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T Regulatory and Primed Uncommitted CD4 T Cells Express CD73, Which Suppresses Effector CD4 T Cells by Converting 5′ -Adenosine Monophosphate to Adenosine This information is current as of October 2, 2021. James J. Kobie, Pranav R. Shah, Li Yang, Jonathan A. Rebhahn, Deborah J. Fowell and Tim R. Mosmann J Immunol 2006; 177:6780-6786; ; doi: 10.4049/jimmunol.177.10.6780

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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 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

T Regulatory and Primed Uncommitted CD4 T Cells Express CD73, Which Suppresses Effector CD4 T Cells by Converting 5؅-Adenosine Monophosphate to Adenosine1

James J. Kobie, Pranav R. Shah, Li Yang, Jonathan A. Rebhahn, Deborah J. Fowell, and Tim R. Mosmann2

CD73 (5؅-ectonucleotidase) is expressed by two distinct mouse CD4 T cell populations: CD25؉ (FoxP3؉) T regulatory (Treg) cells that suppress T cell proliferation but do not secrete IL-2, and CD25؊ uncommitted primed precursor Th (Thpp) cells that secrete IL-2 but do not suppress in standard Treg suppressor assays. CD73 on both Treg and Thpp cells converted extracellular 5؅-AMP to adenosine. Adenosine suppressed proliferation and cytokine secretion of Th1 and Th2 effector cells, even when target cells were activated by anti-CD3 and anti-CD28. This represents an additional suppressive mechanism of Treg cells and a previously unrecognized suppressive activity of Thpp cells. Infiltration of either Treg or Thpp cells at inflammatory sites could potentially Downloaded from convert 5؅-AMP generated by neutrophils or dying cells into the anti-inflammatory mediator adenosine, thus dampening excessive immune reactions. The Journal of Immunology, 2006, 177: 6780–6786.

uring strong inflammatory immune responses, immune at- Treatment of T lymphocytes with adenosine or the A2A agonist tack on pathogens and infected cells also results in damage inhibits proliferation (13, 14), as well as the secretion of cytokines D to host tissues, so that successful immune responses nor- including IL-2, TNF-␣, and IFN-␥ (14, 15). However, the concen- http://www.jimmunol.org/ mally involve a complex balance of pro- and anti-inflammatory me- trations of adenosine required to elicit in vitro effects (25 ␮M) (13) ␤ diators. The cytokines IL-10 (1) and TGF- (2) are both important for were substantially higher than the Kd of various adenosine ana- restraining vigorous inflammatory responses. In addition to cytokine- logues for the A2A receptor (20–50 nM) (16). The Kd of the A2A mediated immune regulation, other factors may contribute to control- receptor for adenosine itself is difficult to determine (17). ling inflammation including PGE2, NO, glucocorticoids, a hypoxic The generation of adenosine at sites of inflammation, hypoxia, environment, and adenosine. organ injury, and traumatic shock is in part mediated by two se- Adenosine has various immunoregulatory activities mediated quential enzymes. The ecto-ATP diphosphohydrolase (CD39) ex- through four adenosine receptors: A1,A2A,A2B, and A3 (3). T pressed by neutrophils