A_2AR signaling promotes LAG-3 expression and regulatory T cells. (A) CD4⁺, 6.5⁺ primary T cells were cultured with irradiated APCs and HA plus or minus 1 μM CGS and IL-2 for 3 days. Total RNA was harvested and assayed for abundance of LAG-3 transcripts by real-time PCR. (B) A_2AR Wt or KO 6.5⁺ T cells were transferred into C3HA mice, harvested 3 days after adoptive transfer, and sorted to more than 98% purity. LAG-3 expression was determined by real-time PCR. (C) Clonotypic 6.5⁺ T cells were transferred into C3HA mice, which were treated with vehicle or CGS for 3 days after the adoptive transfer. The donor T cells were harvested and sorted to more than 98% purity. LAG-3 expression was determined by real-time PCR. For panels A-C, data are representative of 3 independent experiments, 3 mice per group. (D) Survival curve of C3HA mice given Wt or LAG-3 KO T cells and a 4-day treatment with CGS. The “Wt Veh” and “LAG-3 KO CGS” both had 4 mice per group. For the “Wt CGS” and “LAG-3 KO Veh,” both had 5 mice per group. Data are representative of 2 independent experiments. (E) In vivo suppression assay in which vehicle- or CGS-treated C3HA mice (the survivors of Figure 5A; □, [n = 4]; or , [n = 13], respectively) are given a higher dose of 6.5⁺ T cells. Naive mice (▵, [n = 17]) received only this higher dose of 6.5⁺ T cells. No drug was administered during this phase of the experiment (*P < .05).