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 convert 5′-AMP generated by neutrophils or dying cells into the anti-inflammatory mediator adenosine, thus dampening excessive immune reactions.

During strong inflammatory immune responses, immune attack on pathogens and infected cells also results in damage to host tissues, so that successful immune responses normally involve a complex balance of pro- and anti-inflammatory mediators. The cytokines IL-10 (1) and TGF-β (2) are both important for restraining vigorous inflammatory responses. In addition to cytokine-mediated immune regulation, other factors may contribute to controlling inflammation including PGE2, NO, glucocorticoids, a hypoxic environment, and adenosine.

Adenosine has various immunoregulatory activities mediated through four adenosine receptors: A1, A2A, A2B, and A3 (3). T lymphocytes mainly express the high affinity A2AR and the low-affinity A2BR (3). Macrophages and neutrophils can express all four adenosine receptors depending on their activation state, and B cells express A2AR (3). Engagement of A2AR inhibits IL-12 production but increases IL-10 production by human monocytes (4) and dendritic cells (5) and selectively decreases some cytotoxic functions (6) and chemokine production by neutrophils (7). A protective role for adenosine in Con A- or endotoxin-induced inflammatory liver damage was revealed by treatment with adenosine agonists and antagonists or in mice deficient for A2AR (8, 9). In vivo treatment with an A2A agonist decreases intestinal inflammation (10). Conversely, the high levels of adenosine in patients with inherited deficiency of the adenosine-degrading enzyme adenosine deaminase are associated with profound depletion of T, B, and NK lymphocytes (11, 12).

Treatment of T lymphocytes with adenosine or the A2A agonist inhibits proliferation (13, 14), as well as the secretion of cytokines including IL-2, TNF-α, and IFN-γ (14, 15). However, the concentrations of adenosine required to elicit in vitro effects (25 μM) (13) were substantially higher than the Kd of various adenosine analogues for the A2A receptor (20–50 nM) (16). The Kd of the A2A receptor for adenosine itself is difficult to determine (17).

The generation of adenosine at sites of inflammation, hypoxia, organ injury, and traumatic shock is in part mediated by two sequential enzymes. The ecto-ATP diphosphohydrolase (CD39) expressed by neutrophils rapidly converts circulating ATP and ADP to 5′-AMP (18). CD73, expressed on the surface of endothelial cells (19) and a subset of T cells (20, 21, 22), is an ecto-5′-nucleotidase (23) that can convert 5′-AMP to adenosine.

We have recently identified, directly ex vivo, a population of memory CD4 T cells that express CD73 but not Ly-6A/E (22). These cells are similar to the uncommitted primed precursor helper cells (Thpp)3 previously identified in vitro (24). Both cell types secrete IL-2 and chemokines but not IFN-γ or IL-4 when activated (22, 24), both localize preferentially to lymph nodes (22, 25), and both have the flexibility to differentiate further into either Th1 or Th2 phenotypes depending on the polarizing cytokine environment. These Thpp cells may provide an expanded pool of uncommitted Ag-specific T cells without potentially harmful inflammatory functions.

The inhibitory activity of CD4 T cells has primarily been associated with FoxP3+ Treg cells, many of which express CD25 (26, 27). FoxP3 is important in the development and activity of Treg cells, because CD25+ cells isolated from FoxP3-deficient mice lack regulatory activity in vitro and in vivo, and retroviral expression of FoxP3 in nonregulatory T cells confers regulatory activity (28, 29). The mechanism(s) by which Tregs control immune responses in vivo and in vitro may include the suppressive cytokines TGF-β and IL-10, modulation of APC function, and sequestering of IL-2 (30).