rapidly converts circulating ATP and ADP Ј by guest on October 2, 2021 lymphocytes mainly express the high affinity A2AR and the low- to 5 -AMP (18). CD73, expressed on the surface of endothelial Ј affinity A2BR (3). Macrophages and neutrophils can express all cells (19) and a subset of T cells (20–22), is an ecto-5 -nucleoti- four adenosine receptors depending on their activation state, and B dase (23) that can convert 5Ј-AMP to adenosine. cells express A2AR (3). Engagement of A2AR inhibits IL-12 pro- We have recently identified, directly ex vivo, a population of duction but increases IL-10 production by human monocytes (4) memory CD4 T cells that express CD73 but not Ly-6A/E (22). and dendritic cells (5) and selectively decreases some cytotoxic These cells are similar to the uncommitted primed precursor helper functions (6) and chemokine production by neutrophils (7). A pro- cells (Thpp)3 previously identified in vitro (24). Both cell types tective role for adenosine in Con A- or endotoxin-induced inflam- secrete IL-2 and chemokines but not IFN-␥ or IL-4 when activated matory liver damage was revealed by treatment with adenosine (22, 24), both localize preferentially to lymph nodes (22, 25), and agonists and antagonists or in mice deficient for A2AR (8, 9). In both have the flexibility to differentiate further into either Th1 or vivo treatment with an A2A agonist decreases intestinal inflamma- Th2 phenotypes depending on the polarizing cytokine environ- tion (10). Conversely, the high levels of adenosine in patients with ment. These Thpp cells may provide an expanded pool of uncom- inherited deficiency of the adenosine-degrading enzyme adenosine mitted Ag-specific T cells without potentially harmful inflammatory deaminase are associated with profound depletion of T, B, and NK functions. lymphocytes (11, 12). The inhibitory activity of CD4 T cells has primarily been associated with FoxP3ϩ Treg cells, many of which express CD25 (26, 27). FoxP3 is important in the development and activity of Treg ϩ David H. Smith Center for Vaccine Biology and Immunology and Department of cells, because CD25 cells isolated from FoxP3-deficient mice Microbiology and Immunology, University of Rochester Medical Center, Rochester, lack regulatory activity in vitro and in vivo, and retroviral expres- NY 14642 sion of FoxP3 in nonregulatory T cells confers regulatory activity Received for publication May 4, 2006. Accepted for publication August 22, 2006. (28, 29). The mechanism(s) by which Tregs control immune re- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance sponses in vivo and in vitro may include the suppressive cytokines with 18 U.S.C. Section 1734 solely to indicate this fact. TGF-␤ and IL-10, modulation of APC function, and sequestering 1 This work was supported by National Institutes of Health Grants RO1 AI48604 and T32 of IL-2 (30). CA09363-22 (to J.J.K.). 2 Address correspondence and reprint requests to Dr. Tim R. Mosmann, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 609, Rochester, NY 14642. E-mail address: 3 Abbreviations used in this paper: Thpp, primed precursor Th cells; APCP, ␣,␤- [email protected] methylene ADP.

