Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

  • Dongchun Liang,

    Roles Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

    Affiliation Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States of America

  • Hui Shao,

    Roles Conceptualization, Formal analysis, Methodology, Project administration

    Affiliation Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky, United States of America

  • Willi K. Born,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Biomedical Research, National Jewish Health, Denver, CO, United States of America

  • Rebecca L. O'Brien,

    Roles Conceptualization, Formal analysis, Investigation

    Affiliation Department of Biomedical Research, National Jewish Health, Denver, CO, United States of America

  • Henry J. Kaplan,

    Roles Conceptualization, Formal analysis, Investigation

    Affiliation Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky, United States of America

  • Deming Sun

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – review & editing

    dsun@doheny.org

    Affiliation Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States of America

High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU

  • Dongchun Liang, 
  • Hui Shao, 
  • Willi K. Born, 
  • Rebecca L. O'Brien, 
  • Henry J. Kaplan, 
  • Deming Sun
PLOS
x

Correction

8 Nov 2018: Liang D, Shao H, Born WK, O'Brien RL, Kaplan HJ, et al. (2018) Correction: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU. PLOS ONE 13(11): e0207546. https://doi.org/10.1371/journal.pone.0207546 View correction

Abstract

We previously reported that activated γδ T cells greatly enhance autoimmune responses, particularly the Th17 response. To determine the mechanisms involved, we made a series of comparisons between activated and non-activated γδ T cells. Our results showed that activated γδ T cells expressed greatly increased levels of A2A adenosine receptor (A2AR) and decreased amounts of CD73, as well as increased amounts of T cell activation markers such as CD69, CD44 and CD25. We show that A2AR is a major functional molecule in the enhancing activity of γδ T cells. A2AR-/- γδ T cells (isolated from A2AR-/- mouse), lost their Th17-enhancing activity as did A2AR+/+ γδ T cells (isolated from wt-B6 mouse) after treatment with an A2AR antagonist. Since γδ T cells possess either an enhancing or an inhibiting effect, we also tested whether A2AR expression on γδ T cells is essential to their inhibiting effect. Our results showed that the inhibiting effect of A2AR-/- γδ T cells was as potent as that of A2AR+/+ γδ T cells. In a previous report we showed that the expression of different levels of CD73 molecule allowed γδ T cells to adjust their suppressive activity; in the current study, we show that expression of increased amounts of A2AR allows γδ T cells to more effectively exert their enhancing function.

Introduction

γδ T cells can either enhance or inhibit immune responses [1,2], yet the mechanisms by which they do so are unclear. Clarification of these mechanisms should provide a better guide for therapeutic interventions. We previously demonstrated that the enhancing and inhibiting functions of γδ T cells are convertible and that the activation status of the γδ T cell determined the outcome [35]. The enhancing activity is elevated among activated γδ T cells, whereas the suppressive function dominates in non-activated γδ T cells [37]. A large portion (>60%) of the γδ T cells became activated and were a strong driving force on disease progression [3,4,8] during the pre-clinical phases (one week before the clinical appearance of disease) of induced mouse autoimmune uveitis (EAU). We have been searching for contributing molecules in an effort to determine whether the enhancing and inhibiting functions of γδ T cells are associated with the expression of specific surface molecules and to determine the underlying mechanism by which γδ cells switch their regulatory function. Our results showed that in addition to expressing increased amounts of T cell activation markers such as CD69, CD44 and CD25, activated γδ T cells also expressed greatly increased levels of A2A adenosine receptor (A2AR) and decreased amounts of CD73 [5,9]. Both molecules are crucially involved in metabolism, function, and the regulatory effect of extracellular ATP [1012].

In a previous report, we showed that CD73 molecules play an important role in inhibiting the effect of γδ T cells [5]. CD73 converts AMP to adenosine, the expression of decreased amounts of CD73 molecules by activated γδ T cells results in a decreased ATP conversion to adenosine [5]. In the current study, we show that the expression of a high density of A2ARs favors the enhancing effect of γδ T cells, since the binding of increasing amounts of adenosine to γδ T cells diminishes adenosine binding by αβ T cells and dendritic cells (DC). Moreover, A2AR signaling promotes γδ T cell activation, whereas adenosine has an inhibiting effect on αβ T cells [9].

A2AR is a high-affinity adenosine receptor that is predominantly expressed on T cells [1315]. Activation of A2AR suppressed the function of many immune cells such αβ T cells [11,1619] and macrophage/DCs [14,17,18,2027]. We previously reported that adenosine enhanced the responses of γδ and Th17 autoreactive T cell responses, while it inhibited Th1 responses [9]. A better understanding of how adenosine inhibits some immune responses but enhances others would be significant.

To further determine whether increased A2AR expression accounts for the augmented enhancing activity of activated γδ T cells, we compared the regulatory effect of A2AR+/+ and A2AR-/- γδ T cells and assessed A2AR+/+ γδ T cell function, before and after treatment with an A2AR antagonist. Our results showed that γδ T cells lost most, if not all, of their enhancing activity and were less likely to be activated when A2ARs were functionally disabled. In contrast, the inhibiting function was retained. We conclude that a blockade of A2AR on γδ T cells could effectively regulate γδ activation, tipping the balance of the enhancing and inhibiting functions of γδ T cell, and could conceivably become a supplemental therapy for damping augmented autoimmune responses.

Materials and methods

Animals and reagents

All animal studies conformed to the Association for Research in Vision and Ophthalmology statement on the use of animals in Ophthalmic and Vision Research. Institutional approval (Protocol number: ARC#2014-029-03A) was obtained from the Institutional Animal Care and Use Committee of the Doheny Eye Institute, University of California Los Angeles, and institutional guidelines regarding animal experimentation were followed. Veterinary care was provided by IACUC faculty. Immunized animal that displays swelling joints were either be humanely euthanatized or administered an analgesic (buprenorphine, 0.1 mg/kg sc. twice daily or ketoprofen, 2 mg/kg sc. daily) until the swelling resolves. By the end of the study, mice were euthanized by cervical dislocation after an injection of over dosed Ketamine and xylazine prior to tissue collection.

