This study reports on the identification of CD39 together with CD73 as novel cell surface markers of CD4+
T reg cells. In addition to the expression of both ectonucleotidases by T reg cells, the robust generation of adenosine controlled by these ectoenzymes functionally distinguishes classic T reg cells from other CD4+
T cell populations. Our observations concerning extracellular adenosine production by T reg cells have been further developed by applying models conceived by Sitkovsky and others (19
) that show that adenosine receptors on targeted T effector cells mediate suppressive functions. Gavin et al. (8
) predict from gene clustering models that there are at least three potential T reg cell mechanisms inclusive of “suppressive soluble factors, generation of extracellular adenosine and release of reactive oxygen.” We demonstrate in this study that alterations in pericellular levels of adenosine brought about by CD39 (and CD73) associated with T reg cells have a major, albeit not total, suppressive effect on cellular responses in vitro. We also show that CD39 plays a nonredundant role in the suppressive capabilities of T reg cells in vivo, as demonstrated by the relative failure of Cd39
-null T reg cells to block skin allograft rejection. We propose that cell-associated CD39 initiates and regulates the generation of adenosine and comprises an important component of the suppressive machinery of T reg cells.
T reg cells express CD39
Rabbit anti–mouse CD39 polyclonal antibody was raised previously by cDNA immunization (27
) and was used to stain mononuclear cells purified from lymph nodes () and spleen (not depicted) of naive WT mice. CD39 is expressed by the majority of monocytes and by a subset of lymphocytes () in C57BL/6 mice, as inferred by gating on forward scatter and side scatter. Among lymphocytes, the majority of CD39+
cells are B220+
B cells. The remaining CD39+
cells reside within the CD4+
subset, where they consistently range from 8 to 12% of all CD4+
cells in lymph nodes and spleen (). Importantly, no cells within the CD8+
T cell subset express CD39.
Figure 1. CD4+/CD25+ cells express CD39. (A) The expression of CD39 (open profiles) on mouse lymph node cell suspensions is shown in the histograms on the right, contrasted against control IgG (gray). The expression of CD39 on CD4+, CD8 (more ...)
Further characterization of resting C57BL/6 lymphocytes reveals that CD39 is predominantly expressed on those CD4+ cells that concurrently express CD25 (). CD39 is consistently and abundantly expressed by CD4+/CD25high T cells, whereas the CD4+/CD25dim population shows a dichotomic pattern with respect to CD39 expression, with ~50% of the cells being positive. Less than 1% of CD39+ cells are found within the CD4+/CD25− compartment (). Predominant expression of CD39 by CD4+/CD25+ T cells was also observed in other strains of mice such as BALB/c and, additionally, in primates and humans (unpublished data). Nearly all CD4+/CD39+ cells are also CD5high, CD45RBlow and are mostly CD62Llow (). CD4+/CD8−/CD25+ thymocytes express CD39 at equivalent amounts to peripheral CD4+/CD25+ T cells (unpublished data).
RT-PCR analyses of sorted mouse cell populations confirm that CD4+ cells express the highest levels of CD39 mRNA among lymphocytes (unpublished data). Gene expression profiling of CD4+ T cells, which was sorted on the basis of CD39 cell surface expression, indicates that CD4+/CD39+ but not CD4+/CD39− T cells robustly express Foxp3, CD25, glucocorticoid-induced tumor necrosis factor receptor (GITR), and CTLA-4 transcripts, which is in keeping with the recognized T reg cell phenotype ().
When functional properties are tested, CD4+/CD39+ cells are found to suppress CD4+/CD25− proliferation with an efficacy similar to that observed with classic naive CD4+/CD25+ T reg cells (). In concordance with the T reg cell phenotype, CD4+CD39+ cells are nonresponsive to TCR stimulation in the presence of irradiated T-depleted accessory cells and in the absence of IL-2 (unpublished data).
