A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells

Abstract

Tissue-derived adenosine, acting via the adenosine A(2A) receptor (A(2A)R), is emerging as an important negative regulator of T-cell function. In this report, we demonstrate that A(2A)R stimulation not only inhibits the generation of adaptive effector T cells but also promotes the induction of adaptive regulatory T cells. In vitro, antigen recognition in the setting of A(2A)R engagement induces T-cell anergy, even in the presence of costimulation. T cells initially stimulated in the presence of an A(2A)R agonist fail to proliferate and produce interleukin-2 and interferon (IFN)-gamma when rechallenged in the absence of A(2A)R stimulation. Likewise, in an in vivo model of autoimmunity, tissue-derived adenosine promotes anergy and abrogates tissue destruction. Indeed, A(2A)R stimulation inhibits interleukin-6 expression while enhancing the production of transforming growth factor-beta. Accordingly, treating mice with A(2A)R agonists not only inhibits Th1 and Th17 effector cell generation but also promotes the generation of Foxp3(+) and LAG-3(+) regulatory T cells. In this regard, A(2A)R agonists fail to prevent autoimmunity by LAG-3(-/-) clonotypic T cells, implicating an important role for LAG-3 in adenosine-mediated peripheral tolerance. Overall, our findings demonstrate that extracellular adenosine stimulates the A(2A)R to promote long-term T-cell anergy and the generation of adaptive regulatory T cells.

Figure 1

A_2AR expression is up-regulated on T-cell activation and preferentially inhibits IL-2 production. (A) cAMP levels after CGS incubation of A.E7s previously mock stimulated (◇) or stimulated with anti-CD3 in the presence or absence of CSA (▵ or •, respectively). (B) cAMP levels of naive or previously activated primary T cells from A_2AR Wt or null mice after incubation with 0 or 1 μM CGS ( and , respectively, *P < 0.05). (C) IL-2 (top) and IFN-γ (bottom) production of A.E7 T cells during activation with increasing doses of CGS. ID₅₀ for IL-2 is 8 nM, and for IFN-γ 750 nM. (D) Proliferation of A.E7s with 0 or 1 μM of CGS. Data are representative of 3 separate experiments.

Figure 2

A_2AR engagement during activation promotes T cell tolerance. (A,B) Proliferation on rechallenge of A.E7 T cells after 4-day incubation without (A) or with (B) peptide in the absence (□) or presence (♦) of 1 μM CGS. (C) IFN-γ production on rechallenge of A.E7 T cells after priming without or with peptide (left or right side, respectively) in the absence () or presence () of 1 μM CGS. (D,E) Proliferation and IFN-γ production on rechallenge of A.E7s incubated with peptide and exogenous IL-2 in the absence or presence of 1 μM CGS. All rechallenges are done in the absence of CGS or exogenous IL-2. Data are representative of at least 3 independent experiments (*P < .05).

Figure 3

A_2AR signaling promotes decreases signaling of the Ras-MAP-Kinase pathway. (A) Percentage of A_2AR Wt or KO T cells that were IFN-γ positive on rechallenge after incubation with peptide in the absence () or presence () of 1 μM CGS during induction (*P < 0.05). (B) Representative Western blots for phospho-ERK and total ERK (top and bottom, respectively). Activated CD4⁺, 6.5⁺ primary T cells were stimulated with anti-CD3⁺ anti-CD28 in the absence or presence of 1 μM CGS. A vertical line has been inserted to indicate a repositioning of gel lanes from the same experiment. (C) Representative Western blots for junB, and actin (top and bottom, respectively). CD4⁺, 6.5⁺ primary T cells were stimulated with HA and irradiated APCs overnight in the absence or presence of 1 μM CGS. (D) Representative EMSA for AP-1. CD4⁺, 6.5⁺ primary T cells were stimulated with HA and irradiated APCs overnight in the absence or presence of 1 μM CGS. Data are representative of 3 independent experiments.

Figure 4

Endogenous adenosine is required to prevent death by autoimmunity. (A) In vivo A_2AR mRNA expression as determined by Affymetrix microarray analysis for 6.5⁺ TCR transgenic clonotypic T cells specific for hemagglutinin (HA) that had been transferred into B10.D2 mice (NT), vaccinated B10.D2 mice (Vac), or C3HA mice (C3HA). (B) Survival curve of C3HA mice given Wt () or A_2AR^−/− 6.5⁺ T cells (○) (n = 5 each condition). Data are representative of 2 independent experiments.

Figure 5

A_2AR stimulation in vivo prevents death by autoimmunity and promotes T-cell tolerance. (A) Survival curve of C3HA mice given 6.5⁺ T cells and 4 days of vehicle (□) or CGS (♦) (n = 17 mice, each condition). (B-D) In vitro proliferation (B), IFN-γ production (C), and IL-2 (D) of T cells harvested from vehicle- or CGS-treated C3HA mice ( and , respectively). Data are representative of 2 independent experiments, ≥3 mice per group. (E,F) Number of IFN-γ (E) and IL-17 (F) lung-infiltrating 6.5⁺ T cells from C3HA mice given a lethal dose of autoreactive T cells. Data are a combination of 2 independent experiments, 3 mice per group (*P < .05).

Figure 6

A_2AR engagement during inflammation inhibits IL-6 and promotes Foxp3 expression in vivo. (A,B) Primary T cells from 5C.C7 mice were cultured with PCC plus or minus 1 μM CGS for 1 day. Total RNA was harvested and assayed for abundance of IL-6 (A) and TGF-β (B) transcripts by real-time PCR. Data are representative of 3 independent experiments. (C) Splenocytes from 5C.C7 TCR-transgenic Rag2^−/− mice were cultured with antigen (PCC) plus or minus 1 μM CGS for 3 days under Th17-driving conditions (containing TGF-β, IL-6, anti-IFN-γ, and anti-IL-4). The cells were then activated with PMA and ionomycin and assayed for IL-17 by intracellular cytokine staining. Representative FACs plots are shown (left) and bar graph of composite data (right). Data are representative of 3 independent experiments. (D) Primary T cells from 5C.C7 mice were cultured with PCC plus or minus 1 μM CGS for 1 day. Total RNA was harvested and assayed for abundance of Foxp3 transcripts by real-time PCR. Data are representative of 3 independent experiments. (E) 6.5⁺ donor T cells were harvested 3 days after transfer into C3HA hosts treated with vehicle or CGS. Total RNA was harvested and assayed for abundance of Foxp3 by real-time PCR. Data are representative of 2 independent experiments, 3 mice per group. All PCR samples were done in triplicate and evaluated for significance (*P < .05).

Figure 7

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).