Female C57BL/6 (B6) and TCR-δ-/- mice on the B6 background, were purchased from Jackson Laboratory (Bar Harbor, ME). A2AR-/- mice were kindly provided by Dr. Jiang-Fen Chen of Boston University [28]. Animals were housed and maintained in the animal facilities of the University of California, Los Angeles (UCLA). Recombinant murine IL-1, IL-7, and IL-23 were purchased from R & D (Minneapolis, MN). FITC-, PE-, or allophycocyanin-conjugated Abs against mouse CD73 (TY/11.8), CD44 (IM7), αβ T cell receptor (TCR, H57-597), or γδ TCR (GL3) and their isotype control Abs were purchased from Biolegend (San Diego, CA). PE-conjugated anti-mouse A2AR monoclonal Ab (7F6-G5-A2) was purchased from Santa Cruz Biotechnology (Dallas, TX). The non-selective AR agonist 50-N-ethylcarboxamidoadenosine (NECA), selective A2AR agonist 2-p-(2-carboxyethyl) phenethylamino-5’-N-ethylcarboxamidoadenosine (CGS21680), and selective A2AR antagonist (SCH 58261) [29,30] were purchased from Sigma-Aldrich (St. Louis, MO).

Immunization procedure

B6 mice were immunized subcutaneously over 6 spots at the tail base and on the flank with 200 μl of emulsion containing 150 μg of the uveitogenic peptide IRBP1-20 [amino acids 1–20 of human interphotoreceptor retinoid-binding protein (IRBP; Sigma, St. Louis, MO)] emulsified in complete Freund’s adjuvant (CFA; Difco, Detroit, MI). Concurrently, 200 ng of pertussis toxin (PTX) (Sigma, St. Louis, MO) was injected intraperitoneally. At day 13 post-immunization, T cells were isolated from lymph node cells and spleen cells, then 1 x 107 cells in 2 ml of RPMI 1640 medium (Cellgro, VA, USA) containing 10% fetal calf serum were added to each well of a 6-well plate (Costar) and stimulated for 48 h with 10 μg/ml of IRBP1-20 in the presence of 1 x 107 irradiated syngeneic spleen cells as antigen-presenting cells (APCs) in the presence of either IL-12 (Th1 polarized) or IL-23 (Th17-polarized) (10 ng/ml), then activated T cell blasts were separated by Ficoll gradient centrifugation and cultured for another 72 h in the same medium used for stimulation without the peptide.

T cell preparations

αβ T cells were purified from B6 mice immunized with the human interphotoreceptor retinoid-binding protein (IRBP) peptide IRBP1-20, as described previously [4,7,8], while γδ T cells were purified from immunized and control (naïve) B6 or A2AR-/- mice. Nylon wool-enriched splenic T cells from naïve or immunized mice were incubated sequentially for 10 min at 4°C with FITC-conjugated anti-mouse γδ TCR or αβ TCR Abs and 15 min at 4°C with anti-FITC Microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany); the cells were then separated into bound and non-bound fractions on an autoMACSTM separator column (Miltenyi Biotec GmbH). The purity of the isolated cells was >95%, as determined by flow cytometric analysis using PE-conjugated Abs against αβ or γδ T cells. Resting γδ T cells were prepared either by isolation from naïve mice or by incubating activated γδ T cells in cytokine-free medium for 5–7 d, at which time they show down-regulation of CD69 expression [3]. Highly activated γδ T cells were prepared by incubating resting γδ T cells for 2 d with Abs against the γδ TCR (GL3) and CD28 (both 2 μg/ml, both from Bio-Legend, San Diego, CA), or cytokine combination (IL-1,IL-7 and IL-23).

Competitive adenosine binding assay

A two-chamber assay was performed to evaluate the competitive binding ability of γδ T cells. αβ or DCs enriched from immunized TCR-δ-/- mice were seeded in 24-well cell culture plates at a density of 2×106/ml in 500 μl of complete medium, while γδ T cells were seeded at a density of 5×105/ml in a cell culture insertion in 100 μl of complete medium and put into the wells with competitors. Cells were incubated at 37°C for 1 h with H3-adenosine at final concentrations of 1,000 nM in triplicate, then cell-bound and free H3-adenosine were separated by harvesting the cells on a cell harvester (Perkin Elmer) and the cell-associated radioactivity was measured by liquid scintillation. Relative adenosine binding ability of γδ to αβ T cells or to splenic cells was represented by their relative radioactivity of CPM.

CFSE assay

Purified αβ T cells from IRBP1-20-immunized B6 mice were stained with CFSE (Sigma-Aldrich) as described previously [31]. Briefly, the cells were washed and suspended as 5x 106 cells/ml in serum-free RPMI 1640 medium (Corning Cellgro, Manassas, VA), then incubated at 37°C for 10 min with gentle shaking with a final concentration of 5 μM CFSE. The cells were washed twice with RPMI 1640 medium containing 10% fetal calf serum (Atlantic Inc. Santa Fe, CA) (complete medium), suspended in complete medium, stimulated with immunizing peptide in the presence of irradiated syngeneic spleen cells as antigen-presenting cells (APCs), and analyzed by flow cytometry at 5 d after stimulation.

Cytokine assays

Purified αβ T cells (3x104 cells/well; 200 μl) from the draining lymph nodes and spleens of IRBP1-20-immunized B6 mice were cultured in complete medium at 37° C for 48 h in 96-well microtiter plates with irradiated syngeneic spleen APCs (1x105) in the presence of 10 μg/ml of IRBP1-20; a fraction of the culture supernatant was then assayed for IL-17 and IFN-γ using ELISA kits (R & D).

