Adenine, and by extension adenosine, may have been one of the oldest organic compounds on the earth whose appearance preceded the first life form by many 100 million years according to interpretation of life origin experiments (Miller, 1953
; Miller and Urey, 1959
; Oró and Kimball, 1961
). Maybe utilization of adenosine belongs to the oldest and most ancient group of mechanisms regulating immune system in organisms.
Immune cells express A2AR at high levels, and stimulation of A2AR strongly suppresses various immune functions (Ohta and Sitkovsky, 2001
; Lukashev et al., 2004
; Sitkovsky et al., 2004
; Thiel et al., 2005
; Belikoff et al., 2011
). Interaction of A2AR with endogenously produced adenosine serves as a self-limiting mechanism of inflammation. Immunosuppression through the adenosine-A2AR pathway seems to be critical in maintaining inflammation to proper levels. This is because the lack of A2AR led to exaggeration of proinflammatory responses and subsequent inflammatory tissue damage (Ohta and Sitkovsky, 2001
; Thiel et al., 2005
; Ohta et al., 2006
Activation of CD4+
T cells is under control of the adenosine-A2AR pathway. Our previous results have shown A2AR-mediated inhibition of T cell activities such as proliferation, cytokines production and cytotoxicity (Huang et al., 1997
; Ohta et al., 2009
). This inhibitory effect of adenosine and its analogs is based on a direct action via A2AR expressed on T cells. It is consistent with the interruption of T cell receptor signaling by cAMP increase after A2AR stimulation (Vang et al., 2001
; Linnemann et al., 2009
). In addition to the inhibitory effect on T cell priming, A2AR stimulation produced activated T cells with impaired effector function. Indeed, T cells activated in the presence of A2AR agonist showed persistently lower cytokine-producing activity even after the removal of A2AR agonist (Zarek et al., 2008
; Ohta et al., 2009
In our studies on CTL development, we noticed the uniqueness of MLC, where massive expansion of Teff and increase of Treg were observed in the same culture. This may mimic immune responses in vivo: i.e., promotion of proinflammatory activities and compensatory anti-inflammatory responses to prevent excessive tissue destruction. Such responses were not observed in artificial stimulation of T cells with anti-CD3 mAb as Teff strongly overwhelm the culture. The current study revealed that A2AR stimulation inhibited activation of effector T cells and, at the same time, increased the number of Treg (Figures , , and ). CD4+ FoxP3+ cells from the MLC system express CD25, CTLA-4, CD39, and CD73 at high levels and have immunoregulatory activities as it has known for Treg induced by other methods (Figures –). Our data suggests that A2AR not only directly inhibits T cell activation but also produces immunosuppressive cellular environment by inducing massive increase of Treg. Therefore, immunosuppressive concentration of extracellular adenosine may provide a long-lasting immunoregulatory effect even after the disappearance of adenosine.
The A2AR-mediated numerical change of Treg was due to increase of both nTreg and CD4+
cell-derived Treg. MLC after the depletion of nTreg showed development of Treg from CD4+
cells and its enhancement by A2AR stimulation (Figure ). The increase of CD4+
cell-derived Treg in normal MLC, in the presence of nTreg, required distinction for the origin of CD4+
cells in the product. Reconstitution of MLC responders using Thy1.1-expressing nTreg made this distinction possible, and the A2AR-mediated increase of CD4+
cell-derived Treg was confirmed in regular MLC (Figure ). The increase of new Treg is consistent with a previous paper reporting mRNA upregulation of FoxP3 and LAG-3 in CGS-treated T cell culture (Zarek et al., 2008
). Although the increase of Treg from CD4+
cells was significant, these Treg accounted for only a minor portion of A2AR-mediated increase of Treg. Most of Treg after MLC was found to derive from nTreg (Figure ). Purified nTreg was reported to proliferate when cultured with allogenic dendritic cells and IL-2 (Yamazaki et al., 2006
). IL-2 was not added in our culture system, but activated CD4+
cells might have produced IL-2 and supported proliferation of Treg. Indeed, massive proliferation of nTreg was observed in the current study (Figure ). Other possible reasons for A2AR-mediated enhancement of nTreg include prevention of FoxP3 down-regulation and cell death, which have been observed during Treg culture (Strauss et al., 2007
; Hoffmann et al., 2009
). In the current study, a number of nTreg were found to lose their FoxP3 expression after activation, while A2AR stimulation considerably prevented FoxP3 loss (Figure ). This mechanism may be partly responsible for the increase of CD4+
cells treated with A2AR agonists. In addition, A2AR agonists are known to block activation-induced cell death of CD4+
T cells (Himer et al., 2010
Not only A2AR agonist increased the number of Treg, it also enhanced their immunoregulatory activity. T cell suppression assay showed that the same number of Treg from CGS/NECA-treated MLC had 4-times stronger regulatory activity than that from control MLC (Figure ). The mechanism how Treg suppress T cell activation is still unclear, but CTLA-4 and CD25 represent important candidates. Pathogenesis of autoimmune disorders in mice with Treg-specific CTLA-4 deficiency demonstrated the importance of CTLA-4 in their regulatory activity (Wing et al., 2008
; Sakaguchi et al., 2009
). CD25 expression on Treg at high levels is considered to withdraw IL-2 from the microenvironment and induce effector cell death because of IL-2 deprivation (Pandiyan et al., 2007
). The A2AR-mediated upregulation of CTLA-4 and CD25 on Treg (Figures –) may support the enhancement of regulatory activity.