We now show that Treg cells also express CD73. Although Treg Generation of in vitro Thpp, Th1, and Th2 cells and Thpp cells also share other properties and both can differen- Naive CD4 T cells (CD4ϩCD62LϩCD44lowCD25Ϫ) were isolated by cell tiate from naive CD4 T cells under similar conditions in vitro, we sorting as indicated above. Naive CD4ϩ T cells (104) were cultured with show that these are two distinct populations. Adenosine produced 104 irradiated (10,000 rad) allogeneic TA3 cells in 200 ␮l of Alpha MEM by either cell type potently suppresses proliferation of effector containing 10% FBS, 1 ng/ml IL-2, and cytokine/anti-cytokine supple- ments. Thpp cultures contained 2 ng/ml TGF-␤ and 50 ␮g/ml anti-IFN-␥ CD4 T cells and also inhibits their production of cytokines. The 1 Ab (clone XMG1.2). Th1 cultures contained 1 ng/ml IL-12 and 5 ␮g/ml inhibition of effector T cells occurs at low adenosine levels (pro- anti-IL-4 Ab (clone 11B11), and Th2 cultures contained 1 ng/ml IL-4 and vided that adenosine degradation is minimized), and either Thpp or 10 ␮g/ml anti-IFN-␥ Ab (clone XMG1.2). On day 4 of culture, half of the Treg cells derived directly ex vivo can significantly inhibit effector medium was removed and replenished with new medium and cytokines. cells at a ratio of less than 1:1. Thus, the CD73/adenosine pathway Fresh IL-2 and medium were added to cultures weekly. is a potent additional suppressive pathway of Treg cells and also Cytokine detection confers a suppressive anti-inflammatory function on the uncom- ␥ ␣ mitted precursor Thpp cell type. Detection of IL-2, IFN- , and MIP-1 by ELISPOT assay was performed as previously described (22). Detection of cytokines by Fluorospot assay was performed as previously described (32). Briefly, detection of IFN-␥ Materials and Methods and IL-4 by Fluorospot was performed by coating with either 2 ␮g/ml ␥ ␮ ␮ Mice anti-IFN- Ab (clone AN18) or 10 g/ml anti-IL-4 (11B11) with 2 g/ml anti-CD3 and 1 ␮g/ml anti-CD28. Anti-IFN-␥ (clone XMG1.2) or anti- Female C57BL/6 and C57BL/6.PL mice (6–8 wk of age) were obtained IL-4 biotinylated Ab was added at 1 ␮g/ml. After incubation, the plates from The Jackson Laboratory. All animal experiments were approved by were washed in PBS containing 0.1% Tween 20 (PBST) and 2 ␮g/ml the University of Rochester University Committee on Animal Resources streptavidin-conjugated Cy3 in PBST plus 2% BSA was added for 30 min. Downloaded from (Rochester, NY). The plates were washed again with PBST, dried, and scanned using a fluorescent microscope, and the digitized images were quantified by the Abs and cell lines ExploraSpot image analysis program (J. Rebhahn, J. J. Kobie, D. M. Zaiss, T. R. Mosmann, results not shown). Purified anti-mouse IL-2 (clone JES6-1A12) and biotinylated anti-mouse IL-2 (clone JES6-5H4), IL-4 (clone BVD6-2462), and IFN-␥ (clone Coculture of sorted cells with Th1 cells XMG1.2), were obtained from eBioscience. Purified anti-mouse IL-4 ␥ Naive, Thpp, or Treg cell populations were isolated as indicated above and (clone 11B11) and IFN- (clones AN18 and XMG1.2) were obtained from http://www.jimmunol.org/ hybridoma cell lines as previously described (22). Purified anti-mouse cultured in 96-well round-bottom plates in X-VIVO 20 medium (Cambrex) MIP-1␣ (clone 39624.11) and biotinylated anti-mouse MIP-1␣ (clone in the presence of 0.5 ␮M5Ј-AMP (Sigma-Aldrich) and 100 ␮M ␣,␤- BAF450) were obtained from R&D Systems. The following FITC-, R-PE-, methylene ADP (APCP) (Sigma-Aldrich) or 25 ␮M SCH58261 (Sigma- PE-Cy5.5-, PE-Cy7-, PerCP-, PerCP-Cy5.5-, allophycocyanin-, allophyco- Aldrich) as indicated for4hat37°C. After addition of Th1 cells, IFN-␥ cyanin-Cy7, and biotin-conjugated Abs were purchased from BD Pharm- secretion was measured by restimulation in 4-h Fluorospot assays as de- ingen: anti-Ly-6A/E (sca-1) (clone D7), anti-CD73 (clone Y/23), anti-CD4 scribed above. (clone RM4-5), anti-CD44 (clone IM7), anti-CD19 (clone MB19-1), anti- CD8␣ (clone 53-6.7), anti-CD25 (clone PC61.5), and anti-CD62L (clone Statistical