Statistical analysis

The results in the figures are representative of one experiment, which was repeated 3–5 times. The statistical significance of differences between groups in a single experiment was initially analyzed by ANOVA; if statistical significance was detected, the Student–Newman–Keuls post-hoc test was subsequently used. P values less than 0.05 were considered a statistically significant difference and marked with one *; when P<0.01, two ** were used.

Results

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

Our previous studies have established reliable assays for testing the effect of γδ T cells on autoimmune responses, in vitro and in vivo [3,4,3235]. Testing of the adenosine effect used responder αβ T cells prepared from immunized TCR-δ-/- mice because the αβ T cell response to adenosine is strongly affected by <1% of γδ T cells [3,4,3235]. T cell responses to the immunizing antigen were determined in the absence or presence of γδ T cells [3,4,3235]. As expected, the addition of activated γδ T cells, but not of non-activated, γδ T cells, significantly enhanced the Th17 but did not enhance the Th1 response [5,7,8]. We have compared the enhancing effect on autoreactive T cell responses of A2AR+/+ and A2AR-/- γδ T cells, isolated from immunized wt-B6 or A2AR-/- mice, respectively. The αβ T cell responses were assessed under either Th1- (addition of IL-12 and anti-IL-4 antibodies during in vitro stimulation, Fig 1B) or Th17-polarized conditions (Fig 1A, addition of IL-23 during in vitro stimulation) by monitoring intracellular expression of IFN-γ or IL-17 of the responder T cells [7,8]. As shown in Fig 1A, the number of IL-17+ T cells among the responder T cells was significantly increased if A2AR+/+ γδ T cells were added, but was not enhanced if the A2AR-/- cells were added. ELISA tests measuring cytokine production of the responder T cells (Fig 1C) supported the prediction that A2ARs are crucially involved in γδ T cells’ enhancing function; whereas the Th1 response or production of IFN-γ showed no significant difference when either type of γδ T cells was added.

thumbnail
Fig 1. A2AR-/- γδ T cells have a limited enhancing effect on autoimmune responses.

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 IRBP1-20 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 (IRBP1-20), 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) [29,30]. 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 IRBP1-20/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 P ≤ 0.05 (asterisk) and very significant when P ≤ 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 [3,4,8]. The results of two separate experiments are shown.

https://doi.org/10.1371/journal.pone.0199601.g001

We then determined whether treatment of A2AR+/+ γδ T cells with an A2AR antagonist (SCH 58261) would affect their enhancing effect (Fig 1D). A2AR+/+ γδ T cells were added to responder αβ T cells, with or without pre-incubation with an A2AR antagonist. The results showed that pre-treatment of A2AR+/+ γδ T cells with an A2AR specific antagonist significantly decreased the enhancing effect.

We previously reported that injection of a small number (< 1 x 106/mouse) of γδ T cells to TCR-δ-/- mice significantly enhanced the autoreactive T cell responses of the recipients [3,4,32]. To assess the in vivo effect of A2AR+/+ or A2AR-/- γδ T cells (Fig 1E), the TCR-δ-/- mice were injected with either A2AR+/+ or A2AR-/- γδ T cells (1 x 106/mouse) before immunization, and the cytokine levels in the serum as well as the autoreactive T cell responses of the recipients were examined 13 d post-immunization (the time point at which the greatest T cell changes are seen) [4,32,36]. As shown, the number of IL-17+ IRBP-specific αβ T cells of TCR-δ-/- recipients of A2AR+/+ γδ T cells increased 4 fold as compared to non-injected mice and 2–3 fold as compared to the TCR-δ-/- recipients of A2AR-/- γδ T cells. Serum cytokine levels agreed with the prediction that IL-17 levels were significantly increased in recipients of A2AR+/+γδ T cells, but not in recipients of A2AR-/-γδ T cells (Not shown). Adoptive transfer studies (Fig 1F) showed that IRBP-specific T cells isolated from TCR-δ-/- mice injected with A2AR+/+, but not those injected with A2AR-/-, γδ T cells were more susceptible to EAU, suggesting that A2AR is an important molecules on γδ T cells for their enhancing activity.

A2AR agonist showed an increased inhibitory effect in the absence of γδ T cells.

One way to determine the effect of γδ T cells in T cell responses is to compare T cell responses between wt-B6 and TCR-δ-/- mice. Th1 CD3+ responder T cells prepared from immunized wt-B6, but not those prepared from immunized TCR-δ-/- mice, consistently contain approximately 5% γδ T cells [3,7]. The results demonstrated in Fig 2A showed that A2AR agonist (CGS21680, 250nM) more effectively inhibited the response of TCR-δ-/- than it did that of B6 T cells. Cytokine tests showed that in the absence of γδ T cells (T cells of TCR-δ-/- mice) (Fig 2B, lower panel) the αβ T cell response was inhibited; but in the presence of γδ T cell (B6 T cells), the inhibitory effect of the A2A agonist on Th17 response is reversed, suggesting that the γδ T cells among the B6 responder T cells offset the suppressive effect of adenosine, leading to a higher Th17 response. Comparison of the A2AR agonist effect on IFN-γ production demonstrated, however, that the A2AR agonist inhibited the IFN-γ responses regardless of the appearance of γδ T cells (Fig 2B, right panels).

thumbnail
Fig 2. A2AR agonist is more inhibitory in the absence of γδ T cells.

A) A2AR agonist inhibited TCR-δ-/- responder T cells more effectively than it inhibited the B6 responder T cells. The αβ responder T cells (1 x 106/well) were isolated from IRBP1-20-immunized B6 (upper panels) or TCR-δ-/- mice (lower panels). The CFSE-labeled αβ T cells were stimulated for 5 d with immunizing peptide and APCs in the absence or presence of A2AR agonist. Cell proliferation was assessed by FACS analysis. B) In vitro treatment of A2AR agonist enhanced the Th17 responses when added to B6 responder T cells but inhibited the Th17 responses when added to TCR-δ-/- responder T cells. Comparison of IL-17 and IFN-γ production by immunized αβ T cells isolated from immunized B6 or TCR-δ-/- mice after in vitro stimulation, in the absence or presence of A2AR agonist, showing that A2AR agonist inhibited the Th1 response but enhanced the Th17 response of B6 cells, but not of TCR-δ-/- T cells.

https://doi.org/10.1371/journal.pone.0199601.g002

γδ T cells enhance the autoreactive T cell response by competing with the binding of adenosine.

