A2AR^-/- γδ T cells have a limited enhancing effect on autoimmune responses.

<p>A &B) A2AR^+/+ enhanced the Th17 response, but A2AR^-/- γδ T cells did not; and neither enhanced the Th1 response. A2AR^+/+ and A2AR^-/- γδ T cells were isolated from immunized wt-B6 or A2AR^-/- mice, respectively. Responder αβ T cells (1 x 10⁶/well) prepared from IRBP_1-20 immunized TCR-δ^-/- mice on day 13 post-immunization were stimulated for 5 days with immunizing antigen and APCs under Th17 (A) of Th1 (B) polarized conditions with or without addition of 2% (2 x 10⁴/well) A2AR^+/+or A2AR^-/- γδ T cells. The numbers of αβTCR⁺ IL-17⁺ and αβTCR⁺ IFN-γ⁺ cells were calculated by FACS analysis after a 5-day in vitro stimulation. C) Addition of A2AR^+/+ γδ T cells, but not of A2AR^-/- γδ T cells, to αβ responder T cells enhanced IL-17 production but did not affect IFN-γ production. Production of IL-17 and IFN-γ by immunized TCR-δ^-/- mice was assessed by ELISA, after in vitro stimulation with the immunizing peptide (IRBP_1-20), in the presence of A2AR^+/+ or A2AR^-/- γδ T cells (2%). Cytokine amounts were evaluated in the culture cell supernatants by ELISA after a 48 h stimulation. D) Treatment of A2AR antagonist abolished the enhancing effect of A2AR^+/+ γδ T cells. A2AR^+/+ γδ T cells were added to responder αβ T cells in culture, with or without a pre-incubation with SCH58261, a selective A2AR antagonist (100nM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199601#pone.0199601.ref029" target="_blank">29</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199601#pone.0199601.ref030" target="_blank">30</a>]. Results show that blockade of A2AR ligation abolished the enhancing effect of A2AR^+/+ γδ T cells. E) Administration of TCR-δ^-/- mice with A2AR^+/+γδ T cells, but not with A2AR^-/- γδ T cells, enhanced the Th17 response. Groups (n = 4) of TCR-δ^-/- mice were injected with A2AR^+/+ or A2AR^-/- (1 x 10⁶/mouse) γδ T cells, before they were immunized with a pathogenic dose of IRBP_1-20/CFA. At 13 days post-immunization, the Th17 responses were assessed 5 days after in vitro stimulation. The results shown are representative of those from five experiments. Differences were considered significant when <i>P</i> ≤ 0.05 (asterisk) and very significant when <i>P</i> ≤ 0.01 (two asterisks). F.) TCR-δ^-/- mice injected with A2AR^+/+, but not A2AR^-/-, γδ T cells were more susceptible to induced EAU. Groups of TCR-δ^-/- mice were immunized with IRBP _1-20/CFA with or without a prior injection of 1 x 10⁵ (per mouse) A2AR^+/+ or A2AR^-/- γδ T cells. EAU was scored as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199601#pone.0199601.ref003" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199601#pone.0199601.ref004" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199601#pone.0199601.ref008" target="_blank">8</a>]. The results of two separate experiments are shown.