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The purine nucleoside adenosine is an important anti-inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor-mediated mechanisms. Invariant natural killer T (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN-γ production by iNKT cells.
We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR, and A3R, on mouse iNKT cells. We show that IL-4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL-4. These data are supported by A2aR-deficient mice, which exhibit largely decreased levels of IL-4, IL-10 and TGF-β concomitantly with an increase of IFN-γ upon α-GalCer administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL-4 and IL-10 by iNKT cells. These data suggest adenosine:A2aR-mediated mechanisms can control the cytokine secretion pattern of iNKT cells.
Adenosine is an endogenous purine nucleoside present at high concentrations in inflamed, hypoxic and malignant tissues . It is generated from ATP in intracellular and extracellular compartments and is involved in the regulation of a variety of different physiological processes like cell proliferation, vascular regulation and immune functions [2, 3]. To date, four different types of adenosine receptors (A1R, A2aR, A2bR, and A3R) have been described. A1R and A3R belong to the group of Gi-coupled proteins inhibiting adenylate cyclase-mediated production of cAMP. In contrast, A2aR and A2bR are Go/Gs-coupled receptors that raise intracellular levels of cAMP, with A2aR exhibiting a higher affinity for adenosine than A2bR [4, 5]. Adenosine exerts a variety of anti-inflammatory effects mediated by adenosine receptors, adenosine analogs have been proven to inhibit the TCR-mediated activation and cytokine production by T cells [6, 7]. CD8 T cells deficient for A2aR and A2bR conferred increased anti-tumor activity in vivo against B16F10 melanoma  suggesting that adenosine, by adenosine receptor-mediated mechanisms, effectively inhibits immune responses against tumors. Adenosine also inhibits the cell-mediated cytotoxicity of natural killer (NK) cells as well as the maturation and IL-12 production of dendritic cells (DC) [9, 10].
NKT cells represent a subpopulation of T lymphocytes defined by the coexpression of NK-associated molecules such as NK1.1 and the TCR. The majority of NKT cells, termed invariant (iNKT) NKT cells, express a semi-invariant TCR and can be further differentiated based on the expression of the surface molecule CD4 . iNKT cells recognize (glyco-)lipid antigens presented on the monomorphic MHC class I-like transmembrane molecule CD1d . The main function of iNKT cells is to regulate immune responses to either tolerance or inflammation, mainly exerted by secreting copious amounts of different cytokines (e.g. IL-2, IL-4, IL-10, IFN-γ)  upon activation. iNKT cells secrete IL-4 independent of CD40 costimulation whereas the production of IFN-γ by iNKT cells is dependent on CD40:CD40L pathway. The secretion of both cytokines requires costimulation delivered through the CD80/CD86:CD28 pathway . While the contribution of iNKT cells in different immune responses as regulators has been acknowledged, the exact mechanisms polarizing their effector functions are only poorly understood.
NKT cells and regulatory T (Treg) cells share the expression of the ecto-nucleotidases CD39 and CD73, which in two steps generate adenosine from ATP and ADP/AMP. The expression of both enzymes is required for the suppressive function of Tregs [15, 16]. Similarly, iNKT cells express CD73 and CD39. CD39-deficient iNKT cells failed to produce IL-4 upon CD1d-mediated activation , suggesting that endogenous adenosine modulates their cytokine production. Previously, iNKT cells in a mouse model of liver ischemia-reperfusion injury were shown to be sensitive to inhibition be adenosine . Comparable to other cell types, Lappas et al. describe the adenosine-mediated iNKT cell inhibition, as appreciated by a 50% reduction in production of the cytokine IFN-γ. Since the activation of iNKT cells was attributed to only IFN-γ secretion and no other cytokines were measured, it is questionable whether iNKT cells in this model were functionally inhibited by adenosine rather than their cytokine profile being skewed.
The aim of this study was to elucidate whether adenosine regulates the activation of iNKT cells. We expanded on previous studies suggesting that iNKT cells respond and are inhibited by adenosine  and analyzed whether these effects were cell-autonomous or due to adenosine-mediated DC inhibition. We found expression of all four types of adenosine receptors and provide evidence that the cytokine secretion pattern of iNKT cells is controlled by the A2a receptor, showing that production of type-2 cytokines by iNKT cells requires adenosine:A2aR-mediated interaction while adenosine inhibits the production of IFN-γ by iNKT cells.
Adenosine is an important negative regulator of inflammatory processes, and the functions of virtually all types of immune cells are suppressed by adenosine . To assess how adenosine regulates iNKT cells, we first analyzed the adenosine receptor mRNA expression on sorted mouse iNKT cells from spleen and liver (Fig. 1). To compare the expression levels of different genes and exclude differences caused by different amplification efficacies we normalized the expression on standard curves using known copy numbers. iNKT cells from liver and spleen express all four known subtypes of adenosine receptors. The high affinity Gi-protein coupled A2a receptor showed the highest expression in all tested iNKT populations. This is in accordance with previous studies where A2aR was shown to be the predominantly expressed subtype on T cells . We did not observe any significant differences in the expression of adenosine receptors between CD4+ and CD4− iNKT cells (Fig.1). Furthermore, our data are in accordance with previous studies demonstrating that unlike human CD4+ and CD4− iNKT cells where CD4+ iNKT cells preferentially secrete IL-4 , the presence of CD4 on murine iNKT cells is not linked to a cytokine bias .
