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Adenosine A2A Agonist −Mediated Increase in Donor-Derived Regulatory T Cells Suppresses Development of Graft-versus- 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

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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 , 2.3- to 4.7-fold in colon, and 3.8- to 4.6-fold in . 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 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 , 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 , 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, . 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 -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 Mice concentrations in serum samples were measured using mouse For these studies, five mouse strains (with relevant H2 major histocom- cytokine/chemokine multiplex 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). Briefly, liver was scored according to bile duct gifts from Forest Laboratories (New York, NY) (or their fully owned damage, presence of apoptotic bodies, damaged biliary epithelium with subsidiaries) under material transfer agreements. ATL146e (apadenoson; lymphocytic infiltrate, and cell debris in both hepatocytes and Kupffer Downloaded from Stedivaze), the most potent of the three agonists used in our murine studies, cells. Skin was scored by changes in the s.c. fat, an influx of and is in clinical development by Forest Laboratories as a coronary vasodilator mononuclear cells, and increased skin thickening with hyperkeratosis and for use in nuclear single-photon emission computed topography myocar- epidermal hyperplasia. Colon was scored by presence of apoptotic bodies dial perfusion imaging. in the crypt epithelium, damage, and reactive epithelial changes as well as + Murine transplant models used for the study of GVHD in the lumen of the central crypt. Immunostaining for Foxp3 Tregs was performed using an anti-Foxp3 (150D/D4) primary Ab (Santa induction Cruz Biotechnology, Santa Cruz, CA). Cell counts were performed over http://www.jimmunol.org/ Exclusively female mice, 8–12 wk old, were used both as transplant re- five areas chosen randomly in at least three different tissue sections each cipients and as sources of donor hematopoietic stem cells in the form of from a minimum of three mice and analyzed in a blinded manner. + CD3 T cell–depleted bone marrow (TCD-BM) and donor spleen-derived Preparation of from colonic lamina propria or purified CD3+ T lymphocytes. Bone marrow cells were collected from the femurs and tibia of donors and depleted of T cells as previous described skin (24) to yield TCD-BM. Purified T cells were obtained from the spleens + To isolate infiltrating lymphocytes in the skin, 100 mg of skin was pooled, of donors using mouse CD3 T cell enrichment columns (R&D Systems, minced, and then digested with 2.5 mg/ml type I collagenase (Sigma- Minneapolis, MN), resulting in a T cell product of 93–97% purity. Aldrich) and 0.25 mg/ml hyaluronidase (Sigma-Aldrich, St.Louis. MO) Two transplant recipient models of murine GVHD were studied: C57BL/ a for 2 h at 37˚C (28). Colonic lamina propria lymphocytes were isolated as 6J (B6) or B6.PL-Thy1 /CyJ (B6-Thy1.1) donor cells transplanted into described previously (29). Briefly, 100 mg of colonic tissue was washed in