We now show that Treg cells also express CD73. Although Treg and Thpp cells also share other properties and both can differentiate from naive CD4 T cells under similar conditions in vitro, we show that these are two distinct populations. Adenosine produced by either cell type potently suppresses proliferation of effector CD4 T cells and also inhibits their production of cytokines. The inhibition of effector T cells occurs at low adenosine levels (provided that adenosine degradation is minimized), and either Thpp or Treg cells derived directly ex vivo can significantly inhibit effector cells at a ratio of less than 1:1. Thus, the CD73/adenosine pathway is a potent additional suppressive pathway of Treg cells and also confers a suppressive anti-inflammatory function on the uncommitted precursor Thpp cell type.

Female C57BL/6 and C57BL/6.PL mice (6–8 wk of age) were obtained from The Jackson Laboratory. All animal experiments were approved by the University of Rochester University Committee on Animal Resources (Rochester, NY).

Purified anti-mouse IL-2 (clone JES6-1A12) and biotinylated anti-mouse IL-2 (clone JES6-5H4), IL-4 (clone BVD6-2462), and IFN-γ (clone XMG1.2), were obtained from eBioscience. Purified anti-mouse IL-4 (clone 11B11) and IFN-γ (clones AN18 and XMG1.2) were obtained from hybridoma cell lines as previously described (22). Purified anti-mouse MIP-1α (clone 39624.11) and biotinylated anti-mouse MIP-1α (clone BAF450) were obtained from R&D Systems. The following FITC-, R-PE-, PE-Cy5.5-, PE-Cy7-, PerCP-, PerCP-Cy5.5-, allophycocyanin-, allophycocyanin-Cy7, and biotin-conjugated Abs were purchased from BD Pharmingen: anti-Ly-6A/E (sca-1) (clone D7), anti-CD73 (clone Y/23), anti-CD4 (clone RM4-5), anti-CD44 (clone IM7), anti-CD19 (clone MB19-1), anti-CD8α (clone 53-6.7), anti-CD25 (clone PC61.5), and anti-CD62L (clone Mel-14). Alexa Fluor 405-conjugated anti-mouse CD4 (clone RM4-5) was obtained from Caltag Laboratories. Staining with anti-FoxP3-FITC- conjugated Ab (clone FJK-16s) was performed according to the manufacturer’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).

Splenocytes isolated from C57BL/6 mice were evaluated for IL-2 secretion by the Miltenyi cytokine secretion assay (Miltenyi Biotec) according to the manufacturer’s protocol. Twenty million splenocytes were stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) per well in a 12-well culture plate for 12 h. Cells were collected and labeled with the IL-2 capture reagent followed by a 45-min secretion period, and subsequent staining with the PE-conjugated anti-IL2 detection Ab 7AAD and anti-CD4 and anti-CD19 Abs. FoxP3 expression was then determined as indicated above.

Subsets of CD4 T cells were isolated by staining with subsaturating levels of PE-Alexa Fluor 610-anti-CD8α, FITC-anti-CD19, PE-CD73, PE-Cy7-anti-CD25, PerCP-Cy5.5-anti-CD4, Ly-6A/E-allophycocyanin, and allophycocyanin-Cy7-anti-CD44 and collecting the CD4+CD8αCD19CD25CD44lowCD73 (naive), CD4+CD8αCD19CD25CD44highCD73+Ly-6A/E (Thpp), and CD4+CD8αCD19CD25+CD73+ (Treg) populations using a BD FACSAria flow cytometric cell sorter (BD Immunocytometry). Subsaturating levels of Abs, particularly anti-CD4, were used to minimize functional alterations of labeled cells.

CD4+ T cell populations were isolated from the spleens of C57BL/6 (CD90.2+) mice by flow cytometric cell sorting as indicated above. Increasing numbers of CD4+ T cell populations were cultured with 50,000 CFSE-labeled splenocytes isolated from C57BL/6.PL (CD90.1+) in the presence of 1 μg/ml anti-CD3 in round-bottom 96-well plates for 72 h. Following incubation, triplicate wells were pooled, stained with anti-CD4, anti-CD8, anti-CD19, anti-CD90.1, anti-CD90.2, and 7AAD, and analyzed by flow cytometry using a BD LSRII device (BD Biosciences).