We previously reported that γδ T cells expressed significantly greater numbers of A2ARs as compared to αβ T cells and DCs [9]. We wanted to determine whether increased absorption of the AR agonist by γδ T cells via A2ARs accounted for the enhancing effect. Real-time PCR study showed that cytokine activated γδ T cells express an increase of >1000-times the A2AR (Fig 3A, left panels), whereas they expressed only an increase of 10-times the A2BR as compared to naïve γδ T cells (Fig 3A, right panels).

thumbnail
Fig 3. γδ T cells enhance the autoreactive T cell response by competing with the binding of adenosine.

A) Activated γδ T cells express an increase of >1000-times the A2AR but only minimally increased A2BR. Activated γδ T cells and naïve γδ T cells were isolated from immunized from naïve B6 mice, respectively. The expression of A2AR/A2BR was determined by real-time PCR assay. Real-time RT-PCR analysis of A2AR and A2B transcripts among total RNA isolated from αβ and γδ T cells isolated from naïve and IRBP1-20-immunized B6 mice before and after in vitro activation. qPCR was performed with Gapdh as the internal reference. Results were represented as 2-ΔCt. **, p < 0.01. B) Competitive binding test revealed a hierarchical order of adenosine capture by immune cells as γδ T cells >> αβ T cells >> DCs. αβ responder T cells were isolated from IRBP1-20-immunized TCR-δ-/- mice. DCs were from 5 d GM-CSF-cultured bone marrow cells. In a 24-well plate the T cells were seeded at 1 x 106/well and DCs at 5 x 105/well. After incubation with1,000 nM H3-labeled adenosine for 1 h, the cells were harvested, and cell-bound H3-adenosine was counted by liquid scintillation. ** p < 0.01. C) Comparison of adenosine binding of γδ T cells isolated from naïve and immunized mice γδ T cells from immunized or naïve B6 mice, respectively. 1 x 105/well cells were cultured in 96-well plate for 48h. 0–12,000 nM H3-labeled adenosine for 1 h, then the cells were harvested and cell-bound H3-adenosine counted by liquid scintillation. D) A2AR+/+γδ T cells, but not A2AR-/- γδ T cells, possess an enhancing effect, which converted the inhibitory effect to enhancing on the αβ T cell response of an A2AR agonist. αβ and γδ T cells (1 x 105) were isolated from IRBP1-20-immunized TCR-δ-/- mice. The responder αβ T cells were labeled with CFSE and incubated for 5 d with immunizing peptide in the presence of APCs alone (A) or in the presence of the A2AR+/+ or A2AR-/- γδ T cells (B), in the absence or presence of an A2AR agonist, and with or without an addition of A2AR+/+γδ T cells. The results shown are representative of three experiments.

https://doi.org/10.1371/journal.pone.0199601.g003

To further test whether increased adenosine-binding activity of γδ T cells accounts for the observed enhancing effect by removal of adenosine binding to responder αβ T cells, we performed a competitive adenosine-binding test using a radioactively labeled adenosine. For this assay, the γδ T cells were co-cultured, separated by chamber separators, with αβ T cells or macrophages/DCs. H3-labeled adenosine was added to the cultures 6 h before cell harvest and the incorporated amounts of the isotope on γδ, αβ T cells or macrophages/DCs were assessed by a β counter. Binding tests (Fig 3B) revealed that the hierarchical order of adenosine capture by immune cells was γδ T cells >> αβ T cells >> DCs. The αβ or γδ T cells could bind comparable amounts of adenosine if cultured alone; however, the γδ T cells have a much greater adenosine-binding effect than DCs or αβ T cells if they are co-cultured in separated two-chambers, suggesting that the binding of H3-labeled adenosine by αβ T cells and DCs dramatically declined in the presence of activated γδ T cells, and that γδ T cells enhance the autoreactive T cell response by competing with the binding of adenosine. We previously reported that activation confers the increased adenosine-binding activity to γδ T cells [9]. Fig 3C shows that γδ T cells isolated from immunized mice, but not those isolated from naïve mice, expressed a greatly increased adenosine binding activity.

To prove the prediction that expression of A2AR is functionally essential for γδ T cells, responder αβ T cells isolated from immunized TCR-δ-/- mice were CFSE-labeled and stimulated with the immunizing peptide and APCs with or without added A2AR+/+ or A2AR-/- γδ T cells, in the absence or presence of A2AR agonist. The proliferation of CFSE-labeled αβ T cell was assessed by FACS analysis 5 d after stimulation (Fig 3D). The results shown in Fig 3D demonstrated that the addition of A2AR agonist inhibited the proliferation of αβ responders in the absence of γδ T cells and that its inhibitory effect is weakened in the presence of A2AR+/+ γδ T cells, but not in the presence of A2AR-/- γδ T cells. The results suggest that γδ T cells enhance the autoreactive T cell response by competitively binding adenosine via A2AR. We have compared the enhancing effect of A2AR+/+, A2AR-/-, and CD73-/- γδ T cells in promoting cytokine (IL-17) production (Fig 4), our results showed that CD73-/- γδ T cells retained the enhancing activity, implying that altered expression of A2AR, but not CD73, by activated γδ T cells are responsible.

thumbnail
Fig 4. CD73-/- γδ T cells retained the enhancing activity.