b b/d by guest on September 28, 2021 B6D2F1/J (B6-D2) recipients (H2-K into H2-K model); or B6 donor medium (RPMI 1640 supplemented with and streptomycin), cells transplanted into BALB/C recipients (H2-Kb into H2-Kd model). sf then minced. The specimen was then digested in RPMI 1640 medium For some experiments, B6.Cg-Foxp3 /J (B6-Foxp3–deficient) donor containing 2% FBS and 1 mg/ml type IV collagenase (Sigma-Aldrich, St. cells were transplanted into B6-D2 recipients (Foxp3-deficient H2-Kb into b/d Louis. MO) for 2 h at 37˚C with 5% CO2. A single-cell suspension was H2-K ). obtained by passing the digested tissue through a cell strainer. Absolute Transplants were performed by conditioning recipients with 8.5 cGy for cell number was determined as described previously (29). the B6-D2 transplant recipients or with 6.5 cGy for the BALB/C trans- plant recipients. Twenty-four hours later, mice were transplanted by RNA extraction and real-time RT-PCR for Foxp3 lateral tail vein infusion with donor TCD-BM (10 3 106 cells if B6-D2 was the recipient and 5 3 106 cells if BALB/C was the recipient). In- Total RNA from colon and skin was isolated using TRIzol (Invitrogen, duction of GVHD was achieved by also infusing purified T cells (10 3 Grand Island, NY). cDNA was synthesized from 500 ng total RNA using 106 cells for B6-D2 recipients and 5 3 106 cells for BALB/C recipients). Maxima First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD) Transplanted B6-D2 controls that received donor TCD-BM but not pu- according to the manufacturer’s recommendation. For Foxp3 and GAPDH rified T cells served as one type of non-GVHD control. A second control transcripts, real-time PCR was performed with a 7500 Real-Time PCR used B6-D2 mice transplanted with B6-D2 T replete marrow (referred to system (Applied Biosystems, Foster City, CA) based on specific primers as BM only). and general fluorescence detection with SYBR Green. The following Unless alternatively specified in the results, primed Alzet osmotic primer combinations were used: mouse Foxp3, forward 59-AGGAGCCG- minipumps (Durect, Cupertino, CA) were implanted s.c. 24 h before ir- CAAGCTAAAAGC-39 and reverse 59-TGCCTTCGTGCCCACTGT-39; radiation for a constant rate delivery of 10 ng/kg/min ATL146e, 50 ng/kg/ and mouse GAPDH,forward59-CTCATGACCACAGTCCATGC-39 and min ATL370, 200 ng/kg/min ATL1223, or vehicle for 14 d. These specified reverse, 59- CACATTGGGGGTAGGAACAC-39. All PCRs were performed using Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Glen doses of the different A2AR-specific agonists were determined from pre- liminary dose finding experiments used to delineate doses with maximal Burnie, MD). Relative gene expressions of Foxp3 in skin or colonic benefit and minimal . Weights were monitored daily, and ani- tissues were normalized to GAPDH. mals were sacrificed when total weight loss exceeded 25% of their starting + 2 body weight. Generation of Tregs in vitro by culture of CD4 CD25 T cells with TGF-b Flow cytometry for Tregs CD4+CD252 T cells were isolated from single cell suspensions of spleens Peripheral blood was collected from the tail vein or when euthanized, via from naive B6 mice using components of the Miltenyi CD4+CD25+ cardiac puncture. Spleens were also collected from euthanized mice. Finely Regulatory T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) to first minced spleen cell suspensions were passed through a 40-mm nylon cell positively select for CD4+ T cells and then negatively select the purified strainer (BD Biosciences, San Jose, CA) and collected in PBS. RBCs were CD4+ T cells to isolate the CD25 negative subset following manufacturer’s removed with ACK lysis buffer (Quality Biological, Gaithersburg, MD). instructions. The purified CD4+CD252 T cells were cultured in a 24-well Cells were washed and resuspended at 5 3 106 cells/ml in PBS with 0.2% plate (3 3 105 cells/well), stimulated with anti-CD3 and anti-CD28 Ab BSA (Sigma-Aldrich, St. Louis, MO). Aliquots (0.1 ml) were then placed Dynabeads (Invitrogen, Carlsbad, CA) for 3 d in the presence of TGF-b (2 at 4˚C and labeled for 30 min in the dark with fluorochrome-labeled anti- ng/ml) (PeproTech, Rocky Hill, NJ), without or with the addition of mouse CD4 (RM4-5) and anti-mouse CD25 (PC61.5) (eBioscience, San rapamycin (10 nM) (LC Laboratories, Woburn, MA), cyclosporine A Diego, CA). Foxp3 was stained by intracellular analysis after fixation and (CsA, 10 nM) (LC Laboratories, Woburn, MA), the A2AR antagonist 460 A2AR AGONISTS INCREASE Tregs

ZM241385 at 1 mM (Sigma Aldrich), or one of the selective A2AR agonists (ATL370 or ATL1223, 10 nM). The experiment was repeated indepen- dently three times. Statistics Prism software (GraphPad Software, La Jolla, CA) was used for all statistical analyses. Student t tests or one-way ANOVA with post hoc Dunnett’s mul- tiple comparison was used to compare experimental groups with a control group. Histopathological semiquantitative scoring was analyzed using non- parametric Kruskal–Wallis test and Dunn’s multiple comparison test.

