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Figure S1. mRNA expression of adrenergic receptors in naïve CD4+CD62L+ T cells and Foxp3GFP Treg cells by qPCR. Bars show the number of times that target genes are expressed as compared to the housekeeping β2-microglobulin gene in naïve T cells (A) and in Treg cells (B). Data are shown as mean ± SD of triplicates and are representative of three independent experiments. ***p<0.001; *p≤0.05, as determined by t-test.

Figure S2. B2AR is functional in naïve T cells. CD4+CD62L+ naïve T cells were sorted and treated as indicated. (A) CREB phosphorylation in naïve T cells was evaluated by western blot. (B) pCREB quantification was performed by UVIBand software. Data are shown as mean ± SEM of three samples pooled from three experiments. *p ≤ 0.05 as determined by Tukey's multiple comparison test.

Figure S3. B2AR signaling inhibits proliferation of CD4+ CD62L+ naïve T cells. CD4+CD62L+ naïve T cells were sorted, stained with the proliferation dye CellTrace™ Violet and stimulated with anti-CD3 in the presence or not of fenoterol (B2AR agonist). Some fenoterol-treated cells were pretreated with ICI 118,551 (B2AR antagonist). (A) Proliferation of naïve T cells was analyzed by flow cytometry and the division index was calculated using FlowJo software. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. (B) IL-2 levels present in the supernatants were quantified by ELISA. Data are shown as mean ± SD of triplicates and are representative of two independent experiments. (C) ICER mRNA expression in naïve T cells treated with fenoterol for 5 h was evaluated by qPCR. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. ***p<0.001; **p<0.01; *p≤0.05, as determined by Tukey's multiple comparison test or t-test.

Figure S4. Suppression assay titration. (A) Sorted Foxp3GFP Treg cells were cocultured along with naïve T cells in the presence of soluble anti-CD3. The coculture was set at different ratios: 1×105 naïve T cells to either 1 or 0.5 or 0.25 × 105 Treg cells. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. (B) Histogram showing the proliferation of naïve T cells cocultured with Treg cells at different ratios. (C) Sorted Foxp3GFP Treg cells were treated with B2AR agonist for 1h before being cocultured with naïve T cells at different ratios in the presence of soluble anti-CD3. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. ***p<0.001; **p<0.01; *p≤0.05, as determined by Tukey's multiple comparison test or t-test.

Figure S5. Time course of Treg-mediated conversion of Foxp3 naïve T cells into Foxp3+ iTreg cells. After being cocultured for 24 h, 48 h, or 72 h with Foxp3GFP Treg cells the conversion of CellTrace™ Violet stained CD4+ CD62L+ Foxp3 naïve T cells into Foxp3+ cells (iTreg) was analyzed excluding Foxp3GFP Treg cells initially present in the suppression assay cultures, once only naïve T cells were stained with CellTrace. Treg cells were treated (empty circle) or not (black circle) with B2AR agonist for 1 h before being cultured. Data are shown as mean ± SEM of 12 samples pooled from two experiments. ***p≤0.001 as determined by Bonferroni posttests.

Figure S6. B2AR-mediated increase in Treg suppressive function is dependent on PKA. Foxp3GFP Treg cells were sorted, pretreated for 10min with PKA inhibitor H89 and then treated with fenoterol (B2AR agonist) for 1h before being cultured with CD4+ CD62L+ Foxp3 T cells. Up line in the graphic indicates division index of CD4+ CD62L+ Foxp3 T cells cultured without Treg cells. Data are shown as mean ± SD of triplicates and are representative of two independent experiments. ***p< 0.001; **p<0.01, as determined by Tukey's multiple comparison test.

Table S1. Sequence of primers for qPCR analysisa)

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