analysis Mel-14). Alexa Fluor 405-conjugated anti-mouse CD4 (clone RM4-5) was Where statistical difference is indicated two-tailed unpaired Student’s t obtained from Caltag Laboratories. Staining with anti-FoxP3-FITC- tests with a 95% confidence interval were performed using Prism software conjugated Ab (clone FJK-16s) was performed according to the manufac- (GraphPad). by guest on October 2, 2021 turer’s protocol (eBioscience). The cell line TA3, which expresses H-2Id/k on the surface, is a hybrid between a B cell lymphoma and a normal B cell (31). Results Expression of CD73 by distinct uncommitted primed precursor IL-2 secretion and regulatory T cell populations Splenocytes isolated from C57BL/6 mice were evaluated for IL-2 secretion Uncommitted precursor Thpp-like CD4 T cells (secreting IL-2 and by the Miltenyi cytokine secretion assay (Miltenyi Biotec) according to the chemokines) are mainly CD44ϩCD73ϩLy-6A/EϪ (22), whereas manufacturer’s protocol. Twenty million splenocytes were stimulated with naive (CD44low) CD4 T cells (secreting only IL-2) do not express anti-CD3 (2 ␮g/ml) and anti-CD28 (1 ␮g/ml) per well in a 12-well culture plate for 12 h. Cells were collected and labeled with the IL-2 capture significant levels of either CD73 or Ly-6A/E. Effector cells (se- reagent followed by a 45-min secretion period, and subsequent staining creting IFN-␥ or IL-4) express high levels of Ly-6A/E in Ly-6.2 with the PE-conjugated anti-IL2 detection Ab 7AAD and anti-CD4 and (C57BL/6, C57L, PL, C58, SJL, and AKR) mouse strains, but anti-CD19 Abs. FoxP3 expression was then determined as indicated above. most effectors do not significantly express CD73(22). Fig. 1a ϩ ϩ Isolation of CD4 T cells shows that most CD25 CD4 T cells from the spleens of C57BL/6 mice also expressed CD73, raising the possibility that Subsets of CD4 T cells were isolated by staining with subsaturating levels of Treg cells are CD73ϩ. Substantial numbers of CD44ϩ CD25Ϫ PE-Alexa Fluor 610-anti-CD8␣, FITC-anti-CD19, PE-CD73, PE-Cy7-anti- ϩ Ϫ CD25, PerCP-Cy5.5-anti-CD4, Ly-6A/E-allophycocyanin, and allophycocya- CD73 Ly-6A/E cells (potentially Thpp cells) were also present. nin-Cy7-anti-CD44 and collecting the CD4ϩCD8␣ϪCD19ϪCD25ϪCD44low Because activated T cells also express CD25 and some suppres- CD73Ϫ (naive), CD4ϩCD8␣ϪCD19ϪCD25ϪCD44highCD73ϩLy-6A/EϪ sive CD4 T cells do not express CD25 (26), we next determined ϩ Ϫ Ϫ ϩ ϩ (Thpp), and CD4 CD8␣ CD19 CD25 CD73 (Treg) populations using the relationship between the expression of CD73 and FoxP3, a a BD FACSAria flow cytometric cell sorter (BD Immunocytometry). Sub- member of the Forkhead transcription factor family that is strongly saturating levels of Abs, particularly anti-CD4, were used to minimize functional alterations of labeled cells. associated with the Treg suppressor phenotype (28). Spleen cells were stained for CD73, CD44 and Ly-6A/E expression and sub- CFSE proliferation/suppression assay sequently permeabilized and stained for FoxP3 expression. As ex- Ϫ Ϫ CD4ϩ T cell populations were isolated from the spleens of C57BL/6 pected, the FoxP3 CD44 (naive) population stained at low levels (CD90.2ϩ) mice by flow cytometric cell sorting as indicated above. with anti-CD73 (mean fluorescence intensity, 1227) (Fig. 1b). The Increasing numbers of CD4ϩ T cell populations were cultured with 50,000 FoxP3ϩ population expressed higher levels of CD73 (mean fluo- ϩ CFSE-labeled splenocytes isolated from C57BL/6.PL (CD90.1 )inthe rescence intensity, 2171) (Fig. 1b). This was also observed in presence of 1 ␮g/ml anti-CD3 in round-bottom 96-well plates for 72 h. BALB/c and B10.D2 mice (data not shown). Real-time PCR also Following incubation, triplicate wells were pooled, stained with anti-CD4, ϩ ϩ anti-CD8, anti-CD19, anti-CD90.1, anti-CD90.2, and 7AAD, and analyzed showed that FoxP3 was expressed mainly in sorted CD73 CD25 Ϫ by flow cytometry using a BD LSRII device (BD Biosciences). cells but not in CD73 cells (data not shown). 6782 CD73 MEDIATES Thpp AND Treg SUPPRESSION OF Th1/Th2 RESPONSES