Results Selective A2AR agonists augment in vitro TGF-b–stimulated differentiation of Foxp3+ T cells from naive CD4+CD252 T cells CD4+CD252 T cells purified from B6 spleens were stimulated in culture with anti-CD3 and anti-CD28 Ab Dynabeads together with TGF-b, in the absence or presence of the selective A2AR agonists ATL370 or ATL1223 for 3 d. Initially, ,0.1% of the CD4+CD252 T cells were Foxp3 positive. Controls substituted rapamycin Downloaded from or CsA for the selective A2AR agonists. Other controls omitted TGF-b from the culture. At 3 d with TGF-b alone, the proportion of CD4+Foxp3+ T cells increased to 4.9 6 0.3% compared with 0.47 6 0.7% for the control cultures without TGF-b (Fig. 1A, upper group [representative experiment], and Fig. 1C [average of three independent experiments]). The addition of rapamycin (known http://www.jimmunol.org/ to enhance generation of Tregs (30)) enhanced the TGF-b–stimu- lated generation of CD4+Foxp3+ Tcellsto9.76 2.5%. In contrast, addition of CsA (known to interfere with generation of Tregs (30)) inhibited the TGF-b–stimulated generation of CD4+Foxp3+ Tcells to 0.3 6 0.2% (Fig. 1C). The addition of the selective A2AR agonists ATL370 or ATL1223 enhanced the TGF-b–stimulated generation of CD4+ Foxp3+ T cells to 14.5 6 1.0 or 11.5 6 1.3%, respectively (Fig. 1A, lower group, and Fig. 1C, block bar). Thus, when used at by guest on September 28, 2021 equimolar concentrations (10 nM), the two selective A2AR ago- nists, ATL370 or ATL1223, were more effective than rapamycin at enhancing TGF-b–stimulated generation of CD4+Foxp3+ T cells in vitro, increasing generation by 2.3- to 3-fold over TGF-b alone (p = 0.0009 and p = 0.0257 comparing TGF-b alone versus with ATL370 or ATL1223, respectively, over three experiments). This observation in vitro suggested that selective A2AR agonists could act in vivo to directly increase Treg generation as a mechanism to reduce development of GVHD. In addition, use of the A2AR antagonist (ZM 241385) 24 h before the addition of the selective A2AR agonists was used to further confirm that the increase of the T regulatory population was due to the effect of the A2AR agonist. ZM 241385 treatment combined FIGURE 1. The effects of selective A2AR agonists on TGF-b–mediated + + + A with the A2AR agonist decreased the generation of CD4 Foxp3 differentiation of Foxp3 cells from naive CD4 cells. ( ) CD4 cells were T cells in vitro by 2.9- to 3.5-fold compared with ATL370 or isolated from normal B6 mouse spleen by MACS. The cells were stimu- ATL1223 alone (p = 0.0008 and p = 0.0131, respectively, com- lated with Dynabeads mouse T cell activator (anti-CD3/anti-CD28 Ab) in paring ATL370 or ATL1223 alone versus with the antagonist over the presence of no TGF-b, TGF-b (2 ng/ml), TGF-b plus rapamycin (rapa; + + 10 nM), TGF-b plus CsA (10 nM), TGF-b plus ATL370 (10 nM), or TGF-b three experiments; Fig. 1B). In fact the number of CD4 Foxp3 B 6 plus ATL1223 (10 nM) and analyzed for Foxp3 expression. ( ) Addition of T cells with both ZM 241385 and A2AR agonist treatment (4.1 A 6 A2AR antagonist ZM241385 (ZM; 1 mM)24-hpretreatmenton( )and 1.5% with ATL370 or 3.9 1.2% with ATL1223; Fig. 1C) was experiment (C). The data shown are representative of three independent 6 similar to treatment with TGF-b alone (4.9 1.3%; Fig. 1C). Inter- experiments and gives the mean percentage of positive cells 6 SEM. + + estingly,ZM241385didnotaffectCD4 Foxp3 T cell generation The p values were calculated, comparing TGF-b only versus TGF-b plus in cells treated with either TGF-b or rapamycin (Fig. 1C). ATL370 or TGF-b plus ATL1223 and comparing TGF-b plus ATL370 versus TGF-b plus ATL370 plus ZM241385 or TGF-b plus ATL1223 Selective A R agonists ameliorates weight loss and mortality 2A versus TGF-b plus ATL1223 plus ZM241385 by Student t tests. due to acute GVHD

We previously reported that the A2AR specific agonist, ATL146e, nism for this effect. In the current study, we also studied the can decrease the incidence and severity of GVHD-induced weight effects of additional A2AR-specific agonists, ATL370 and ATL1223, loss as well as improve survival of mice in a B6 into B6-D2 model as well as used a second model with a greater degree of mismatch, of transplant-related GVHD (20) but did not delineate a mecha- that is the B6 into BALB/C model. The Journal of Immunology 461

In Fig. 2A and 2B, we confirmed our previous observation that weight loss, and they did not have significant mortality over the continuous administration of ATL146e by s.c. osmotic pump for indicated observation periods (data not shown). All of the A2AR 14 d, beginning 2 d before transplant of B6 TCD-BM plus T cells agonist treated mice had reduced GVHD-associated weight loss into B6-D2 mice reduces both GVHD-associated weight loss and mortality as compared with vehicle only-treated mice. Fur- and mortality compared with mice treated with vehicle control. thermore, when using ATL370 or ATL1223 (Fig. 2D, 2F) .50% of In addition, the protective effect of ATL146e administered for the A2AR agonist-treated animals survived beyond 30 d post 12 d posttransplant is observable up to 40 d posttransplant as transplant as compared with 0% survival of vehicle-treated con- shown in this experiment. In Fig. 2C and 2D, both ATL370 and trols in either transplant model. This effect was dose dependent ATL1223 similarly delivered for 14 d by osmotic pump beginning because osmotic pump administration of ATL370 at 10 ng/kg/min 2 d before transplant of B6 TCD-BM plus T cells into B6-D2 mice and ATL1223 at 50 ng/kg/min did not at all inhibit GVHD- reduces both GVHD-associated weight loss and mortality com- associated weight loss in either model (data not shown), but the pared with mice treated with vehicle control. Furthermore, the use of 50 ng/kg/min ATL370 and 200 ng/kg/min ATL1223 as effect is again observable out to 30 d posttransplant as shown in shown in Fig. 2C–F significantly reduced weight loss and mor- this experiment. Finally, using the more severe GVHD model of tality. B6 into BALB/C, we examined the ability of ATL370 or ATL1223 again delivered for 14 d by osmotic pump beginning 2 d before Inhibitory effect of selective A2AR agonists on proinflammatory transplant to reduce the development of severe GVHD-associated cytokine and chemokine production in the acute GVHD mouse weight loss and accelerated mortality compared with mice treated model with vehicle control (Fig. 2E, 2F). In the setting of GVHD, G-CSF, IL-6, IFN-g, and TNF-a appear to Downloaded from Mice in all groups that received donor TCD-BM only (without be proinflammatory cytokines that correlate with disease severity, additional donor T cells) did not develop acute GVHD-associated whereas increased levels of IL-10 and TGF-b may be protective or http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. A2AR agonists ameliorate GVHD after allogeneic HST. Weight loss and survival are shown in mice receiving TCD-BM plus donor T cells after treatment with either ATL146e (A, B) or ATL370 or ATL1223 (C, D) in the B6 into B6-D2 model, with ATL370 or ATL1223 (E, F) in the B6 into BALB/C model. Data shown are from three independent experiments (n = 15); error bars indicate SEM. Mortality was defined as mice that were found dead or had reached a moribund state because of excessive body weight loss, necessitating euthanasia. 462 A2AR AGONISTS INCREASE Tregs

