Adenosine is an endogenous purine nucleoside present at high concentrations in inflamed, hypoxic and malignant tissues [1
]. 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
]. 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
-coupled receptors that raise intracellular levels of cAMP, with A2aR exhibiting a higher affinity for adenosine than A2bR [4
]. 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
]. CD8 T cells deficient for A2aR and A2bR conferred increased anti-tumor activity in vivo
against B16F10 melanoma [8
] 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
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 [11
]. iNKT cells recognize (glyco-)lipid antigens presented on the monomorphic MHC class I-like transmembrane molecule CD1d [12
]. 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) [13
] 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 [14
]. 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
]. Similarly, iNKT cells express CD73 and CD39. CD39-deficient iNKT cells failed to produce IL-4 upon CD1d-mediated activation [17
], 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 [18
]. 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 [18
] 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.