The enhancing effect of A2AR+/+, A2AR-/-, and CD73-/- γδ T cells were compared for promoting cytokine (IL-17) production of responder T cells obtained form TCR-δ-/- mice. Responder T cells were stimulated with the immunizing antigen and APCs in the absence or presence of indicated γδ T cells. The 48h cultured cell supernatants were sampled for testing IL-17 production by ELISA.

https://doi.org/10.1371/journal.pone.0199601.g004

Failure to express A2AR did not affect γδ T cells’ suppressive activity.

Since γδ T cells can either enhance or inhibit an immune response [5,6,3739], we wished to determine whether γδ T cells with disabled A2AR function have altered suppressive function. Our previous studies determined that the inhibitory effect of γδ T cells was enhanced when exogenous AMP was provided and that increased degradation of AMP to adenosine by the CD73 molecules on γδ T cells accounted for the effect [5,10,40]. Inhibiting tests were done using a CFSE assay, in which the responder αβ T cells, isolated from immunized TCR-δ-/- mice, were pre-labeled with CFSE and stimulated with the immunizing peptide and APCs (Fig 4). In the presence of AMP, both A2AR+/+ and A2AR-/- γδ T cells showed a comparable effectiveness in suppression, whereas both cells showed a significantly diminished suppressive activity if tested in the absence of AMP. Importantly, the A2AR+/+ and A2AR-/- γδ T cells demonstrated a comparable suppressive effect.

A2AR dysfunction compromised γδ T cells’ activation.

Previous studies have shown that cytokines are potent stimulators of γδ T cell [37,38,41,42]. We were able to show that adenosine has an amplifying effect on the cytokine-mediated γδ activation [5,9]. To determine whether dysfunction of A2AR affects γδ activation, we exposed A2AR+/+ and A2AR-/- γδ T cells to a determined amount of pooled cytokines (IL-1+IL7+IL-23) [5,9], with or without the addition of an adenosine analogue NECA (300 ng/ml). Fig 5 shows that NECA and the A2AR-specific agonist CGS21680 significantly amplified A2AR+/+ γδ T cells’ IL-17 production induced by cytokine Mixture. No such synergistic effect could be found in the cytokine response of A2AR-/- γδ T cells (Fig 6A), which reacted less to cytokines; and the addition of A2AR agonist showed no enhancing effect, further supporting the notion that A2AR on γδ T cells crucially regulates the activation of γδ T cells and thus the enhancing effect of γδ T cells. We also compared the activating response of γδ T cells, before and after A2AR antagonist treatment. The wt-γδ T cells treated with A2AR antagonist expressed significantly decreased amounts of activation markers when activated by cytokines (Fig 5B, upper panels). However, when A2ARs were blocked by an A2AR antagonist, cytokine-induced CD25 expression on γδ T cells was decreased, indicating that A2ARs on γδ T cells are crucially involved in γδ T cell activation. Fig 6B shows the result of CD25 staining. The same results were observed on the expression of additional T cell activation markers such as CD44 and CD69 (data not shown).

thumbnail
Fig 5. The suppressive effects of A2AR-/- and A2AR+/+ γδ T cells are not distinguishable.

Proliferative responses of CFSE-labeled αβ T cells isolated from immunized TCR-δ-/- B6 mice (1 x 105/well) were assessed after in vitro incubation for 1 h with immunizing peptide and APCs alone (upper panels) or in the presence of 10 μM AMP (lower panels), 5% A2AR+/+ γδ T cells (Middle panels), or 5% A2AR-/- γδ T cells (right panels). The results shown are representative of those obtained in three separate experiments.

https://doi.org/10.1371/journal.pone.0199601.g005

thumbnail
Fig 6. A2AR dysfunction compromised γδ T cells’ activation threshold.

A) Failure to express A2AR rendered γδ T cells less activated. In a 24-well plate, 2 x 105 A2AR+/+ or A2AR-/- γδ T cells were incubated with medium only (Control), with pooled cytokines (IL-1, IL-7, and IL-23), and with or without or NECA (300 nM) or the A2AR-specific agonist CGS21680. At 48 h after the culture, IL-17 amounts in the culture supernatants were assessed by ELISA. B) Blockade of A2AR expression prevented γδ T cell activation. Surface staining for the T cell activation marker CD25 showing that A2AR antagonist treated A2AR+/+ γδ T cells express low amounts of CD25 after exposure to cytokines. The results shown are from a single experiment and are representative of those obtained in >5 experiments. **, p < 0.01.

https://doi.org/10.1371/journal.pone.0199601.g006

Discussion

Studies in mouse models of autoimmune diseases (EAU & EAE) have demonstrated that the development of autoimmune diseases is closely associated with increased activation of γδ T cells [7,43]. It was hypothesized that activated γδ T cells exacerbate the autoimmune development [35]; however, the mechanism remains to be clarified. In previous reports we have demonstrated that activated γδ T cells exacerbate EAU development by enhancing Th17 autoimmune responses. Our studies have repeatedly shown that this enhancing effect was due to activated γδ T cells. In addition, we have revealed that the γδ T cells’ inhibiting and enhancing functions are both strongly affected by extracellular ATP metabolism [44]. To define structural and functional change between activated and non-activated γδ T cells in correlation to their functional switch, our previous studies demonstrated that activated γδ T cells expressed elevated amounts of A2AR but decreased amounts of CD73 molecules [5,9]. Both molecules are crucially involved in metabolism, function, and the regulatory effect of extracellular ATP [1012].

Adenosine is an endogenous purine nucleoside that diffuses to surrounding cells, where it binds to specific cell surface receptors after its release from cells and after being formed by the breakdown of nucleotides. Early studies have shown that extracellular adenosine accumulation protects cells and tissues from injury caused by an augmented inflammatory response and immune-mediated damage [12] [18,27,4548]. Supporting data include that lymphocytes from A2AR-/- mice show higher rates of cell proliferation and produce high amounts of IFN-γ levels upon stimulation [49]. Engagement of A2AR inhibits IL-12 production but increases IL-10 production by human monocytes [50,51]. In vivo treatment with an A2A agonist decreases intestinal inflammation [48]. Treatment of T lymphocytes with adenosine or the A2AR agonist inhibits proliferation [21,52] and inhibits the secretion of cytokines, including IL-2, TNF-α, and IFN-γ [21,53]. Nevertheless, aberrant adenosine signaling has been implicated in various pathological conditions [11,18,54].