The chemokine receptor expression pattern and memory phenotype [22, 23] suggests that iNKT cells mainly migrate and function in peripheral tissues that have been shown to harbor elevated concentrations of adenosine . We therefore asked whether the TCR-mediated activation and cytokine secretion of iNKT cells is sensitive to adenosine. iNKT cells were stimulated in the presence of the stable adenosine analogue CADO. Comparable to suppressive effects of CADO and related compounds on T cells, the CD1d-induced cytokine secretion of iNKT cells was substantially inhibited by CADO (Fig.2). These data show that like other lymphocyte populations, including NK cells, iNKT cells are sensitive to the immunosuppressive effects of adenosine.
Adenosine-related compounds cause the simultaneous engagement of Gs- and Gi-coupled adenosine receptors. We therefore asked whether ligation of the predominant high-affinity A2aR during TCR-mediated stimulation would modulate activation or effector functions, i.e. cytokine production, of iNKT cells. To exclude a lack of costimulatory molecules accounting for a lack of IFN-γ secretion, we next used BMDC, at day of culture typically consisting of both immature and mature cells. To exclude responses of the BMDC to A2aR modulation, cells were fixed upon α-GalCer–loading. Enriched iNKT cell preparations were thus stimulated in the presence of a specific A2aR agonist or antagonist.
Comparable to the effects of the stable adenosine analogue, the exposure to A2aR agonist CGS21680 during the stimulation period inhibited the production of IFN-γ by iNKT cells. In striking contrast, CGS21680 led to a significant increased in IL-4 production. Conversely, the A2aR antagonist ZM241485 inhibited the iNKT cell-mediated secretion of IL-4 and concomitantly increased the production of IFN-γ (Fig. 2B), markedly skewing the Th1/Th2 ratio of cytokines produced by iNKT cells towards IFN-γ. These data were corroborated by a similar analysis of a human iNKT cell line (Fig. 2C). The requirement of A2aR signaling for IL-4 production clearly is in opposition to the effects of A2aR ligation on conventional T cells, which are inhibited non-selectively . These data also provide an explanation for the phenomenon Beldi et al. recently described , in which iNKT cells lacking the ecto-enzyme CD39 and hence unable to generate adenosine, were not able to produce IL-4 upon CD1d mediated activation.
To determine the physiological in vivo significance of these findings, we asked whether iNKT cells in mice lacking the predominant A2aR would be functionally altered. We injected A2aR k.o. mice or WT mice with α-GalCer and tested the cytokine production 90 min and 5 h later, reflecting the time of appearance in serum. The production of IL-4 and IL-10 upon α-GalCer administration can be observed early after activation, whereas IFN-γ secretion by iNKT cells requires IL-12 produced by DC upon maturation and hence are detectable later after injection.
We detected increases in all four tested cytokines (IL-4, IL-10, TGF-β, and IFN-γ) in the serum of α-GalCer injected WT mice compared to uninjected mice (data not shown). Comparable with the in vitro results, iNKT cells in the absence of A2aR produced significantly lower levels of IL-4 upon α-GalCer injection (Fig.3A). The expression of another Th2 cytokine IL-10 was also markedly decreased in the A2aR k.o. mice. In marked contrast, but also comparable with the in vitro results, IFN-γ was increased in the A2aR-deficient mice. Taken together, these data suggest that adenosine via A2aR is enhancing iNKT cell secretion of Th2 cytokines. Interestingly, we also noted TGF-β secretion, which was lost in A2aR k.o. mice, suggesting that TGF-β may be produced by iNKT cells and enhanced through adenosine stimulating A2aR. However, TGF-β production has not been described in iNKT cells and could have been indirectly from other cells. We therefore activated sorted iNKT cells with plate-bound CD1d molecules and assessed their TGF-β production. As Fig.3B shows, iNKT cells directly produced TGF-β in the active form in response to CD1d-mediated activation.
To further confirm that the cytokines observed in sera were from NKT cells, we injected WT and A2aR k.o. mice with α-GalCer and tested NKT and NK cells for their intracellular cytokine content. NKT cells from A2aR k.o. mice produced significantly more IFN-γ compared to stimulated WT counterparts. Additionally, NK cells known to be transactivated by NKT cells produced significantly more IFN-γ in the absence of an A2aR (Fig.3C, bottom), however, no IL-4 could be detected in these cells (data not shown). Supporting the serum data (Fig. 3A), we observed a clear trend to a lower IL-4 production in A2aR−/− NKT cells, although not reaching statistical significance (n=3).