may correlate with control or resolution of GVHD (31–33). Serum of the A2AR can reduce systemic levels of proinflammatory cyto- levels of G-CSF, IL-6, IFN-g, TNF-a, IL-10, and TGF-b were kines and increase immunotolerogenic cytokines in the setting of thus analyzed as an indicator of inflammatory activity of acute transplant-related GVHD.

GVHD at 16–20 d in the B6 into B6-D2 model of TCD-BM plus + high + T cell transplant-related GVHD. We examined the effect of ve- Selective A2AR agonist induces CD4 CD25 Foxp3 Tregs in hicle, ATL146e (10 ng/kg/min), ATL370 (50 ng/kg/min), or acute GVHD mouse model ATL1223 (200 ng/kg/min) delivered for 14 d by osmotic pump In this study, we showed in vitro that A2AR activation significantly beginning 2 d prior to transplant. For these studies, we used the augments TGF-b–induced generation of mouse CD4+Foxp3+ levels from B6-D2 mice transplanted with cells from B6-D2 donors T cells. Furthermore, our in vivo studies in the transplant-related (BM only) as our baseline control. GVHD mouse model demonstrated that the A2AR activation- The serum levels of TNF-a, IFN- g, IL-6, and G-CSF were associated amelioration of GVHD is associated with significant significantly lower in mice treated with the A2AR agonists com- reduction in levels of serum proinflammatory cytokines and sig- pared with the vehicle-treated controls (Fig 3A–D). Furthermore, nificant increase in serum levels of IL-10 and TGF-b, which are when we measured serum levels of the anti-inflammatory, tolerance- known to be factors in the generation of Tregs. In addition, there is inducing cytokines IL-10 and TGF-b (Fig3E,3F),thesewere further evidence in vivo from a mouse model of that significantly increased by 1.5- or 1.5- to 2.5-fold, respectively, A2AR-induced amelioration of autoimmunity is associated with when compared with vehicle only. It should be noted that in the generation of Tregs (23). BM only control, baseline levels of proinflammatory cytokines were We therefore next sought to determine whether specific A2AR very low, whereas the levels of the anti-inflammatory cytokines agonist treatment in the mouse transplant-related GVHD models Downloaded from were much higher compared with any of the experimental values. was associated with changes in Tregs in lymphoid organs, pe- Treatment of the experimental mice with A2AR agonists resulted ripheral blood, and at anatomic target sites of GVHD. In the in all cases in reversion of the cytokine levels toward the values analysis shown in Fig. 4 we used our mouse GVHD models (B6 seen in the BM only controls. These data indicate that activation into B6-D2 shown in Fig. 4A‑F; B6 into BALB/c shown in Fig. 4G‑I) http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. GVHD-induced elevation of proinflammatory cytokine and chemokine levels is reduced by A2AR agonists administration in the B6 into B6-D2 model. Blood was collected 16–20 d after transplant, and serum concentrations of G-CSF (A), IL-6 (B), IFN-g (C), TNF-a (D), IL-10 (E), and TGF-b (F) were measured using multiplex immunoassay kits with the BioPlex system and ELISA. Data shown are from three independent experiments (n = 9); error bars indicate SEM. The p values were calculated comparing agonist to vehicle control by Student t tests. The Journal of Immunology 463 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. A2AR agonists increase Tregs in acute GVHD mouse models. The percentages or number of CD4, CD25, and Foxp3-positive cells in various tissues posttransplant are shown. (A) Data from a representative experiment using the B6-D2 into B6 model. (B and C) Cumulative number of CD4, CD25, and Foxp3-positive cells data from three experiments analyzing 100 mg spleen and 100 ml peripheral blood, respectively. (D) Data from a representative experiment using the same model but treated with either ATL370 or ATL1223. (E and F) Cumulative data from three independent experiments for both agonists. Data from the B6 into BALB/C recipients is shown in (G–I), where (G) is the data from one representative experiment, and (H)and(I)arethecumulativedataforspleen and peripheral blood from three separate experiments. For all experiments, p values were calculated comparing vehicle control to drug using a Student t test.