duction profiles were tested by an ELISPOT assay. The putative Thpp-enriched population produced IL-2 and MIP-1␣ but not IFN-␥, consistent with the Thpp cytokine profile (35). In contrast, negligible numbers of the putative Treg population produced IL-2 or IFN-␥, and only a low frequency produced MIP-1␣ (Fig. 2a). Naive (CD25ϪCD44low) cells produced primarily IL-2 (Fig. 2a). Although these results strongly suggested that Treg and Thpp cells were separate populations, a minority of FoxP3ϩ cells are CD25Ϫ and therefore are present in the putative Thpp population (data not shown). To exclude the possibility that these cells were contributing to IL-2 production, IL-2 secretion and FoxP3 expression were measured by flow cytometry in splenocytes following anti- TCR stimulation. As the fixation methods for intracellular cytokine staining and FoxP3 expression were incompatible, the Miltenyi cytokine secretion assay was used to detect IL-2-producing cells. The FoxP3ϩ and IL-2ϩ splenocyte cell populations were separate, indicating that FoxP3ϩ cells within the CD4ϩCD25ϪCD73ϩLy- 6A/EϪ population do not contribute to the observed IL-2 production (Fig. 2b). A subpopulation of FoxP3ϩ cells was also present in cultures of Downloaded from cells differentiated in vitro under Thpp-polarizing conditions (TGF-␤ and anti-IFN-␥), raising the possibility that the “Thpp” cultures also contained some Treg cells. This was supported by the observation that FoxP3ϩ cells were also distinct from IL-2-secreting cells in Thpp populations derived in vitro (Fig. 2b)asintheex vivo populations. Similar IL-2 and FoxP3 expression patterns were http://www.jimmunol.org/ observed following the stimulation of splenocytes and Thpp cells with alloantigen or staphylococcal enterotoxin B (data not shown). As expected, cells differentiated in vitro in Th1-polarizing conditions (IL-12) did not contain a FoxP3ϩ population (Fig. 2b). We next tested whether the functions of ex vivo Thpp (CD25ϪCD73ϩLy-6A/EϪ) and Treg (CD25ϩCD73ϩLy-6A/Eϩ/Ϫ) cells matched their FoxP3 and cytokine expression patterns. Suppres- sion of lymphocyte proliferation was measured by soluble anti-CD3 by guest on October 2, 2021 stimulation of CFSE-labeled splenocytes in the presence of sorted Thpp, Treg, or naive cells (and the absence of the CD73 substrate 5Ј-AMP). Treg cells markedly suppressed proliferation of CD4ϩ and CD8ϩ T cells (Ͼ50% of the cells remained undivided); however, naive or Thpp cells had minimal effects on target cell proliferation (Fig. 3). When Thpp cells were present at the highest amount (1:2), a slight inhibition of CD4 proliferation was observed that may have been a result of the FoxP3ϩ cells present in the Thpp population. No significant difference in suppressive activity was observed between FIGURE 1. CD73 is expressed by T regulatory and Thpp cells. Spleno- CD25ϩCD73ϩLy-6A/EϪ and CD25ϩCD73ϩLy-6A/Eϩ cells (data cytes from C57BL/6 mice were isolated and analyzed by flow cytometry not shown). for cell surface marker expression (a and b) and intracellular expression Together, these results demonstrate that although CD73 is ex- of FoxP3 (b). Data are representative of at least three independent pressed by both Thpp and Treg populations, each has distinct char- experiments. acteristics; only the Thpp cells secrete IL-2, and only the Treg population is capable of suppression in the anti-CD3-stimulated splenocyte proliferation assay. Both Thpp-like and CD25ϩ FoxP3ϩ cells expressed CD73 di- CD73 expression by Treg cells is consistent with their overall rep- rectly ex vivo, and the in vitro derivation of both Thpp and Treg ertoire of suppressive functions, as CD73 generates adenosine, which cells from naive CD4 T cell populations can be enhanced by suppresses some T cell effector functions. Expression of CD73 (and TGF-␤ (24, 33). Despite these similarities, the lack of expression hence adenosine) may also be consistent with Thpp cell functions, as of CD25 on most Thpp-like cells ex vivo (34) suggested that Thpp Thpp may represent a restrained T cell response that allows Ag-spe- and Treg cells are separate populations, defined mainly by the cific T cell expansion without strong, potentially harmful effector surface marker patterns CD73ϩLy-6A/ElowCD25ϪFoxP3Ϫ (Thpp) functions. Thus, Thpp may not only lack strong effector functions but and CD73ϩLy-6A/Eϩ/Ϫ CD25ϩFoxP3ϩ (Treg). Two functional may also dampen the effector functions of Th1 or Th2 cells in the properties of Thpp and Treg cells were used to test this hypothesis: vicinity. To determine whether the amount of CD73 expressed by Thpp cells secrete IL-2 on stimulation, and Treg cells suppress T Treg and Thpp populations could convert sufficient 5Ј-AMP to aden- cell responses. osine to suppress Th1 and Th2 responses, we first determined the CD25ϪCD73ϩLy-6A/EϪ (potential Thpp) and CD25ϩCD73ϩ sensitivity of Th1 and Th2 responses to adenosine and then analyzed (potential Treg) populations were isolated by cell sorting from the suppression of Th1 responses by adenosine generated by Thpp or splenocytes, stimulated through the TCR, and their cytokine pro- Treg cells. The Journal of Immunology 6783 Downloaded from http://www.jimmunol.org/

