An Adora2b-specific agonist enhances Treg abundance in vitro following activation of murine T cells.

<p>Bulk splenocytes from either <i>Adora2b</i>+/+ (C57BL/6J) or <i>Adora2b</i>−/− mice were cultured with soluble anti-CD3 for three days with or without the Bay60-6583 compound, at which time the relative abundance of Tregs was assessed. (A) Fold change in Treg abundance relative to <i>Adora2b</i>+/+ cultures without the Bay compound control, where Tregs were defined as viable, CD4+ FoxP3+ cells by flow cytometry. (B) Flow cytometry plots shown from <i>Adora2b</i>+/+ cultured splenocytes. Bay60-6583, an Adora2b-specific agonist was added to a final concentration of 4 nM. Data from two independent experiments, each containing 1–3 independent replicates. The numbers present on each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate, with FoxP3 expressing cells defined relative to isotype control-stained samples (not shown). Statistically significant differences are indicated and were calculated by one-way ANOVA followed by Bonferroni's post-test correction. ns indicates a comparison that is not statistically significantly different.</p

Relative expression levels of adenosine receptor genes in T cell subsets.

<p>Real-time PCR analysis of mRNA for the four adenosine receptor genes in FACS purified CD4 T cell subsets of naïve CD4 T cells or Tregs, with Tregs isolated from FoxP3GFP or DEREG mice. Values were standardized to bulk spleen mRNA, with each value showing expression relative to actin. High level expression of FoxP3 mRNA is consistent with a highly purified population of Tregs. Data representative of two to three independent experiments, analyzing at least three independently isolated populations for both naïve CD4 T cells and Tregs. Data depict mean ± SEM for each transcript. Statistically significant differences were calculated by unpaired t test comparing expression in naïve CD4 T cells relative to Tregs, as indicated.</p

Adenosine receptor activation enhances the abundance of Tregs following in vitro stimulation of primary mouse T cells.

<p>(A) Adenosine receptor activation enhances the abundance of Tregs following antibody-mediated stimulation of T cells (using anti-CD3 antibody, 1 µg/mL combined with 10 ng/mL of IL-2, in the absence of TGF-β). Samples were either untreated (+Vehicle) or treated with 10 µM of NECA (+NECA), a potent adenosine receptor agonist, analyzed three days post-stimulation for the relative abundance of FoxP3-expressing Tregs. The numbers present in the upper left-hand corner of each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate. Background staining with an isotype control antibody is indicated in the leftmost panel. (B) Quantitation of FoxP3-expressing cells following either control (vehicle treated, white bars) or NECA treated (black bars), with data indicating mean +/− SEM of triplicate cultures done in two separate experiments. (C) Adenosine receptor activation enhances the abundance of Tregs following antibody-mediated stimulation of T cells in the presence of TGF-β, a known inducer of Tregs (using anti-CD3 antibody, 1 µg/mL combined with 10 ng/mL of IL-2, combined with 0.75 ng/mL TGF-β), showing flow cytometric analysis (C) and quantitation (D). The numbers present in the upper left-hand corner of each flow cytometry plot indicate the percentage of cells that are FoxP3+ as defined by the square gate. Relative abundance of Tregs within cultures were defined by flow cytometry, with Tregs defined as live, MHC class II negative, CD8−, CD4+ cells that express the transcription factor FoxP3. Data indicate mean +/− SEM of triplicate cultures, representative of two independent experiments. (E) Tregs generated by TGF-β with NECA (solid black line) relative to Tregs generated by TGF-β treatment alone (indicated in gray) have a comparable cell surface expression of CD25, CD39 and CD73. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032416#s2" target="_blank">Results</a> representative of results from three independent cultures, done in two independent experiments. Statistical analysis was performed using unpaired t test, with statistically significant differences as indicated.</p