+ high + to examine the absolute number of CD4 CD25 Foxp3 Tregs cific A2AR agonists delivered by route, dose, and time noted in appearing in the spleen and peripheral blood 16–20 d post- the previous results and as described in Materials and Methods. transplant. We compared treatment with vehicle versus spe- Again, “BM only” refers to the B6-D2 into B6-D2 control 464 A2AR AGONISTS INCREASE Tregs transplanted with unmanipulated marrow and no additional creased 8.1- to 9.4-fold with ATL370 (p = 0.0338; Fig. 4I) or T cells. ATL1223 (p , 0.0001; Fig. 4I). These results suggest that specific + high + As expected, splenic CD4 CD25 Foxp3 Tregs were signifi- activation of A2AR inhibits acute GVHD through decreased proin- cantly decreased in both of the vehicle-treated acute GVHD mouse flammatory cytokine production, induction of IL-10 and TGF-b models compared with “BM only” controls. Conversely, the number andanincreaseinCD4+CD25highFoxp3+ immunosuppressive of Tregs in the “BM only” groups was similar to that measured in Tregs in the spleen and peripheral blood. the spleens of untransplanted/untreated mice (data not shown). Treatment of mice from both GVHD models with any of the three Tregs increased by the selective A2AR agonist in the GVHD + mouse model were of donor origin specific A2AR agonists resulted in a profound increase in CD4 high + CD25 Foxp3 Tregs in the spleen compared with the vehicle- We determined the origin of A2AR induced Tregs by substituting treated GVHD mice (Fig. 4A, 4D, 4G). With the B6 into B6-D2 donor cells from B6-Thy1.1 instead of B6 for transplant into B6- model, this increase in number compared with vehicle treatment D2 recipients to allow detection of Thy1.1 on Tregs appearing in averaged ∼10-fold with ATL146e (p = 0.0093) treatment and 5- to the transplanted mice. This B6-Thy1.1 into B6-D2 model also 7-fold with ATL370 (p = 0.0164) or ATL1223 (p = 0.0258) treat- replicated the acute GVHD symptoms similar to the B6 into B6- ment (Fig. 4B, 4E). In the greater mismatched B6 into BALB/C D2 model, and also responded to treatment with ATL146e, model, the increase in splenic Tregs averaged ∼5- to 6-fold with resulting in less weight loss and improved survival (data not shown). ATL370 (p = 0.0393) or ATL1223 (p = 0.0616) treatment (Fig. 4H). As shown in Fig. 5A and 5B, measuring Tregs in the spleen and In addition to the spleen, we also measured the absolute number peripheral blood, we confirmed again the severe depletion of Tregs of CD4+CD25highFoxp3+ Tregs in the peripheral blood finding a in the vehicle treated GVHD model (2.91 6 2.52% of CD4+ cells Downloaded from 5.4-fold increase with ATL146e (p = 0.0262) treatment and 3.6- to in the spleen, 0.88 6 0.51% of CD4+ cells in peripheral blood) 4.6-fold increase with ATL370 (p = 0.0271) or ATL1223 (p = compared with “BM only” control (12.06 6 3.43% of CD4+ cells 0.0200) treatment in the B6 into B6-D2 model (Fig. 4C, 4F). In in the spleen, 15.64 6 6.55% of CD4+ cells in peripheral blood), the B6 into BALB/C model, peripheral blood Tregs were in- but significant restoration of the level of Tregs almost to the level http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Tregs that are increased by A2AR agonists are donor derived in acute GVHD. Shown are the percentages of CD4, CD25, and Foxp3-positive cells found in B6-D2 recipients using B6-Thy1.1 donors and treated with either vehicle or ATL146e or receiving T replete marrow from B6-D2 donors (BM only) found in the spleen (A) or peripheral blood (B) with the data from three separate experiments shown in (C) and (D). The p values were calculated comparing treatment to vehicle control by a Student t test (n = 9). (E and F) Weight loss and mortality of B6-D2 recipients despite using either ATL 370 or