FIGURE 3. Treg and not Thpp cells suppress CD4 and CD8 proliferation. CFSE-labeled splenocytes (5 ϫ 104) isolated from B6.PL (Thy1.1) mice were stimulated with anti-CD3 stimulated for 72 h in the presence of naive, Thpp, or Treg cells isolated from spleens of C57BL/6 mice. a, Rep- resentative CFSE histograms of CD4 proliferation from cultures that were unstimulated (Ϫ), stimulated with anti-CD3 alone (ϩ), or stimulated in the presence of 2.5 ϫ 104 naive, Thpp, or Treg cells. Plots were gated on Ϫ Ϫ ϩ

7AAD CD19 CD90.1 cells. b, CD4 and CD8 proliferation following by guest on October 2, 2021 culture of CFSE-labeled B6.PL splenocytes in the presence of various numbers of naive, Thpp, or Treg cells was evaluated by ﬂow cytometry by gating on 7AADϪCD19ϪCD90.1ϩ cells. Data are representative of three independent experiments.

Adenosine inhibits the cytokine production and proliferation of Th1 and Th2 cells Adenosine has been demonstrated to inhibit T cell production of IFN-␥ (13), although this required concentrations (50 ␮M) (13, 36)

that were considerably higher than the Kd of the A2A receptor for various adenosine analogues (16). The use of hydrolysis-resistant

A2A receptor agonists and A2A receptor antagonists has demonstrated that suppression of IFN-␥ production by effector CD4 T

cells is mediated through the A2A receptor (15). In preliminary experiments we confirmed that IFN-␥ synthesis by Th1 cells was inhibited only at high adenosine concentrations, and so we ad- dressed the possibility that high concentrations were required because adenosine was rapidly degraded in culture. Th1 cells, pre- pared by in vitro differentiation (35), were stimulated with anti- CD3 and anti-CD28 Abs in the presence of varying concentrations of adenosine, and the number of IFN-␥-secreting cells was mea- FIGURE 2. Cytokine production by Thpp and Treg cells. a, Naive sured by a Fluorospot assay (32). In medium containing serum, (CD44ϪCD25ϪCD73Ϫ), Thpp (CD44ϩCD25ϪCD73ϩLy-6A/EϪ), and ϩ ϩ ϩ adenosine minimally reduced the number of IFN-␥-secreting cells Treg (CD25 CD73 ) CD4 T cells were isolated from C57BL/6 spleno- ␮ cytes and stimulated with plate-bound anti-CD3 and anti-CD28 in IL-2, even at a concentration of 10 M (data not shown), whereas in ␮ Ͻ MIP-1␣, and IFN-␥ ELISPOT assays for 48 h. b, Splenocytes from serum-free medium even 0.1 M adenosine significantly ( p C57BL/6 mice, in vitro derived Thpp, or in vitro derived Th1 cultures were 0.0001) inhibited IFN-␥ production (Fig. 4). The sensitivity of the stimulated with anti-CD3 and anti-CD28 for 12 h and evaluated for IL-2 Th1 cells to suppression by Ͻ0.1 ␮M adenosine in serum-free ϩ secretion and FoxP3 expression by flow cytometry. Plots are gated on CD4 medium (Fig. 5) is consistent with the Kd of the A2A receptor for CD19Ϫ cells. Data are representative of two independent experiments. several adenosine analogues (16). Serum did not significantly alter 6784 CD73 MEDIATES Thpp AND Treg SUPPRESSION OF Th1/Th2 RESPONSES

FIGURE 5. Suppression of IFN-␥ production by adenosine is mediated ␥ through the A2a receptor. Th1 cells were stimulated in IFN- Fluorospot assays as indicated above with increasing doses of adenosine or 5Ј-AMP in

the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxan- ␮ ␮ thine (DP-CPX; 5 M) or the A2A receptor antagonist SCH58261 (5 M). Means Ϯ SEM of triplicate wells are shown. Data are representative of three independent experiments.

