Activation of the Adenosine A_2A Receptor Promotes the Development of Donor-Derived T Regulatory Cells in a Graft-Versus Host Disease Mouse Model.

Graft versus host disease (GVHD) remains a significant complication of allogenic stem cell transplantation and is a considerable cause of transplant morbidity and mortality. The recognition of the role of Foxp3⁺ regulatory T cells in immunomodulation has given rise to interest in using these cells to abrogate or modify the severity of graft versus host disease as well as in methods to increase their development during transplant. A less well known avenue of research is the targeting of the adenosine A_2A receptor (A_2AR). In ischemia models, activation of the Gs-coupled adenosine receptors play a role in terminating inflammation and improving survival of damaged and/or transplanted organs by directly down regulating the activity of the receptor bound T cell. We have shown previously that the use of a specific A_2AR agonist known as ATL146e decreases the incidence and severity of GVHD as well as improves survival of mice in a GVHD transplant model (paper in submission).

In order to further understand the role of the agonist in GVHD abrogation we performed studies looking at the possible role of T regulatory cells in relation to the use of the agonist. Using a parental into irradiated F1 offspring transplant model (C57BL/6J [B6, H-2^b] → B6D2F1/J [BDF1, H-2^b/d]) we can induce GVHD manifested by weight loss and mortality in 100% of mice by infusing an additional 10 million donor T cells into mice previously engrafted with 10 million bone marrow donor cells and 800cGy of radiation. We administered the ATL146e or a PBS control by osmotic mini pumps resulting in continuous subcutaneous infusion for 14days starting one day before the donor T cell infusion. Mice that received only the congenic donor bone marrow transplant and no donor T cells did not develop GVHD and served as an additional control. Post transplant, splenocytes and peripheral blood cells were collected and stained for CD4, CD25 and FoxP3 and were analyzed by flow cytometry. The level of Foxp3 mRNA expression in lymphocytes was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and the concentration of IL-10 in serum was measured by enzyme-linked immunosorbent assay (ELISA). To identify the origin of activated T regulatory cells, we also performed transplants used a B6.PL-Thy1/CyJ (B6-Thy1.1, H-2^b) mouse strain as the donor.

From these studies we confirmed that the A_2AR agonist, ATL146e, inhibited the weight loss and mortality associated with acute GVHD progression and seen in the PBS treated controls. More notably, treatment with ATL146e resulted in a 7 fold increase in CD4⁺CD25⁺FoxP3⁺ T regulatory cells in both the spleen and peripheral blood compared to our PBS treated group at days 14 to 20 after hematopoietic stem cell transplantation. From our Thy-1 disparate transplants we determined that the increased T regulatory cells were of donor origin. We also found that the expression of Foxp3 mRNA in splenocytes and the level of IL-10 in the serum was increased 3 fold and 2.4 fold respectively in the ATL146e treated mice. ATL146e agonist activity is very specific to A_2AR. When using an alternate adenosine agonist less specific to A_2AR, we did not see the same increases of Foxp3 or IL-10, nor did we see any decrease in the severity of the graft versus host disease.

Thus we believe that the specific activation of A_2AR inhibits acute GVHD through the increase of donor-derived CD4⁺ CD25⁺ FoxP3⁺ immunosuppressive T regulatory cells. Our observation provides an additional mechanistic basis for the anti-inflammatory capacity of A_2AR agonist in acute GVHD. Additional studies are ongoing to elucidate further the mechanism of the agonist's ability to increase the T regulatory population as well as the effects of combination therapies.

No relevant conflicts of interest to declare.