1223 when Foxp3-deficient mice are used as transplant donors. (G–L)A2AR agonist administration does not effect GVHD-induced elevation of proin- flammatory cytokine and chemokine levels in the B6-Foxp3–deficient mice into B6-D2 model. Blood was collected 16–20 d after transplant, and serum concentrations of G-CSF (G), IFN-g (H), IL-6 (I), TNF-a (J), IL-10 (K), and TGF-b (L) were measured using multiplex immunoassay kits with the BioPlex system and ELISA. Data are from two independent experiments (n = 10); error bars indicate SEM. The Journal of Immunology 465 of the “BM only” control in the B6-Thy1.1 into B6-D2 GVHD (Fig. 6A). Vehicle-treated mice group demonstrated severe in- model treated with ATL146e (10.66 6 5.18% of CD4+ cells in flammation, necrosis, , and amyloid-like material in the spleen, 12.10 6 4.28% of CD4+ cells in peripheral blood; Fig. 5C, lamina propria of the colon (Fig. 6B), and inflammation with 5D). Furthermore, gating on Tregs and assessing expression of the thickness, ulceration, and pustule formation in the skin (Fig. 6C). Thy1.1 donor marker versus Thy1.2 recipient marker (Fig. 5A, However, ATL370 or ATL1223 mice had minimal focal ulcera- 5B, right panels) demonstrated that in the “BM only” control all tion, a less-pronounced submucosal edema in the colon (Fig. 6B) Tregs expressed only Thy1.2, as expected, whereas no Thy1.2 and mostly normal-appearing skin (Fig. 6D). Tregs are detected in the vehicle or ATL146e-treated B6-Thy1.1 As shown in Fig. 6D (colon) or Fig. 6E (skin), immunohisto- into B6-D2 GVHD model transplant groups. Virtually all of the chemical staining for Foxp3 showed that transplanted mice re- + Tregs arising in the GVHD model transplant mice were of Thy1.1 ceiving the A2AR agonists appeared to have more Foxp3 cells donor origin. This was seen in both the vehicle-treated GVHD than those treated with vehicle. We quantified this observation by model, where there is a relative paucity of Tregs compared with having a blinded observer count the number of Foxp3+ cells in the “BM only” control, and in the ATL146e-treated GVHD model, a random sampling of high power fields as noted in Materials and where relative numbers of Tregs are restored closer to levels seen Methods. The results of this analysis for colon and for skin are in the “BM only” control. We also observed the same results when shown in Fig. 6F and 6G, respectively. In mice treated with ve- we used either ATL370 or ATL1223 (data not shown). hicle control and with active GVHD, the number of Foxp3+ cells Because the Tregs arising in the GVHD model of transplant are detectable in colon was 14 6 6 per high-power field. However, in all of donor origin, we next performed experiments to determine the ATL370-treated mice, there were 64 6 14 Foxp3+ cells per whether functional Foxp3 in donor cells was required for A2AR high-power field in colon, a difference that was statistically sig- Downloaded from agonist efficacy. B6-Foxp3–deficient mice (scurfy mouse on B6 nificant compared with the vehicle control group (p , 0.0001; background) have been shown to be incapable of generating Fig. 6F, middle bar). The trend was similar in the colon of functional Tregs and suffer from a variety of autoimmune prob- ATL1223-treated mice, although the increase in Foxp3+ cells also lems. When B6-Foxp3–deficient donors were transplanted into reached statistical significance (p = 0.0078; Fig. 6F, right bar). In + B6 recipients, the two groups of A2AR agonist treatment mice contrast, the number of Foxp3 T cells per high-power field in skin

(ATL370 and ATL1223) experienced the same accelerated body was only 8 6 6 in the vehicle control but increased to a highly http://www.jimmunol.org/ weight loss and mortality as the vehicle-treated mice (Fig. 5E, 5F; significant 30 6 7 in the ATL370-treated mice (p = 0.0013; Fig. compare with Fig. 2C, 2D). We measured serum levels of G-CSF, 6G, middle bar) and to 36 6 14 in the ATL1223-treated mice IL-6, IFN-g, TNF-a, IL-10, and TGF-b at 16–20 d in the B6- (p = 0.0030; Fig. 6G, right bar). We also determined the number Foxp3–deficient mice into B6-D2 model. Proinflammatory cyto- of infiltrating Tregs in colon and skin by flow cytometry analysis kines (G-CSF, IL-6, IFN-g, and TNF-a) were not significantly after extraction of lymphocytes from the tissues. In mice treated + decreased in two groups of A2AR agonist treatment mice (ATL370 with vehicle control and with active GVHD, the number of CD4 and ATL1223; Fig 5G–J). This result can be compared with the Foxp3+ cells in 100 mg colon was 432 6 101. However, we serum levels of proinflammatory cytokine in the B6 into B6-D2 measured 1256 6 365 of CD4+Foxp3+ cells in the ATL370-treated model (Fig. 3A–D). Moreover, serum levels of IL-10 and TGF-b mice and 732 6 110 of CD4+Foxp3+ cells in ATL1223-treated by guest on September 28, 2021 were also no different between the vehicle control group and mice, which is statistically significantly higher than the vehicle A2AR agonist treatment groups (ATL370 and ATL1223 [Fig. 5K, control group (p = 0.0001, p = 0.0154; Fig. 6H). As well, the 5L] compared with [Fig. 3E, 3F]). This then confirms the absolute number of Tregs in the skin was 287 6 97 in the vehicle control requirement for the donor graft to develop functional Tregs in mice but increased to 786 6 132 in the ATL370-treated mice order for A2AR agonist treatment to provide protection against (p = 0.0055) and to 865 6 115 in the ATL1223-treated mice increased mortality from GVHD in this model. (p = 0.0004). Therefore, the number of infiltrating Tregs in skin or

+ colonic tissues was increased 1.7- to 3-fold after treatment with an Mice treated with the A2AR have increased numbers of Foxp3 A2AR agonist for acute GVHD. Furthermore, we observed Foxp3 cells in both skin and colon increased 3.21-fold (p = 0.0069; Fig. 6I) in the As the skin and are target organs of GVHD, colonic tissues of ATL370-treated mice as measured by increased we performed histopathological analysis by H&E staining and mRNA levels, although we did not see a difference when using immunohistochemical staining on liver, and colon biopsies ATL1223. In the skin, Foxp3 gene expression also increased 2- obtained at day 16–20 in our B6 into B6-D2 GVHD mouse model fold (p = 0.0078 for ATL370, p = 0.0099 for ATL 1223 compare recipients after treatment with either of the two A2AR agonists with vehicle control; Fig. 6I) for each of the A2AR agonist-treated (ATL370 or ATL1223) or Vehicle only. Pathological scoring for groups. evaluation of GVHD in the target tissues (liver, skin, and colon) was performed by a pathologist blinded to the treatment status of Discussion the mouse (Table I). Animals treated with either agonists had Allogeneic transplantation is being increasingly used to cure a wide significantly lower scores than vehicle-treated mice and in some variety of cancers and monogenetic disorders of blood and other cases was similar to that of bone marrow only transplanted mice organs. However, acute and chronic GVHD remain as some of