Collectively, our data suggest that the secretion of type-2 cytokines IL-4, IL-10 and TGF-β by iNKT cells requires signaling through the A2aR since blocking or genetic ablation of this receptor efficiently abrogates their secretion. In contrast, ligation of the same receptor abrogates the production of IFN-γ. Pharmacological ligation of the high-affinity A2aR might reflect the situation in vivo with low adenosine concentrations skewing the cytokine production of iNKT cells towards a Th2-type phenotype. Increased levels of adenosine, such as found in tumors might then additionally ligate the low-affinity A2bR and thus inhibit the activation of iNKT cells, comparable to other cell types.
Conceivably, the manipulation of the A2aR on iNKT cells might control their activation and support host defense and immunotherapeutic approaches in both malignancy and tolerance.
C57BL/6J were purchased from Jackson Laboratories (Bar Harbor, MA). Mice deficient the A2aR were previously described and backcrossed to C57BL/6 background . Mice were housed under specific pathogen-free conditions. Animal experiments were performed in accordance to protocols approved by Institutional Animal Care and Use Committee. Six- to 8-wk-old C57BL/6J mice were used for experiments.
PBS57-loaded or empty CD1d monomers and tetramers were provided by the NIH tetramer facility (Emory Vaccine Center, Atlanta, GA). CADO, CGS21680, and ZM241485 were purchased from Tocris (Ellisville, MO). Cells were cultured in RPMI-1640 supplemented with Penicillin, Streptomycin (Mediatech, Manassas, VA), and 5% FBS (Hyclone, Logan, UT).
DC were generated from mouse BM in the presence of GM-CSF as described in  with modifications. Briefly, BM cells were cultured in complete medium supplemented with 5% supernatants of GMCSF-producing B16F10 (kind gift of Dr. G. Dranoff, Dana-Farber Cancer Institute, Boston, MA), replaced every other day. On day 6 BMDC were detached with enzyme-free digestion buffer (Sigma-Aldrich, St. Louis, MO). BMDC pulsed with α-GalCer (200 ng/ml, Kirin Ltd.) or vehicle (Tween-20) in medium for 3 h at 37°C. BMDC were subsequently washed with PBS, fixed with 0.02% Glutaraldehyde (Sigma-Aldrich) for 1 min. before used in experiments.
Single cell suspensions from spleens were prepared by standard techniques. Liver MNC were isolated as previously described  without prior Collagenase digestion. Briefly, livers were perfused with PBS, minced and iNKT cells were enriched by centrifugation in a two-step Percoll gradient. Enriched populations typically contained 20-30% iNKT cells.
Human iNKT cell lines were established by sorting PBMC with iNKT-mAb 6B11 and expanding with mitogen as described . Lines were maintained by periodic re-stimulations and purity checked with Vα24 mAb .
iNKT cells from livers were stimulated in the presence of either plate-bound PBS57-loaded CD1d monomers or α-GalCer-pulsed and Glutaraldehyde-fixed BMDC. PBS57-loaded CD1d monomers were plate-bound overnight in PBS at 4°C, blocked and washed with complete culture medium before cells were added.
Cytokine-specific ELISA assays (eBioscience, San Diego, CA) were performed following the manufacturers instructions. Sera were diluted 1:10 in PBS/1% BSA.
RNA isolations using TRIzol (Invitrogen, Carlsbad, CA) and RT reactions were performed as described . Real-time PCR using 1/20 volume of reverse transcription reactions and primers specific for adenosine receptors A1R (F, 5′- CATTGGGCCACAGACCTACT-3′, R, 5′- CAAGGGAGAGAATCCAGCAG-3′), A2aR (F, 5′- CACGCAGAGTTCCATCTTCA-3′, R, 5′- ATGGGTACCACGTCCTCAAA-3′), A2b (F, 5′- TGCTCACACAGAGCTCCATC-3′ R, 5′- AGTCAATCCAATGCCAAAGG-3′), A3R (F 5′-GCTGATCTTCACCCATGCTT-3′, R, 5′- ATCCAAACTGACCACGGAAC-3′), and GAPDH (F, 5′- AACTTTGGCATTGT-3′, 5′-ACACATTTGGGGGTA-3′) were performed using Quantitect SYBR Green in a Corbett (Qiagen, Valencia, CA). Target gene expression was normalized against levels of GAPDH and normalized against standards with known copy numbers (102-105/reaction) of adenosine receptors.
Subsequent to blocking with anti-CD16/32 mAbs cells were stained with CD3-FITC, NK1.1-PE and CD1d tetramer-APC. NKT cells were gated as CD3+NK1.1+CD1d-tetramer+ and sorted to purities >95% using a FACSAria (all BD Biosciences, San Jose, CA). Intracellular stainings for IL-4 and IFN-γ were performed using Cytofix/cytoperm (BD Biosciences) according to manufacturers instructions.
Results are expressed mean ± SD. For statistical analyses, the One-way-ANOVA with Newman-Keuls post-test was used. Values of p<0.05 were considered as significant.
We thank Bettina Jux for critically reading this manuscript and fruitful discussions, and the BIDMC flow cytometry core facility for their technical support. This work was supported by National Institutes of Health grants NIH R01 DK 066917 and a Dana-Farber/Harvard Cancer Center Prostate Cancer SPORE P50CA090381 Development Award (MAE).
Conflict of interest
The authors declare no conflicts of interest.