with physiological dilutions of 5Ј-AMP (38) and measuring the Downloaded from number of Th1 cells secreting IFN-␥ in response to anti-CD3 and anti-CD28 stimulation. In the absence of 5Ј-AMP, none of these three T cell populations inhibited the synthesis of IFN-␥ by anti- CD3- and anti-CD28-stimulated Th1 cells (Fig. 6). The lack of Treg-mediated suppression of IFN-␥ synthesis in the absence of FIGURE 4. Adenosine inhibits the cytokine production and prolifera- 5Ј-AMP is consistent with previous reports indicating that stimu- http://www.jimmunol.org/ tion of Th1 and Th2 cells. a, Th1 and Th2 cells were cultured in X-VIVO ␮ Ј lation through CD28 can block Treg-mediated suppression (39– 20 medium with the absence or presence of 1 M adenosine or 5 -AMP Ј and stimulated with plate-bound anti-CD3 and anti-CD28 in either IFN-␥ 41). However, the presence of 5 -AMP at concentrations as low as ␮ ␥ or IL-4 Fluorospot assays for 4 and 16 h, respectively. Data are represen- 0.5 M resulted in the inhibition of Th1 IFN- synthesis in cul- ϩ Ϫ p Ͻ 0.01 (significant differ- tures containing CD73 Thpp or Treg cells, but not CD73 naive ,ء .tative of mean Ϯ SEM of triplicate wells ence). b, CFSE-labeled Th1 or Th2 cells were stimulated in X-VIVO 20 cells. The role of CD73 in inhibiting IFN-␥ production was con- medium with plate-bound anti-CD3 and anti-CD28 with IL-2 in the pres- firmed by showing that suppression was prevented by a specific ence of 10 ␮M adenosine for 72 h and stained with 7AAD, anti-CD4, and inhibitor of CD73 enzymatic activity, APCP (42). An A2A receptor anti-CD19 and evaluated for proliferation by flow cytometry. Data are antagonist (SCH58261) also prevented suppression, confirming Ͻ by guest on October 2, 2021 ء Ϯ mean SD of triplicate wells. , p 0.05 (significant difference). that the effect was mediated through adenosine (Fig. 6). The adenosine-mediated inhibitory effect of Thpp and Treg cells was ex- erted at ratios of less than 1:1 of suppressor to target cells. the number of IFN-␥-secreting Th1 cells in the absence of adenosine (data not shown). 5Ј-AMP did not inhibit IFN-␥ production Discussion in serum-free medium (Fig. 4a). Adenosine also significantly ( p ϭ Taken together, these results strongly support a model (Fig. 7) in 0.006) reduced the number of IL-4-secreting cells in stimulated which the expression of CD73 by the two distinct CD4 T cell subsets, Th2 cell populations at a similar concentration to that required for Thpp and Treg, confers a potential anti-inflammatory function. In this Th1 cell inhibition (Fig. 4a). model, 5Ј-AMP released at a site of ongoing inflammation (43, 44) is The effects of adenosine on proliferation of Th1 and Th2 cells cleaved by CD73 on local Treg or Thpp cells, and the resulting aden-

were tested by stimulating CFSE-labeled Th1 or Th2 cells with osine acts directly through the A2A receptor on effector T cells such as anti-CD3, anti-CD28, and IL-2 in serum-free medium in the pres- Th1 cells to inhibit their proliferation and secretion of cytokines. This ence or absence of adenosine. Proliferation of Th1 or Th2 cells, as in turn will mitigate ongoing inflammation, providing negative feed- measured by dilution of CFSE, was significantly ( p ϭ 0.0006 and back regulation between cell damage (and release of 5Ј-AMP) and 0.0003, respectively) inhibited by adenosine (Fig. 4b). inflammatory cytokine production. Consistent with previous reports, the suppressive activity of This potential suppressive pathway is distinct from the conven- adenosine on Th1 cytokine secretion was abrogated by the addition tional suppressive mechanism of Treg cells, as revealed in different

