Adenosine A2A Receptor Agonist −Mediated Increase in Donor-Derived Regulatory T Cells Suppresses Development of Graft-versus-Host Disease This information is current as of September 28, 2021. Kyu Lee Han, Stephenie V. M. Thomas, Sherry M. Koontz, Cattlena M. Changpriroa, Seung-Kwon Ha, Harry L. Malech and Elizabeth M. Kang J Immunol 2013; 190:458-468; Prepublished online 7 December 2012; Downloaded from doi: 10.4049/jimmunol.1201325 http://www.jimmunol.org/content/190/1/458 http://www.jimmunol.org/ References This article cites 52 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/190/1/458.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Adenosine A2A Receptor Agonist–Mediated Increase in Donor-Derived Regulatory T Cells Suppresses Development of Graft-versus-Host Disease Kyu Lee Han,* Stephenie V. M. Thomas,* Sherry M. Koontz,* Cattlena M. Changpriroa,* Seung-Kwon Ha,† Harry L. Malech,* and Elizabeth M. Kang* Graft-versus-host disease (GVHD) remains a significant complication of allogeneic transplantation. We previously reported that the adenosine A2A receptor (A2AR) specific agonist, ATL146e, decreases the incidence and severity of GVHD in a mouse transplant model. There is increasing interest in treatments that increase CD4+CD25highFoxp3+ regulatory T cells (Tregs) to suppress GVHD. Our current study found in vitro that A2AR selective agonists enhanced TGF-b–induced generation of mouse Tregs 2.3- to 3-fold. We demonstrated in vivo suppression of GVHD with specific A2AR agonists in two different murine GVHD transplant models Downloaded from associated with profound increases in both circulating and target tissue Tregs of donor origin. Three different A2AR agonists of differing potency, ATL146e, ATL370, and ATL1223, all significantly inhibited GVHD-associated weight loss and mortality. At the same time, Tregs shown to be of donor origin increased 5.1- to 7.4-fold in spleen, 2.7- to 4.6-fold in peripheral blood, 2.3- to 4.7-fold in colon, and 3.8- to 4.6-fold in skin. We conclude that specific activation of A2AR inhibits acute GVHD through an increase of donor-derived Tregs. Furthermore, the increased presence of Tregs in target tissues (colon and skin) of A2AR-specific agonist- treated mice is likely the mechanistic basis for the anti-inflammatory effect preventing acute GVHD. The Journal of Immunology, http://www.jimmunol.org/ 2013, 190: 458–468. raft-versus-host disease (GVHD) continues to be a sig- cAMP-elevating agents are known to induce alloreactive T cell nificant cause of morbidity and mortality after allogeneic tolerance and prevent GVHD lethality in murine models (10). G hematopoietic stem cell transplantation (1). Targeted Because the activation of the Gs-coupled adenosine A2A receptor methods to prevent or treat GVHD are in high demand. Regulatory (A2AR) appears to terminate inflammation through the regulation T cells (Tregs) are a subset of CD4+CD25high T cells that express of cells that mediate both innate and adaptive immunity, it is a the FOXP3 and have been shown to suppress the proliferation of promising pharmacological target for the treatment of GVHD by guest on September 28, 2021 T conventional cells and help promote tolerance (2, 3). There is (11). The selective activation of the A2AR has been shown to increasing recognition that Tregs are important in preventing the potently limit inflammation and injury in various inflammatory development, reducing the severity, and/or mediating the resolu- disease models, and the data suggest a central role for this receptor tion of GVHD (4, 5). It has been reported that donor Treg infusions involves a feedback mechanism that inhibits the inflammation as- will suppress the development of GVHD in a mouse model (6–8) sociated with activation of either innate or acquired immunity. A2AR and that the number of Tregs in the peripheral blood and affected agonists have significant protective effects in multiple models of tissues is decreased during the development of acute GVHD in ischemia–reperfusion injury and inhibit the progression of inflam- humans (9). It is likely that Tregs act to modulate immune matory bowel disease and reduce joint destruction because of responses at anatomic sites of GVHD inflammation but also may septic arthrosis (12–16). Furthermore, it has been shown that the act to modulate immunity in central and peripheral lymphoid anti-inflammatory effects of methotrexate are mediated in part via organs as well as in peripheral blood. the