Table I. Semiquantitative histopathological analysis

BM Only Vehicle ATL370 ATL1223 Liver 1.250 6 0.957 3.125 6 0.354 0.500 6 0.548 1.400 6 0.894 Skin 1.200 6 0.837 3.000 6 0.577 1.429 6 0.535 1.750 6 0.463 Colon 0.600 6 0.548 2.286 6 0.756 0.600 6 0.894 1.600 6 0.548 The pathologist was blinded to the samples and graded for GVHD in multiple samples from animals treated with bone marrow transplant only (a non-GVHD control), vehicle, or either A2A agonist. The mean score (6SEM) is given for each tissue type. 466 A2AR AGONISTS INCREASE Tregs Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 6. ATL370 and ATL1223 increase Tregs in colon and skin of acute GVHD mouse model. (A) The cumulative results of the histopathological scoring are represented in bar graph form (*p , 0.05, **p , 0.01, ***p , 0.001 comparison with vehicle). Colon (B, D) or skin (C, E) was obtained and placed in 4% paraformaldehyde 16–20 d after transplantation, and paraffin embedded and H&E staining (B, C) and immunohistochemistry (IHC) by mouse Foxp3 Ab (D, E) were performed. (F, G) The numbers of Foxp3+ cells (dark brown stained cells) were counted per high power field (HPF) for colon or skin, respectively, with original magnification of 340. Data shown are the cells counted from a random area (n = 5); error bars indicate SEM. (H) FACS analysis of infiltrating Treg numbers per 100 mg skin or colon tissues in B6-D2 recipients. Skin or colon tissues were digested as described in Materials and Methods and stained with Abs for CD4, CD25, and Foxp3. Data are shown mean (6 SEM) from five independent skin or colon samples from each group. (I) Relative gene expression of Foxp3 in skin or colon tissues in B6-D2 recipients. Total RNA was extracted from skin or colon tissues and analyzed by real- time RT-PCR. Levels were normalized using GAPDH mRNA. Data represent the mean (6 SEM) from four independent skin or colon samples from each group. The p value is versus vehicle control as assessed by a Student t test. the most significant adverse effects of this potentially life-saving presence of an A2AR agonist in vitro are anergic (23), and it has procedure. Current treatment for GVHD involves the use of high- been shown that activation of the A2AR can prevent T cell pro- dose immunosuppressants including , various in- liferation or production of IL-2 or IFN-g upon restimulation. Our hibitors of T cell function, and mAbs directed at T and Ags study suggests that in vivo these effects of A2AR agonist treatment or proinflammatory signaling pathways, which in themselves are likely augmented or mediated by an increased production of impair innate and acquired immunity, may impair engraftment or Tregs. In this study, we examined the selective A2AR agonists of impede antitumor effects and lead to a significantly increased risk differing potency (ATL146e, ATL370, and ATL1223) in the re- of . There is a need for new agents with alternate mech- duction of GVHD in two models of transplant related anisms of action to prevent GVHD, particularly those that act to GVHD in mice. We showed a profound protection against weight enhance tolerance rather than merely suppress T cell function. loss and mortality, which was associated with a decrease in pro- More targeted prevention and treatment of GVHD is therefore a inflammatory cytokines, an increase in protective anti-inflamma- goal of many transplanters. Our study demonstrates that specific tory cytokines, and enhanced production of protective Tregs in activation of A2AR mediates reduction of acute GVHD pathology spleen, peripheral blood, and target tissues (colon and skin). We and symptoms and improves survival rate in mouse mismatch demonstrated that these A2AR agonist induced protective Tregs transplant models in large part by enhancing development of Tregs were of donor origin and that failure to produce Tregs (using that act to increase graft tolerance to host Ags. Foxp3-deficient donor cells) resulted in failure to provide pro- In addition, A2AR blocks the function of effector T lymphocytes tection against GVHD-mediated mortality. Tregs unequivocally by suppressing proinflammatory cytokine production and de- have been shown to play an important role in development of donor creasing expression of activation markers and proliferation (34–36). tolerance to host, with protection against development of GVHD It has been reported that activated conventional T cells in the (37–41). In a recent clinical study, it was shown that infusion of The Journal of Immunology 467