of the A2A receptor antagonist SCH58261(14), whereas the A1 assays (Figs. 3 and 6). If target T cells are stimulated only with receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (37) had anti-CD3 (without anti-CD28 or 5Ј-AMP), the resulting prolifer- minimal effects on the suppressive activity (Fig. 5). ation can be suppressed by Treg (but not Thpp) cells. In this conventional Treg assay, costimulation with anti-CD28 Abs prevents Coculture of Treg and Thpp with Th1 cells in the presence of suppression by Tregs. However, even in the presence of both anti- Ј ␥ 5 -AMP suppresses IFN- production CD3 and anti-CD28, Treg and Thpp cells can suppress by the The suppression of T cell cytokine production by even low con- CD73-dependent pathway if the substrate 5Ј-AMP is added. centrations of adenosine raised the possibility that the CD73 ex- Because both Thpp and Treg cells can be found in peripheral tissue pressed on Thpp and Treg cells could generate sufficient adenosine (22, 26, 45, 46), it is likely that, at an inflammatory site, adenosine (by cleaving 5Ј-AMP) to inhibit effector T cell function. This was generated by these cell types could contribute to other anti- tested by coculturing Th1 cells with ex vivo isolated Thpp inflammatory activities including suppression of B cell, neutrophil, (CD25ϪCD73ϩLy-6A/EϪ), Treg (CD25ϩCD73ϩ), or naive monocyte, macrophage, and dendritic cell functions (3). Adenosine- (CD73ϪCD25ϪCD44Ϫ) CD4 T cells that had been precultured mediated suppression of IFN-␥ production occurred at a ratio of less The Journal of Immunology 6785

FIGURE 7. Model of CD73-mediated suppression of inflammation. ATP released during inflammation from dying cells or activated neutrophils is converted by CD39 to 5Ј-AMP. The resulting 5Ј-AMP is dephos- phorylated by CD73 expressed on the surface of Thpp or Treg cells to adenosine. Adenosine suppresses the production of IFN-␥ and TNF-␣ by effector CD4 T cells, including Th1 cells, thus limiting inflammation. Downloaded from well as CD73/adenosine-mediated suppression of IFN-␥ synthesis (Fig. 6) can occur in the presence of costimulation through CD28. T cell susceptibility to adenosine-mediated suppression would

likely be a function of A2A receptor expression, which is low on naive cells, increases as cells progress to an effector stage, and is

decreased on memory cells (3). This fine tuning would render http://www.jimmunol.org/ those cells with the greatest inflammatory potential (effector cells) the most sensitive to adenosine suppression. FIGURE 6. Thpp and Treg suppress IFN-␥ production in the presence of 5Ј-AMP. Naive, Thpp, and Treg cells (5 ϫ 103) isolated as indicated Although both Treg and Thpp cells have the potential to sup- above were cultured in X-VIVO 20 medium with increasing doses of 5Ј- press inflammatory responses by the CD73/adenosine mechanism, AMP alone or in the presence of either 100 ␮M APCP (CD73 antagonist) these two cell types may be most advantageous during different ␮ or 5 M SCH58261 (A2A receptor antagonist) for 4 h and then added with responses. Thpp cells may be appropriate for temporarily suppress- 7.5 ϫ 103 Th1 cells to IFN-␥ Fluorospot assays and stimulated for 4 h with ing acute antipathogen responses that are causing excessive pa- plate-bound anti-CD3 and anti-CD28. Data (mean Ϯ SEM of triplicates) thology and, in addition, the Thpp cells would retain the potential are representative of three independent experiments. to differentiate later into strong effector (e.g., Th1 or Th2) pheno- by guest on October 2, 2021 types (24, 59). In contrast, Treg cells would be more suitable for long-term suppression of autoimmune anti-inflammatory responses, without the risk of future differentiation into effector cells. than one Thpp or Treg per effector T cell, suggesting that this could be an effective regulatory mechanism of inflammatory reactions. Even after more than a decade of heightened attention, the Acknowledgment mechanism(s) by which Tregs mediate suppression of immune re- We thank Nathan Laniewski for performing flow cytometric cell sorting. sponses remains unclear. In particular, the relationship between their activity in conventional in vitro suppressor assays and their Disclosures ability to mitigate autoimmune pathology in vivo has been difficult The authors have no financial conflict of interest. to define. TGF-␤ has been implicated as a potential mechanism by which Treg cells suppress (47–49). Although extensive studies References ␤ ␤ 1. Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, using both TGF- deficient (50) and TGF- unresponsive Treg R. Kuhn, W. Muller, G. Trinchieri, and A. Sher. 1996. In the absence of endog- (51) and effector T cells (52) have been performed, TGF-␤ is im- enous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal ϩ portant for Treg activity in some but not all studies. Although Treg immune response dependent on CD4 T cells and accompanied by overproduc- tion of IL-12, IFN-␥ and TNF-␣. J. 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