Naive CD4 T cells (CD4+CD62L+CD44lowCD25) were isolated by cell sorting as indicated above. Naive CD4+ T cells (104) were cultured with 104 irradiated (10,000 rad) allogeneic TA3 cells in 200 μl of Alpha MEM containing 10% FBS, 1 ng/ml IL-2, and cytokine/anti-cytokine supplements. Thpp cultures contained 2 ng/ml TGF-β1 and 50 μg/ml anti-IFN-γ Ab (clone XMG1.2). Th1 cultures contained 1 ng/ml IL-12 and 5 μg/ml anti-IL-4 Ab (clone 11B11), and Th2 cultures contained 1 ng/ml IL-4 and 10 μg/ml anti-IFN-γ Ab (clone XMG1.2). On day 4 of culture, half of the medium was removed and replenished with new medium and cytokines. Fresh IL-2 and medium were added to cultures weekly.

Detection of IL-2, IFN-γ, and MIP-1α 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-γ and IL-4 by Fluorospot was performed by coating with either 2 μg/ml anti-IFN-γ Ab (clone AN18) or 10 μg/ml anti-IL-4 (11B11) with 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28. Anti-IFN-γ (clone XMG1.2) or anti-IL-4 biotinylated Ab was added at 1 μg/ml. After incubation, the plates were washed in PBS containing 0.1% Tween 20 (PBST) and 2 μg/ml streptavidin-conjugated Cy3 in PBST plus 2% BSA was added for 30 min. The plates were washed again with PBST, dried, and scanned using a fluorescent microscope, and the digitized images were quantified by the ExploraSpot image analysis program (J. Rebhahn, J. J. Kobie, D. M. Zaiss, T. R. Mosmann, results not shown).

Naive, Thpp, or Treg cell populations were isolated as indicated above and cultured in 96-well round-bottom plates in X-VIVO 20 medium (Cambrex) in the presence of 0.5 μM 5′-AMP (Sigma-Aldrich) and 100 μM α,β-methylene ADP (APCP) (Sigma-Aldrich) or 25 μM SCH58261 (Sigma-Aldrich) as indicated for 4 h at 37°C. After addition of Th1 cells, IFN-γ secretion was measured by restimulation in 4-h Fluorospot assays as described above.

Where statistical difference is indicated two-tailed unpaired Student’s t tests with a 95% confidence interval were performed using Prism software (GraphPad).

Uncommitted precursor Thpp-like CD4 T cells (secreting IL-2 and chemokines) are mainly CD44+CD73+Ly-6A/E (22), whereas naive (CD44low) CD4 T cells (secreting only IL-2) do not express significant levels of either CD73 or Ly-6A/E. Effector cells (secreting IFN-γ or IL-4) express high levels of Ly-6A/E in Ly-6.2 (C57BL/6, C57L, PL, C58, SJL, and AKR) mouse strains, but most effectors do not significantly express CD73(22). Fig. 1 a shows that most CD25+CD4+ T cells from the spleens of C57BL/6 mice also expressed CD73, raising the possibility that Treg cells are CD73+. Substantial numbers of CD44+ CD25 CD73+ Ly-6A/E cells (potentially Thpp cells) were also present.

FIGURE 1.

CD73 is expressed by T regulatory and Thpp cells. Splenocytes from C57BL/6 mice were isolated and analyzed by flow cytometry for cell surface marker expression (a and b) and intracellular expression of FoxP3 (b). Data are representative of at least three independent experiments.

FIGURE 1.

CD73 is expressed by T regulatory and Thpp cells. Splenocytes from C57BL/6 mice were isolated and analyzed by flow cytometry for cell surface marker expression (a and b) and intracellular expression of FoxP3 (b). Data are representative of at least three independent experiments.