induction of adenine nucleotide release from injured tissue and the subsequent activation of A2ARs on local immune cells (17, 18). Notably, T cell tolerance by T cell anergy can also be induced by selective A2AR agonist treatment (19). *Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, We have previously shown that treatment with the specific A2AR National Institutes of Health, Bethesda, MD 20892; and †Life Science Research and Development Center, SK Chemicals, Inc., Sungnam-Si, Gyeonggi-Do 463-410, Re- agonist, ATL146e, decreases the incidence and severity of GVHD public of Korea as well as improves survival of mice in a GVHD transplant model Received for publication May 10, 2012. Accepted for publication October 31, 2012. (20). However, the mechanism of action of ATL146e mediating This work was supported by intramural funds from the National Institute of Allergy this reduction of GVHD mortality was not clearly determined. and Infectious Diseases. There had been a few reports exploring the relationship between K.L.H., S.V.M.T., S.M.K., S.-K.H., and C.M.C. performed experiments; K.L.H., Tregs and A2AR (15, 21–23), but there was no prior evidence to S.-K.H., and S.V.M.T. collected and analyzed data; K.L.H., H.L.M., and E.M.K. show that activation of A R could actually induce immunosup- designed the research; and K.L.H., E.M.K., and H.L.M. wrote the manuscript. 2A pressive Tregs in the setting of GVHD. In our current study, we Address correspondence and reprint requests to Dr. Elizabeth M. Kang, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National In- found that selective A2AR agonists potently enhanced the TGF-b– stitutes of Health, Building 10, Room 6-3752, 10 Center Drive, MSC-1456, Bethesda, induced generation of mouse Tregs in vitro. In vivo in two GVHD MD 20892-1456. E-mail address: [email protected] mouse transplant models, treatment with selective A2AR agonists + high + Abbreviations used in this article: A2AR, A2A receptor; B6, C57BL/6J; B6-D2, a greatly increased the number of CD4 CD25 Foxp3 Tregs in B6D2F1/J; BM, bone marrow; B6-Thy1.1, B6.PL-Thy1 /CyJ; CsA, cyclosporine A; GVHD, graft-versus-host disease; mTOR, mammalian target of rapamycin; peripheral blood and lymphatic tissue such as the spleen, as well TCD, T cell–depleted; Treg, regulatory T cell. as locally in tissues (skin and colon) that are the target of GVHD www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201325 The Journal of Immunology 459 in these models. Our findings in vitro and in vivo strongly link the permeabilization using a PE-conjugated anti-mouse Foxp3 (FJK-16s) Ab (eBioscience). Control samples were labeled with isotype-matched control action of selective A2AR agonists to the induction of Tregs that act to reduce the development of GVHD in target tissues. Abs. The samples were run on a benchtop flow cytometer (FACSort; BD Biosciences, San Diego, CA) with a minimum of 10,000 events collected. Analysis was performed with Cell Quest (Tree Star, Ashland, OR). Materials and Methods Measurement of serum cytokines Mice Cytokine concentrations in serum samples were measured using mouse For these studies, five mouse strains (with relevant H2 major histocom- cytokine/chemokine multiplex immunoassay kits (Linco, St. Charles, MO) patibility type indicated) were purchased from The Jackson Laboratory (Bar with the BioPlex system (Bio-Rad, Hercules, CA) using Luminex tech- Harbor, ME): C57BL/6J (H2-Kb); B6.PL-Thy1a/CyJ (congenic to C57BL/ b d nology. Serum levels of TGF-b were measured by ELISA (R&D Systems, 6J carrying the Thy 1.1 allele [H2-K ]); BALB/c (H2-K ); B6D2F1/J (F1 Minneapolis, MN). hybrid cross between C57BL/6J female 3 DBA/2J male [H2-Kb/d]); and B6. Cg-Foxp3sf/J (Foxp3 deficient scurfy on C57BL/6J background [H2-Kb]). Histopathological analysis and immunohistochemistry The National Institute of Allergy and Infectious Disease Animal Care and Use Committee (Bethesda, MD) approved all animal studies (Institutional Skin and colon were harvested from euthanized mice and fixed in 4% Animal Care and Use Committee–approved protocol LHD-3E). paraformaldehyde in PBS (pH 7.4) and embedded in paraffin. Sections (4 mm) were subjected to standard H&E staining and immunochemical Specific A2AR agonists staining. Each section was then scored separately by a board-certified toxicologic pathologist, blinded to animal status, according to the pub- The highly specific A2AR agonists ATL146e, ATL370, and ATL1223 were lished criteria (25–27).