Since most previous studies examined the adenosine effect on Th1-type immune responses, we sought to determine whether Th17-type immune responses are similarly affected. To our surprise, adenosine had many different effects on αβ and γδ T cells [5,9,35], and it differs greatly with respect to its activation of Th1 and Th17 autoimmune responses. Moreover, unlike its suppressive effect on αβ T cells and DCs [27,48,55], adenosine enhanced the γδ T cell response and Th17 autoreactive T cell responses [9]. We have found that adenosine is an important factor in γδ T cell activation; that γδ T cells participate in the conversion of extracellular ATP to adenosine [5,9]; and that γδ T cells bind adenosine much more strongly compared to immune cells such as αβ T cells and DCs so that these cells were less affected by adenosine in the presence of γδ T cell [9,35]. Further study on the issues of how ATP/adenosine metabolites and γδ T cells regulate each other’s function should improve our understanding of autoimmune pathogenesis and our understanding of how γδ T cells exert their regulation effects, which will lead to better treatment of the disease.

In this study we determined whether increased expression and increased adenosine binding ability of A2AR on activated γδ T cells is accountable for their enhancing or inhibiting function. The availability of A2AR-/- mice [28] gave us the opportunity to test whether the A2AR+/+ and A2AR-/- γδ T cells were functionally distinct in their enhancing and inhibiting effects. We also tested whether blockade of A2ARs on A2AR+/+ γδ T cells using a specific A2AR antagonist affected their enhancing or inhibiting function. The results of both approaches led to the conclusion that A2ARs on γδ T cells are critical regulatory molecules; in the absence of functional A2AR, the enhancing effect of γδ T cells was mostly abolished.

We have shown that expression of a high density of A2AR is essential for activated γδ T cells to exert their enhancing effect; however, the inhibitory effect of the γδ T cells is not affected by this molecule. As a consequence, the net effect of enhancing and inhibiting was balanced toward the former. Our results demonstrated therefore that blockade of A2AR might be a successful strategy for damping augmented autoimmune responses.

Our results demonstrated that an augmented enhancement of autoimmune responses by γδ T cells relies on expression of higher levels of A2ARs. Overexpression of the high-affinity A2ARs enables γδ T cells to effectively remove excess adenosine, which would otherwise bind to αβ T cells and DCs. Given that adenosine is a co-stimulating molecule in γδ activation [9], elevated A2AR on γδ T cells would decrease the activation threshold of γδ T cells and augment γδ T cell activation, leading to enhanced autoimmune responses.

In a previous report, we demonstrated that CD73 molecules expressed on γδ T cells are functionally important for γδ T cells’ suppressive function [5]. As an effective enzyme that converts AMP to adenosine, the expression of decreased amounts of CD73 molecules by activated γδ T cells results in a decreased ATP conversion to adenosine [5], consequently leading to augmented immune responses.

In summary, Purinergic signaling plays a central role in inflammation [10,5658]. The incomplete understanding of varied roles of the different purinergic signaling events in physiological and pathophysiological processes has been hindering the application. In a previous study we demonstrated that expression of downregulated CD73 allowed activated γδ T cells to exhibit downgraded suppression and thus shift the enhancement and inhibition toward the latter. In the current study, we show that A2ARs are important molecules that are required for γδ T cells to exert their enhancing activation. Blockade of A2AR function on γδ T cells should allow us to tip the enhancing and inhibiting function of γδ T cells toward the latter.