Tregs derived in vitro from umbilical cord blood can safely reduce nation treatment studies in our GVHD mouse models in vivo the incidence of GVHD (42, 43). These reports provided an im- are ongoing. portant basis for initiating our assessment of the effect of A2AR Finally, we observed that A2AR agonists, but not cyclosporine activation on generation of Tregs in our acute GVHD mouse A, promoted TGF-b–mediated differentiation of Tregs in vitro models. We showed that CD4+CD25highFoxp3+ Tregs are increased (Fig. 1). Treg development and homeostasis are controlled by by all three of the A2AR agonists that we studied, although in TCR engagement, cytokines such as IL-2, IL-10, and TGF-b, and general, the most potent and specific agonist, ATL146e, had the costimulatory receptors such as CD28 and CTLA-4 (51). It has most profound effect. We also documented significant reduction of also been reported that TCR signaling through PI3K, ki- peripheral blood proinflammatory cytokine levels, including IL-6, nase B (Akt), and mammalian target of rapamycin (mTOR) reg- IFN-g, IL-2, and TNF-a and increased levels of the immuno- ulates Foxp3 expression in activated CD4 lineage thymoctyes and suppressive cytokine IL-10 and TGF-b from administration of peripheral T cells. Conversely, constitutive activation of the PI3K/ A2AR agonist relative to vehicle controls. AKT/mTOR signaling pathway in chromosome 10 (PTEN, ma- There have also been some reports suggesting that adenosine jor negative regulator of PI3K/Akt signaling)–deficient T cells activation, via the A2AR, not only can enhance generation of reduces Foxp3 induction, which can be restored by PI3K inhibi- Tregs, but that the effectiveness of the tolerogenic action of those tor (52). The fact that we do not see a synergistic effect in com- Tregs can be increased by engaging the A2AR on the target ef- bination with rapamycin suggests that there is some overlap fector T cells (21). In fact, some have suggested that generation of through the mTOR pathway as a mechanism for Treg develop- adenosine by Tregs suppresses effector T cells via the A2AR. In an ment; however, this needs to be explored further. There is also data

low Downloaded from immune-mediated colitis model, it was found that CD45RB or suggesting that A2AR stimulation enhances Foxp3 transcription by + 2/2 high CD25 Tregs from A2AR mice failed to suppress CD45RB decreasing IL-6 expression and enhancing TGF-b expression (23). cells and prevent disease induced by pathogenic CD45RBhigh As seen in the scurfy model, the level of proinflammatory cyto- high 2/2 T cells, and that CD45RB effector T cells from A2AR mice kines remained high, and there was no impact by the agonists in are not suppressed by suppressor Treg cells derived from wild- the severity of GVHD, insinuating that the generation of Tregs type mice (15). Furthermore, the Powell laboratory group has may be dampened by the high cytokine levels and lack of IL-6 reported that extracellular adenosine stimulates the A2AR to pro- production. However, we still have questions as to whether acti- http://www.jimmunol.org/ + + mote T cell anergy and the generation of Foxp3 and LAG3 vation of the A2AR is directly involved in TGF-b–induced Foxp3 Tregs by reducing the production of IL-6 and enhancing the expression on GVHD mouse model or if this is an indirect effect. production of TGF-b after Ag stimulation (23). It has also been Additional studies are needed to further elucidate the mechanism shown that mice lacking the CD73/ecto-59-nucleotidase appear by which A2AR-specific agonist increases Tregs. to have increased GVHD due to the inability to convert AMP In summary, we have demonstrated that specific activation of to adenosine (44). Furthermore, autoimmunity in ADA-SCID A2AR enhances development of Tregs in vitro, and that treatment patients has been linked to a dysregulation of the CD39/73 ade- with specific agonist of A2AR inhibits the development of acute nosinergic pathways resulting in aberrant T regulatory function GVHD in two types of HLA mismatched mouse transplant models (45). Finally, methotrexate, a commonly used drug in the man- through the increase of donor-derived Tregs in the spleen, blood, by guest on September 28, 2021 agement and treatment of GVHD as well as autoimmune disorders and target tissues of GVHD (colon and skin). Our preclinical studies such as has been shown to increase levels of in these mouse models, along with clinical studies using A2A ago- AMP and extracellular adenosine and administration of adenosine nists in normal volunteers documenting their low toxicity profile, A2A receptor agonists reversed its anti-inflammatory effects in provide strong support for bringing highly specific agonists of A2AR a mouse model of inflammation (46, 47). However binding of to the for the prevention and treatment of GVHD. adenosine to other receptors can also trigger proinflammatory effects perhaps explaining some of the failures seen with metho- Acknowledgments trexate use (48). We thank Dr. So Yoon Kim for help with immunohistochemistry and we In our current study, we not only observed that A2AR specific also thank the Comparative Branch animal care facility techni- agonists improved survival rate and inhibited weight loss, but we cians. We also acknowledge the gift of the adenosine agonists from Forrest also observed a profound reduction in clinical manifestations of LLC. skin sores and in the treated mice. Although these man- ifestations of clinical improvement in the treated mice were dif- Disclosures ficult to quantify compared with measures of weight loss and sur- The authors have no financial conflicts of interest. vival, the amelioration of skin sores and diarrhea was clearly evident to the investigators and animal care team. A number of studies have documented that rapamycin mediates References GVHD prevention in part through the induction of Tregs, and that 1. Reddy, P. 2003. Pathophysiology of acute graft-versus-host disease. Hematol. Oncol. 21: 149–161. these cells act within the affected tissues (49, 50). Our current 2. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. 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