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Because activated T cells also express CD25 and some suppressive CD4 T cells do not express CD25 (26), we next determined the relationship between the expression of CD73 and FoxP3, a member of the Forkhead transcription factor family that is strongly associated with the Treg suppressor phenotype (28). Spleen cells were stained for CD73, CD44 and Ly-6A/E expression and subsequently permeabilized and stained for FoxP3 expression. As expected, the FoxP3CD44 (naive) population stained at low levels with anti-CD73 (mean fluorescence intensity, 1227) (Fig. 1,b). The FoxP3+ population expressed higher levels of CD73 (mean fluorescence intensity, 2171) (Fig. 1 b). This was also observed in BALB/c and B10.D2 mice (data not shown). Real-time PCR also showed that FoxP3 was expressed mainly in sorted CD73+CD25+ cells but not in CD73 cells (data not shown).

Both Thpp-like and CD25+ FoxP3+ cells expressed CD73 directly ex vivo, and the in vitro derivation of both Thpp and Treg cells from naive CD4 T cell populations can be enhanced by TGF-β (24, 33). Despite these similarities, the lack of expression of CD25 on most Thpp-like cells ex vivo (34) suggested that Thpp and Treg cells are separate populations, defined mainly by the surface marker patterns CD73+Ly-6A/ElowCD25FoxP3 (Thpp) and CD73+Ly-6A/E+/− CD25+FoxP3+ (Treg). Two functional properties of Thpp and Treg cells were used to test this hypothesis: Thpp cells secrete IL-2 on stimulation, and Treg cells suppress T cell responses.

CD25CD73+Ly-6A/E (potential Thpp) and CD25+CD73+ (potential Treg) populations were isolated by cell sorting from splenocytes, stimulated through the TCR, and their cytokine production profiles were tested by an ELISPOT assay. The putative Thpp-enriched population produced IL-2 and MIP-1α but not IFN-γ, consistent with the Thpp cytokine profile (35). In contrast, negligible numbers of the putative Treg population produced IL-2 or IFN-γ, and only a low frequency produced MIP-1α (Fig. 2,a). Naive (CD25CD44low) cells produced primarily IL-2 (Fig. 2,a). Although these results strongly suggested that Treg and Thpp cells were separate populations, a minority of FoxP3+ cells are CD25 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 FoxP3+ and IL-2+ splenocyte cell populations were separate, indicating that FoxP3+ cells within the CD4+CD25CD73+Ly-6A/E population do not contribute to the observed IL-2 production (Fig. 2 b).

FIGURE 2.

Cytokine production by Thpp and Treg cells. a, Naive (CD44CD25CD73), Thpp (CD44+CD25CD73+Ly-6A/E), and Treg (CD25+CD73+) CD4+ T cells were isolated from C57BL/6 splenocytes and stimulated with plate-bound anti-CD3 and anti-CD28 in IL-2, MIP-1α, and IFN-γ ELISPOT assays for 48 h. b, Splenocytes from C57BL/6 mice, in vitro derived Thpp, or in vitro derived Th1 cultures were stimulated with anti-CD3 and anti-CD28 for 12 h and evaluated for IL-2 secretion and FoxP3 expression by flow cytometry. Plots are gated on CD4+ CD19 cells. Data are representative of two independent experiments.

FIGURE 2.

Cytokine production by Thpp and Treg cells. a, Naive (CD44CD25CD73), Thpp (CD44+CD25CD73+Ly-6A/E), and Treg (CD25+CD73+) CD4+ T cells were isolated from C57BL/6 splenocytes and stimulated with plate-bound anti-CD3 and anti-CD28 in IL-2, MIP-1α, and IFN-γ ELISPOT assays for 48 h. b, Splenocytes from C57BL/6 mice, in vitro derived Thpp, or in vitro derived Th1 cultures were stimulated with anti-CD3 and anti-CD28 for 12 h and evaluated for IL-2 secretion and FoxP3 expression by flow cytometry. Plots are gated on CD4+ CD19 cells. Data are representative of two independent experiments.

