Adenosine analogue (NECA) enhanced Foxp3 T cell response at very low doses.

A) NECA (3–30 nM) significantly enhanced Foxp3 T cell response. Titrated doses of NECA were added to TCR-δ^-/- responder T cells cultured in 1 ng/ml of recombinant IL-2. Five days later the Foxp3⁺ cells were assessed as described earlier. B) The enhancing effect of NECA was offset by A2AR antagonist, but not by antagonists specific for other adenosine receptors (A1R, A2BR, and A3R). TCR-δ^-/- responder T cells were cultured in medium containing 1 ng/ml of recombinant IL-2 and 300 nM NECA, in the presence of indicated adenosine receptor antagonists: A1R antagonist (DPCPX, 50nM); A2AR antagonist (SCH58261, 100nM); A2BR antagonist (MRS1754, 100nM); and A3R antagonist (MRS 1220, 5 μM). Five days later the Foxp3⁺ cells were assessed as described earlier. C) Adenosine-mediated enhancing effect on Foxp3 T cell response is regulated by γδ T cells. Responder T cells isolated from immunized wt-B6 and TCR-δ^-/- mice, 13 days post-immunization were cultured in medium containing 1 ng/ml of IL-2 and indicated doses of NECA. Five days later the Foxp3⁺ cells were assessed as described earlier. D) Supernatants of cultured γδ T cells contained higher amounts of adenosine. 25 μl of supernatant samples of indicated immune cells (αβ/γδ T cells and DCs) were mixed with assay buffer, adenosine convertor, adenosine detector, adenosine developer and adenosine probe in room temperature and protected from exposure to light for 15 minute. Fluorescence amounts generated as a result of reaction was read in a spectraMax iD5 multi-mode microplate reader (Molecular Devices,LLC. USA) at Ex/Em = 535/587 nm.