOVA with Tukey’s multiple comparison posttest) and cytokine secretion as assessed in supernatants (mean ± SD, technical replicates, CD85k and Lag-3 n = 3, Tim-3 and TIGIT n = 5–6, naïve (C,D) n = 3 (E) n = 4, (F) n = 7, representative experiment of ≥2 independent experiments, one-way ANOVA with Tukey’s multiple comparison post test) is shown (n.a., not applicable). [(C–F), right] Representative FACS Plots of indicated Treg subsets co-expressing multiple co-inhibitory receptors.