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A subpopulation of FoxP3+ cells was also present in cultures of cells differentiated in vitro under Thpp-polarizing conditions (TGF-β and anti-IFN-γ), raising the possibility that the “Thpp” cultures also contained some Treg cells. This was supported by the observation that FoxP3+ cells were also distinct from IL-2-secreting cells in Thpp populations derived in vitro (Fig. 2,b) as in the ex vivo populations. Similar IL-2 and FoxP3 expression patterns were 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 FoxP3+ population (Fig. 2 b).

We next tested whether the functions of ex vivo Thpp (CD25CD73+Ly-6A/E) and Treg (CD25+CD73+Ly-6A/E+/−) cells matched their FoxP3 and cytokine expression patterns. Suppression of lymphocyte proliferation was measured by soluble anti-CD3 stimulation of CFSE-labeled splenocytes in the presence of sorted Thpp, Treg, or naive cells (and the absence of the CD73 substrate 5′-AMP). Treg cells markedly suppressed proliferation of CD4+ and CD8+ T cells (>50% 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 FoxP3+ cells present in the Thpp population. No significant difference in suppressive activity was observed between CD25+CD73+Ly-6A/E and CD25+CD73+Ly-6A/E+ cells (data not shown).

FIGURE 3.

Treg and not Thpp cells suppress CD4 and CD8 proliferation. CFSE-labeled splenocytes (5 × 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, Representative CFSE histograms of CD4 proliferation from cultures that were unstimulated (−), stimulated with anti-CD3 alone (+), or stimulated in the presence of 2.5 × 104 naive, Thpp, or Treg cells. Plots were gated on 7AADCD19CD90.1+cells. b, CD4 and CD8 proliferation following 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 7AADCD19CD90.1+ cells. Data are representative of three independent experiments.

FIGURE 3.

Treg and not Thpp cells suppress CD4 and CD8 proliferation. CFSE-labeled splenocytes (5 × 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, Representative CFSE histograms of CD4 proliferation from cultures that were unstimulated (−), stimulated with anti-CD3 alone (+), or stimulated in the presence of 2.5 × 104 naive, Thpp, or Treg cells. Plots were gated on 7AADCD19CD90.1+cells. b, CD4 and CD8 proliferation following 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 7AADCD19CD90.1+ cells. Data are representative of three independent experiments.

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Together, these results demonstrate that although CD73 is expressed by both Thpp and Treg populations, each has distinct characteristics; only the Thpp cells secrete IL-2, and only the Treg population is capable of suppression in the anti-CD3-stimulated splenocyte proliferation assay.

CD73 expression by Treg cells is consistent with their overall repertoire of suppressive functions, as CD73 generates adenosine, which suppresses some T cell effector functions. Expression of CD73 (and hence adenosine) may also be consistent with Thpp cell functions, as Thpp may represent a restrained T cell response that allows Ag-specific T cell expansion without strong, potentially harmful effector functions. Thus, Thpp may not only lack strong effector functions but may also dampen the effector functions of Th1 or Th2 cells in the vicinity. To determine whether the amount of CD73 expressed by Treg and Thpp populations could convert sufficient 5′-AMP to adenosine to suppress Th1 and Th2 responses, we first determined the sensitivity of Th1 and Th2 responses to adenosine and then analyzed the suppression of Th1 responses by adenosine generated by Thpp or Treg cells.

