Fig 1: Adenosine regulates Treg stability(A, C, D, and H–J) 2 × 105 Foxp3GFP tTregs added to the upper well of a Boyden chamber with or without LECs. tTregs were migrated or incubated for 16 h and analyzed from upper and/or lower wells. Cells were gated on Annexin-V-7-AAD-/CD4+. Bar diagrams are a summary of 3–5 independent experiments. (A) Expression of indicated cell surface molecules on migrated (lower well) and non-migrated (upper well) Tregs. Numbers are geometric MFI (gMFI).(B) Treg ectonucleotidase activity of CD39 (left panel, ATP conversion to AMP) and CD73 (right panel, consumption of AMP).(C) Supplementation with adenosine at indicated doses prevented exTreg conversion. Representative plots and bar diagram summarizing 5 independent experiments shown.(D) Effect of adenosine (10 µM) plus A2aR-blocker CPI 444 (1 µM) or A2bR-blocker MRS 1754 (1 µM) on inhibition of exTreg conversion.(E) Sorted tTregs (CD4+Foxp3GFP+) injected into hind footpads of naive mice along with indicated adenosine receptor blocker (1 µM) with or without adenosine. exTreg conversion of non-migrated Tregs in the footpads was analyzed 16 h later.(F and G) Effect of adenosine and/or A2aR-blocker CPI 444 pre-treatment on (F) a LEC monolayer grown on inverted Boyden chamber or on (G) mouse ears pretreated first with an A2aR blocker for 1 h and then adenosine for 4 h. Data are representative of 3 independent experiments involving 3 mice per group, with 2–3 ear pinnae for each staining condition, and 5–10 fields/sample, acquired at 20× (scale bar, 42 µm) or 60× (scale bar, 20 µm) magnification.(H) LEC monolayer pre-treated, as in (F), and analyzed for dye permeability.(I) Mouse footpads injected with A2aR blocker and 1 h later with adenosine. Four h after adenosine, Evans blue dye was administered, and 16 h later, dye transport from dLNs was measured for indicated conditions.(J and K) Effect of adenosine pre-treatment of Tregs or LECs on (J) inhibition of exTreg conversion and (K) IL-6 secretion. Results were pooled from duplicate wells in 5 independent experiments.(L) Effect of supplementation with adenosine and/or IL-6 (10 ng/mL) on exTreg conversion in the upper chamber.(M and N) Effect of LEC pre-treatment with LTßR decoy peptides (40 µM) on adenosine inhibition of (M) exTreg conversion and (N) IL-6 secretion.(B–D, G, and I–N) Bar diagrams summarize 2–5 independent experiments. (E and I) Results of pooled footpads (6–12 footpads/3–6 mice) per treatment condition from 2 (I) to 3 (E) independent experiments. Repeated measures by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.See also Figures S3, S4, S8, and S9.