Supporting information

References

  1. 1. Chien Y-h, Meyer C, Bonneville M. γδ T Cells: First Line of Defense and Beyond. Ann Rev Immunol. 2014. 32: 121–155.
  2. 2. Paul S, Shilpi Lal G. Role of γδ T cells in the autoimmunity. J Leukoc Biol. 2014. 97: 295–271.
  3. 3. Nian H, Shao H, O'Brien BA, Born WK, K H.J, Sun D. Activated γδ cells promote the activation of uveitogenic T cells and exacerbate EAU development. Invest Ophthalmol Vis Sci. 2011. 52: 5920–5927. pmid:21296823
  4. 4. Liang D, Zuo A, Shao H, Born WK, O’Brien RL, Kaplan HJ, et al. IL-23 Receptor Expression on γδ T Cells Correlates with Their Enhancing or Suppressive Effects on Autoreactive T Cells in Experimental Autoimmune Uveitis. J Immunol. 2013. 191: 1118–1125. pmid:23797670
  5. 5. Liang D, Zuo A, Zhao R, Shao H, Born WK, O'Brien RL, et al. CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis. PLoS ONE. 2016. 11: e0150078. pmid:26919582
  6. 6. Liang D, Nian H, Shao H, Kaplan HJ, Sun D. Functional Conversion and Dominance of γδ T Subset in Mouse Experimental Autoimmune Uveitis. J Immunol. 2017. 198: 1429–1438. pmid:28069804
  7. 7. Cui Y, Shao H, Lan C, Nian H, O'Brien RL, Born WK, et al. Major Role of γδ T Cells in the Generation of IL-17+ Uveitogenic T Cells. J Immunol.2009. 183: 560–567. pmid:19542467
  8. 8. Nian H, Shao H, Zhang G, Born WK, O'Brien R, Kaplan HJ, et al. Regulatory effect of γδ T cells on IL-17+ uveitogenic T cells. Invest Ophthalmol Vis Sci. 2010. 51: 4661–4667. pmid:20375337
  9. 9. Liang D, Zuo A, Shao H, Chen M, Kaplan HJ, Sun D. Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells. PLoS ONE. 2014. 9: e108932. pmid:25268760
  10. 10. Antonioli L, Pacher P, Vizi ES, Haskó G. CD39 and CD73 in immunity and inflammation. Trend Mol Med. 2013. 19: 355–367.
  11. 11. Sitkovsky MV, Lukashev D, Apasov S, Kojima H, Koshiba M, Caldwell C, et al. Physiological Control of Immune Response and Inflammatory Tissue Damage by Hypoxia-Inducible Factors and Adenosine A2A Receptors. Ann Rev Immunol. 2004. 22: 657–682.
  12. 12. Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov. 2008. 7: 759–770. pmid:18758473
  13. 13. Manohar M, Hirsh MI, Chen Y, Woehrle T, Karande AA, Junger WG. ATP release and autocrine signaling through P2X4 receptors regulate γδ T cell activation. J Leuk Biol. 2012. 92: 787–794.
  14. 14. Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a Extracellular Adenosine Receptor-Mediated Signaling in Adenosine-Mediated Inhibition of T-Cell Activation and Expansion. Blood. 1997. 90: 1600–1610. pmid:9269779
  15. 15. Koshiba M, Rosin DL, Hayashi N, Linden J, Sitkovsky MV. Patterns of A2A extracellular adenosine receptor expression in different functional subsets of human peripheral T cells. Flow cytometry studies with anti-A2A receptor monoclonal antibodies. Mol Pharmacol. 1999. 55: 614–624. pmid:10051547
  16. 16. Zarek PE, Huang C-T, Lutz ER, Kowalski J, Horton MR, Linden J, et al. A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells. Blood. 2008. 111: 251–259. pmid:17909080
  17. 17. Naganuma M, Wiznerowicz EB, Lappas CM, Linden J, Worthington MT, Ernst PB. Cutting Edge: Critical Role for A2A Adenosine Receptors in the T Cell-Mediated Regulation of Colitis. J Immunol. 2006. 177: 2765–2769. pmid:16920910
  18. 18. Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 2001. 414: 916–920. pmid:11780065
  19. 19. Jin D, Fan J, Wang L, Thompson LF, Liu A, Daniel BJ, et al. CD73 on Tumor Cells Impairs Antitumor T-Cell Responses: A Novel Mechanism of Tumor-Induced Immune Suppression. Cancer Res. 2010. 70: 2245–2255. pmid:20179192
  20. 20. Lappas CM, Rieger JM, Linden J. A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells. J Immunol. 2005. 174: 1073–1080. pmid:15634932
  21. 21. Erdmann AA, Gao Z-G, Jung U, Foley J, Borenstein T, Jacobson KA, et al. Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo. Blood. 2005. 105: 4707–4714. pmid:15746085
  22. 22. Sevigny CP, Li L, Awad AS, Huang L, McDuffie M, Linden J, et al. Activation of Adenosine 2A Receptors Attenuates Allograft Rejection and Alloantigen Recognition. J Immunol. 2007. 178: 4240–4249. pmid:17371980
  23. 23. Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MKK, et al. A2A adenosine receptor protects tumors from antitumor T cells. Pro Nat Acad Sci USA. 2006. 103: 13132–13137.
  24. 24. Schnurr M, Toy T, Shin A, Wagner M, Cebon J, Maraskovsky E. Extracellular nucleotide signaling by P2 receptors inhibits IL-12 and enhances IL-23 expression in human dendritic cells: a novel role for the cAMP pathway. Blood. 2005. 105: 1582–1589. pmid:15486065
  25. 25. Panther ELIS Idzko MARC, Herouy YARE Rheinen HENR, Gebrick-Haerter PJ Mrowietz ULRI, et al. Expression and function of adenosine receptors in human dendritic cells. FASEB J. 2001. 15: 1963–1970. pmid:11532976
  26. 26. Sitkovsky MV, Ohta A. The 'danger' sensors that STOP the immune response: the A2 adenosine receptors? Trend. Immunol. 2005. 26: 299–304.
  27. 27. Alam MS, Kurtz CC, Wilson JM, Burnette BR, Wiznerowicz EB, Ross WG, et al. A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence. Mucosal Immunol. 2009. 2: 232–242. pmid:19262506
  28. 28. Chen JF, Huang Z, Ma J, Zhu J, Moratalla R, Standaert D, et al. A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice. J Neurosci. 1999. 19: 9192–9200. pmid:10531422
  29. 29. Zocchi C, Ongini E, Conti A, Monopoli A, Negretti A, Baraldi PG, et al. The non-xanthine heterocyclic compound SCH 58261 is a new potent and selective A2a adenosine receptor antagonist. J Pharmacol Exp Therap. 1996. 276: 398–404.