Adenosine has been demonstrated to inhibit T cell production of IFN-γ (13), although this required concentrations (50 μM) (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-γ production by effector CD4 T cells is mediated through the A2A receptor (15). In preliminary experiments we confirmed that IFN-γ synthesis by Th1 cells was inhibited only at high adenosine concentrations, and so we addressed the possibility that high concentrations were required because adenosine was rapidly degraded in culture. Th1 cells, prepared 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-γ-secreting cells was measured by a Fluorospot assay (32). In medium containing serum, adenosine minimally reduced the number of IFN-γ-secreting cells even at a concentration of 10 μM (data not shown), whereas in serum-free medium even 0.1 μM adenosine significantly (p < 0.0001) inhibited IFN-γ production (Fig. 4). The sensitivity of the Th1 cells to suppression by <0.1 μM adenosine in serum-free medium (Fig. 5) is consistent with the Kd of the A2A receptor for several adenosine analogues (16). Serum did not significantly alter the number of IFN-γ-secreting Th1 cells in the absence of adenosine (data not shown). 5′-AMP did not inhibit IFN-γ production in serum-free medium (Fig. 4,a). Adenosine also significantly (p = 0.006) reduced the number of IL-4-secreting cells in stimulated Th2 cell populations at a similar concentration to that required for Th1 cell inhibition (Fig. 4 a).

FIGURE 4.

Adenosine inhibits the cytokine production and proliferation of Th1 and Th2 cells. a, Th1 and Th2 cells were cultured in X-VIVO 20 medium with the absence or presence of 1 μM adenosine or 5′-AMP and stimulated with plate-bound anti-CD3 and anti-CD28 in either IFN-γ or IL-4 Fluorospot assays for 4 and 16 h, respectively. Data are representative of mean ± SEM of triplicate wells. ∗, p < 0.01 (significant difference). b, CFSE-labeled Th1 or Th2 cells were stimulated in X-VIVO 20 medium with plate-bound anti-CD3 and anti-CD28 with IL-2 in the presence of 10 μM adenosine for 72 h and stained with 7AAD, anti-CD4, and anti-CD19 and evaluated for proliferation by flow cytometry. Data are mean ± SD of triplicate wells. ∗, p < 0.05 (significant difference).

FIGURE 4.

Adenosine inhibits the cytokine production and proliferation of Th1 and Th2 cells. a, Th1 and Th2 cells were cultured in X-VIVO 20 medium with the absence or presence of 1 μM adenosine or 5′-AMP and stimulated with plate-bound anti-CD3 and anti-CD28 in either IFN-γ or IL-4 Fluorospot assays for 4 and 16 h, respectively. Data are representative of mean ± SEM of triplicate wells. ∗, p < 0.01 (significant difference). b, CFSE-labeled Th1 or Th2 cells were stimulated in X-VIVO 20 medium with plate-bound anti-CD3 and anti-CD28 with IL-2 in the presence of 10 μM adenosine for 72 h and stained with 7AAD, anti-CD4, and anti-CD19 and evaluated for proliferation by flow cytometry. Data are mean ± SD of triplicate wells. ∗, p < 0.05 (significant difference).

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FIGURE 5.

Suppression of IFN-γ production by adenosine is mediated through the A2a receptor. Th1 cells were stimulated in IFN-γ Fluorospot assays as indicated above with increasing doses of adenosine or 5′-AMP in the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DP-CPX; 5 μM) or the A2A receptor antagonist SCH58261 (5 μM). Means ± SEM of triplicate wells are shown. Data are representative of three independent experiments.

FIGURE 5.

Suppression of IFN-γ production by adenosine is mediated through the A2a receptor. Th1 cells were stimulated in IFN-γ Fluorospot assays as indicated above with increasing doses of adenosine or 5′-AMP in the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DP-CPX; 5 μM) or the A2A receptor antagonist SCH58261 (5 μM). Means ± SEM of triplicate wells are shown. Data are representative of three independent experiments.

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The effects of adenosine on proliferation of Th1 and Th2 cells were tested by stimulating CFSE-labeled Th1 or Th2 cells with anti-CD3, anti-CD28, and IL-2 in serum-free medium in the presence or absence of adenosine. Proliferation of Th1 or Th2 cells, as measured by dilution of CFSE, was significantly (p = 0.0006 and 0.0003, respectively) inhibited by adenosine (Fig. 4 b).