We assumed that it would be very effective immunosuppression if the activities of Treg were additive to or synergistic with other immunosuppressive mechanisms in the microenvironment. Accordingly, it was hypothesized that the immunosuppressive cytokines and molecules such as CTLA-4 would be increased on Treg by the same mechanism that mediates the hypoxia-adenosinergic immunosuppression (Sitkovsky, 2009
). The detailed studies of hypoxia response element (HRE)/hypoxia-inducible factor-1α (HIF-1α) and cAMP response element (CRE)/cAMP response element binding protein (CREB) have been implicated in Treg activities only by circumstantial set of data (Sitkovsky, 2009
) and the direct studies of CRE and HRE in Treg are still to be performed.
Tumors contain high levels of extracellular adenosine (Blay et al., 1997
; Ohta et al., 2006
). Adenosine in tumor microenvironment may be one of the important immunosuppressive mechanisms, which discourage anti-tumor immune responses, because A2AR-deficient mice could efficiently eradicate tumors, while wild-type mice could not (Ohta et al., 2006
). One important reason for adenosine-mediated inactivation of anti-tumor immune responses would be a direct action at A2AR on T cells. However, the present data also suggest that the adenosine-A2AR signaling may enhance Treg in tumors. Tumors contain overwhelming number of Treg to suppress effector T cells (Turk et al., 2004
; Antony et al., 2005
; Sitkovsky et al., 2008
; Facciabene et al., 2011
). There might be a contribution from intratumoral high concentration of adenosine to the increase of Treg. Moreover, A2AR stimulation may enhance the regulatory activity of Treg in tumors and further inactivate anti-tumor immune responses. Physiological control of Treg activity in vivo
by extracellular adenosine is yet to be determined.
While Treg is a target to be discouraged for the improvement of tumor immunotherapy, transfusion of Treg is a promising approach for the treatment of autoimmune diseases and allogenic reaction after transplantation (Riley et al., 2009
; Matsuoka et al., 2010
). Because large doses of Treg are necessary to suppress GVHD, Treg require massive expansion ex vivo
before transfer, but the expansion of Treg is somewhat challenging. It is difficult to start the expansion from a large number of Treg because of low frequency of Treg in peripheral blood. In addition, since both Treg and activated effector T cells share CD4+
phenotype, polyclonal activation of T cell could result in considerable contamination by effector T cells (Riley et al., 2009
). Treatment with A2AR agonist induces expansion of Treg, while it suppresses activation of effector T cells. Such a culture condition favoring Treg outgrowth may be suitable for the expansion of Treg. Effects of A2AR agonists on human Treg will need to be examined for numerical increase and enhancement of regulatory activity. Dependent on the nature of human Treg, optimization of culture condition is expected to improve the recovery of functionally enhanced Treg.
In conclusion, we found that T cell activation in the presence of A2AR agonist resulted in expansion of Treg. After the A2AR-mediated expansion, Treg acquired stronger immunoregulatory activity. The quantitative and qualitative enhancement of Treg by the adenosine-A2AR pathway may be relevant to the establishment of longer-lasting immunomodulation. This mechanism may be utilized in the expansion of Treg for treatment of autoimmune diseases and GVHD.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.