  30. 30. Feoktistov I, Biaggioni I. Adenosine A2B Receptors. Pharmcol Rev. 1997. 49: 381–402.
  31. 31. Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods.1994. 171: 131–137. pmid:8176234
  32. 32. Liang D, Zuo A, Shao H, Born WK, O'Brien RL, Kaplan HJ, et al. Retinoic Acid Inhibits CD25+ Dendritic Cell Expansion and γδ T-Cell Activation in Experimental Autoimmune Uveitis. Invest.Ophthal.Vis.Sci. 2013. 54: 3493–3503. pmid:23611991
  33. 33. Liang D, Zuo A, Shao H, Born WK, O'Brien RL, Kaplan HJ, et al. Role of CD25+ Dendritic Cells in the Generation of Th17 Autoreactive T Cells in Autoimmune Experimental Uveitis. J Immunol.2012. 188: 5785–5791. pmid:22539790
  34. 34. Liang D, Zuo A, Shao H, Chen M, Kaplan HJ, Sun D. Anti- or pro-inflammatory effect of an adenosine receptor agonist on the Th17 autoimmune response is inflammatory environmental-dependent. J Immunol. 2014. 193: 5498–5505. pmid:25367119
  35. 35. Liang D, Zuo A, Zhao R, Shao H, Kaplan HJ, Sun D. Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis. J Immunol. 2016. 196: 2646–2654. pmid:26856700
  36. 36. Zuo A, Liang D, Shao H, Born WK, Kaplan HJ, Sun D. In vivo priming of IL-17+ uveitogenic T cells is enhanced by Toll ligand receptor (TLR)2 and TLR4 agonists via γδ T cell activation. Mol Immunol.2012. 50: 125–133. pmid:22301006
  37. 37. Petermann F, Rothhammer V, Claussen MC, Haas JD, Blanco LR, Heink S, et al. γδ T Cells Enhance Autoimmunity by Restraining Regulatory T Cell Responses via an Interleukin-23-Dependent Mechanism. Immunity. 2010. 33: 351–363. pmid:20832339
  38. 38. Sutton CE, Lalor SJ, Sweeney CM, Brereton CF, Lavelle EC, Mills KHG. Interleukin-1 and IL-23 Induce Innate IL-17 Production from gd T Cells, Amplifying Th17 Responses and Autoimmunity. Immunity. 2009. 31: 331–341. pmid:19682929
  39. 39. Huang Y, Yang Z, Huang C, McGowan J, Casper T, Sun D, et al. γδ T Cell–Dependent Regulatory T Cells Prevent the Development of Autoimmune Keratitis. J Immunol. 2015. 195: 5572–5581. pmid:26566677
  40. 40. Beavis PA, Stagg J, Darcy PK, Smyth MJ. CD73: a potent suppressor of antitumor immune responses. Trend Immunol. 2012. 33: 231–237.
  41. 41. Michel M-L, Pang DJ, Haque SFY, Potocnik AJ, Pennington DJ, Hayday AC. Interleukin 7 (IL-7) selectively promotes mouse and human IL-17–producing γδ cells. Pro Nat Acad Sci. 2012. 109: 17549–17554.
  42. 42. Riol-Blanco L, Lazarevic V, Awasthi A, Mitsdoerffer M, Wilson BS, Croxford A, et al. IL-23 Receptor Regulates Unconventional IL-17-Producing T Cells That Control Bacterial Infections. J Immunol. 2010. 184: 1710–1720. pmid:20083652
  43. 43. Cheng L, Cui Y, Shao H, Han G, Zhu L, Huang Y, et al. Mouse γδ T cells are capable of expressing MHC class II molecules, and of functioning as antigen-presenting cells. J Neuroimmunol. 2008. 203: 3–11. pmid:18774183
  44. 44. Zhao R, Liang D, Sun D. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU). PLoS ONE.2016. 11: e0155953. pmid:27196432
  45. 45. Hasko G, Cronstein BN. Adenosine: an endogenous regulator of innate immunity. Trend Immunol. 2004. 25: 33–39.
  46. 46. Csoka B, Himer L, Selmeczy Z, Vizi ES, Pacher P, Ledent C, et al. Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function. FASEB J.2008. 22: 3491–3499. pmid:18625677
  47. 47. Himer L, Csóka B, Selmeczy Z, Koscsó B, Pócza T, Pacher P, et al. Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death. The FASEB J. 2010. 24: 2631–2640. pmid:20371613
  48. 48. Odashima M, Bamias G, Rivera-Nieves J, Linden J, Nast CC, Moskaluk CA, et al. Activation of A2A Adenosine Receptor Attenuates Intestinal Inflammation in Animal Models of Inflammatory Bowel Disease. Gastroenterology. 2005. 129: 26–33. pmid:16012931
  49. 49. Mills JH, Kim DG, Krenz A, Chen JF, Bynoe MS. A2A adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis. J Immunol. 2012. 188: 5713–5722. pmid:22529293
  50. 50. Khoa ND, Montesinos MC, Reiss AB, Delano D, Awadallah N, Cronstein BN. Inflammatory Cytokines Regulate Function and Expression of Adenosine A2A Receptors in Human Monocytic THP-1 Cells. J Immunol. 2001. 167: 4026–4032. pmid:11564822
  51. 51. Hasko GY, KUHEL DG, Chen JF, SCHWARZSCHILD MA, Deitch EA, MABLEY JG, et al. Adenosine inhibits IL-12 and TNF-a production via adenosine A2a receptor-dependent and independent mechanisms. FASEB J.2000. 14: 2065–2074. pmid:11023991
  52. 52. Xie KP, Wang YF, Huang SY, Xu L, Bielenberg D, Salas T, et al. Nitric oxide-mediated apoptosis of K-1735 melanoma cells is associated with downregulation of Bcl-2. Oncogene.1997. 15: 771–779. pmid:9266963
  53. 53. Lappas CM, Sullivan GW, Linden J. Adenosine A2A agonists in development for the treatment of inflammation. Exp Opin Invest Drugs. 2005. 14: 797–806.
  54. 54. Eltzschig HK, Sitkovsky MV, Robson SC. Purinergic Signaling during Inflammation. N Eng J Med. 2012. 367: 2322–2333.
  55. 55. Blackburn M, Vance C, Morschl E, Wilson C (2009) Adenosine Receptors and Inflammation. In: Wilson CN, Mustafa SJ, editors. Adenosine Receptors in Health and Disease: Springer Berlin Heidelberg. pp. 215–269.
  56. 56. Bours MJL, Swennen ELR, Di Virgilio F, Cronstein BN, Dagnelie PC. Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol & Therap. 2006. 112: 358–404.
  57. 57. Eltzschig HK, Carmeliet P. Hypoxia and Inflammation. N.Eng.J.Med. 2011. 364: 656–665.
  58. 58. Junger WG. Immune cell regulation by autocrine purinergic signalling. Nat Rev Immunol. 2011. 11: 201–212. pmid:21331080