Consistent with previous reports, the suppressive activity of adenosine on Th1 cytokine secretion was abrogated by the addition of the A2A receptor antagonist SCH58261(14), whereas the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (37) had minimal effects on the suppressive activity (Fig. 5).

The suppression of T cell cytokine production by even low concentrations of adenosine raised the possibility that the CD73 expressed on Thpp and Treg cells could generate sufficient adenosine (by cleaving 5′-AMP) to inhibit effector T cell function. This was tested by coculturing Th1 cells with ex vivo isolated Thpp (CD25CD73+Ly-6A/E), Treg (CD25+CD73+), or naive (CD73CD25CD44) CD4 T cells that had been precultured with physiological dilutions of 5′-AMP (38) and measuring the number of Th1 cells secreting IFN-γ in response to anti-CD3 and anti-CD28 stimulation. In the absence of 5′-AMP, none of these three T cell populations inhibited the synthesis of IFN-γ by anti-CD3- and anti-CD28-stimulated Th1 cells (Fig. 6). The lack of Treg-mediated suppression of IFN-γ synthesis in the absence of 5′-AMP is consistent with previous reports indicating that stimulation through CD28 can block Treg-mediated suppression (39, 40, 41). However, the presence of 5′-AMP at concentrations as low as 0.5 μM resulted in the inhibition of Th1 IFN-γ synthesis in cultures containing CD73+ Thpp or Treg cells, but not CD73 naive cells. The role of CD73 in inhibiting IFN-γ production was confirmed by showing that suppression was prevented by a specific inhibitor of CD73 enzymatic activity, APCP (42). An A2A receptor antagonist (SCH58261) also prevented suppression, confirming that the effect was mediated through adenosine (Fig. 6). The adenosine-mediated inhibitory effect of Thpp and Treg cells was exerted at ratios of less than 1:1 of suppressor to target cells.

FIGURE 6.

Thpp and Treg suppress IFN-γ production in the presence of 5′-AMP. Naive, Thpp, and Treg cells (5 × 103) isolated as indicated above were cultured in X-VIVO 20 medium with increasing doses of 5′-AMP alone or in the presence of either 100 μM APCP (CD73 antagonist) or 5 μM SCH58261 (A2A receptor antagonist) for 4 h and then added with 7.5 × 103 Th1 cells to IFN-γ Fluorospot assays and stimulated for 4 h with plate-bound anti-CD3 and anti-CD28. Data (mean ± SEM of triplicates) are representative of three independent experiments.

FIGURE 6.

Thpp and Treg suppress IFN-γ production in the presence of 5′-AMP. Naive, Thpp, and Treg cells (5 × 103) isolated as indicated above were cultured in X-VIVO 20 medium with increasing doses of 5′-AMP alone or in the presence of either 100 μM APCP (CD73 antagonist) or 5 μM SCH58261 (A2A receptor antagonist) for 4 h and then added with 7.5 × 103 Th1 cells to IFN-γ Fluorospot assays and stimulated for 4 h with plate-bound anti-CD3 and anti-CD28. Data (mean ± SEM of triplicates) are representative of three independent experiments.

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Taken together, these results strongly support a model (Fig. 7) in which the expression of CD73 by the two distinct CD4 T cell subsets, Thpp and Treg, confers a potential anti-inflammatory function. In this model, 5′-AMP released at a site of ongoing inflammation (43, 44) is cleaved by CD73 on local Treg or Thpp cells, and the resulting adenosine acts directly through the A2A receptor on effector T cells such as Th1 cells to inhibit their proliferation and secretion of cytokines. This in turn will mitigate ongoing inflammation, providing negative feedback regulation between cell damage (and release of 5′-AMP) and inflammatory cytokine production.

FIGURE 7.

Model of CD73-mediated suppression of inflammation. ATP released during inflammation from dying cells or activated neutrophils is converted by CD39 to 5′-AMP. The resulting 5′-AMP is dephosphorylated by CD73 expressed on the surface of Thpp or Treg cells to adenosine. Adenosine suppresses the production of IFN-γ and TNF-α by effector CD4 T cells, including Th1 cells, thus limiting inflammation.

FIGURE 7.

Model of CD73-mediated suppression of inflammation. ATP released during inflammation from dying cells or activated neutrophils is converted by CD39 to 5′-AMP. The resulting 5′-AMP is dephosphorylated by CD73 expressed on the surface of Thpp or Treg cells to adenosine. Adenosine suppresses the production of IFN-γ and TNF-α by effector CD4 T cells, including Th1 cells, thus limiting inflammation.

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This potential suppressive pathway is distinct from the conventional suppressive mechanism of Treg cells, as revealed in different assays (Figs. 3 and 6). If target T cells are stimulated only with anti-CD3 (without anti-CD28 or 5′-AMP), the resulting proliferation can be suppressed by Treg (but not Thpp) cells. In this conventional Treg assay, costimulation with anti-CD28 Abs prevents suppression by Tregs. However, even in the presence of both anti-CD3 and anti-CD28, Treg and Thpp cells can suppress by the CD73-dependent pathway if the substrate 5′-AMP is added.

Because both Thpp and Treg cells can be found in peripheral tissue (22, 26, 45, 46), it is likely that, at an inflammatory site, adenosine generated by these cell types could contribute to other anti-inflammatory activities including suppression of B cell, neutrophil, monocyte, macrophage, and dendritic cell functions (3). Adenosine-mediated suppression of IFN-γ production occurred at a ratio of less 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 mechanism(s) by which Tregs mediate suppression of immune responses remains unclear. In particular, the relationship between their activity in conventional in vitro suppressor assays and their ability to mitigate autoimmune pathology in vivo has been difficult to define. TGF-β has been implicated as a potential mechanism by which Treg cells suppress (47, 48, 49). Although extensive studies using both TGF-β deficient (50) and TGF-β unresponsive Treg (51) and effector T cells (52) have been performed, TGF-β is important for Treg activity in some but not all studies. Although Treg cells from IL-10 deficient mice were suppressive in vitro, they failed to suppress immunity to Leishmania (53) and intestinal inflammation (54). Treg cell-mediated suppression may also occur indirectly through modulation of APC function (55, 56, 57) and through an unknown mechanism requiring cell-to-cell contact or close proximity (58).

Thus, Treg cells inhibit responses by multiple mechanisms, and inhibition by CD73/adenosine may be an additional Treg suppressive mechanism, absent in the standard Treg in vitro assays due to lack of 5′-AMP but contributing to the overall suppressive activity of Tregs in vivo. The in vitro ability of Tregs to suppress proliferation and IL-2 production is abrogated by stimulation through CD28, high Ag dose, or addition of IL-2 (39, 40, 41) when 5′-AMP is absent. Our results demonstrate that adenosine-mediated suppression of proliferation and cytokine synthesis (Figs. 4 and 6) as well as CD73/adenosine-mediated suppression of IFN-γ 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 those cells with the greatest inflammatory potential (effector cells) the most sensitive to adenosine suppression.

Although both Treg and Thpp cells have the potential to suppress inflammatory responses by the CD73/adenosine mechanism, these two cell types may be most advantageous during different responses. Thpp cells may be appropriate for temporarily suppressing acute antipathogen responses that are causing excessive pathology and, in addition, the Thpp cells would retain the potential to differentiate later into strong effector (e.g., Th1 or Th2) phenotypes (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.

We thank Nathan Laniewski for performing flow cytometric cell sorting.

The authors have no financial conflict of interest.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants RO1 AI48604 and T32 CA09363-22 (to J.J.K.).

3

Abbreviations used in this paper: Thpp, primed precursor Th cells; APCP, α,β-methylene ADP.

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