The incomplete overlap between CD4+
subsets is interesting given recent data demonstrating that Foxp3 is expressed in a significant percentage of CD4+
cells that is potentially independent of CD25 expression (7
). Further work suggests that a combination of surface markers, including CD127, provides a more accurate definition of T reg cells than CD25 alone (14
). Lastly, CD39 is one of 67 genes, as shown by gene profiling, to be highly associated with Foxp3 expression, even more so linked than CD25 (7
). Indeed, Foxp3 amplifies and stabilizes the expression of genes encoding cell surface or secreted molecules (e.g., Fgl2, CD73, CD39, TRAIL, or CTLA4; reference 8
To discern the relationship between CD39 expression, CD73, and the T reg cell phenotype-function relationships, we studied purified T cells from mice with the GFP reporter gene introduced into the endogenous Foxp3 locus (designated as Foxp3+
) knock-in cells; reference 28
). The great majority (~80%) of Foxp3+
) cells from spleen and lymph nodes strongly express CD39 (). CD39 is a more consistent and reliable marker for T reg cells than CD25; importantly, only ~50% of Foxp3+
) knock-in cells also express CD25 (). However, a limited fraction of Foxp3−
) cells are also CD39+
cells are rarely seen and have been excluded from further analyses.
Figure 2. Foxp3+ cells express CD39. (A) CD4+ T cells from Foxp3+ (GFP+) knock-in mice stained with CD25-PE or CD39-PE. (B) Real-time PCR analysis of Foxp3+/CD39+ (black bars), Foxp3−/CD39+ (shaded (more ...)
Therefore, three substantive populations can be defined on the basis of differential CD39 and Foxp3 expression: Foxp3+/CD39+, Foxp3−/CD39+, and Foxp3−/CD39−. These populations were sorted, and gene expression profiles together with functional suppressive properties were determined. The Foxp3+/CD39+ fraction was shown to mirror the recognized genetic profile of T reg cells, as defined by the presence of Foxp3, GITR, CTLA-4, and IL-10 transcripts (). These Foxp3+/CD39+ cells also efficiently suppress CD4+/CD25− proliferation in vitro (). Conversely, the Foxp3−/CD39+ subset contains T lymphocytes that appear to be associated with the memory compartment, pointing to a comparable derivation of T reg cells and other activated memory cells (unpublished data). As expected, the Foxp3−/CD39− population expresses neither phenotypic nor functional features of T reg cells ().
We next sought to determine the exact functional relationship between the capacity for adenosine generation and Foxp3 expression within recognized mouse CD4+ T cell populations. Using Foxp3 knock-in mice, Foxp3+(GFP+) T reg cells were found to coexpress both ectonucleotidases CD39 and CD73. This is a unique situation among T lymphocytes and provides precise phenotypic markers and designates biochemical functions for T reg cells (). Consistent with the phenotypic data, RT-PCR analysis confirms that Foxp3+/CD39+ cells exhibit high levels of the gene expression of both CD39 and CD73 (). Moreover, CD4+/CD39+/CD73+ cells have comparable suppressive properties to classic CD4+/CD25+ cells in vitro (not depicted and ).
T reg cells generate extracellular adenosine
CD39 and CD73 coexpression are associated with Foxp3+
T cells (). However, CD39 and CD73 are also typically expressed by monocytes and endothelial cells, in which altered expression can be linked to disordered circulatory homeostasis, thrombosis, and platelet activation (29
). Moreover, CD39 expression encompasses different cellular lineages, including B lymphocytes and Langerhans cells, where it may play a role in modulating immune cell–cell contacts by catalyzing local changes in extracellular nucleotide-triggered responses (25
). CD73 is more generally expressed on the vasculature by select T and B cell subsets, on follicular dendritic cells, and on thymic medullary reticular fibroblasts together with various epithelial cells (24
The finding that a population of CD4+ lymphocytes endowed with suppressive functions selectively coexpresses CD39 and CD73 illustrates potential functional roles of extracellular nucleotide-metabolizing enzymes within the T cell pool. In the biochemical systems that we propose for T cell regulation, a degree of specificity could be dictated by three essential modulatory components: titrated and various nucleotide release into the extra- or pericellular milieu, specific receptors for these mediators, and, finally, a varied repertoire of ectonucleotidases upon regulatory and effector cells.
CD39 biochemical activity is the rate-limiting component of the ectoenzymatic chain that metabolizes extracellular nucleoside di- and triphosphates, ultimately to the respective nucleosides, such as adenosine. Nucleoside triphosphate diphosphohydrolase (NTPDase) biochemical activity levels of intact WT CD4+/CD25+ T cells are 8–10 times greater than that of the CD4+/CD25− counterparts, constitutively not expressing CD39 (). Furthermore, using 14C-radiolabeled nucleotides, we demonstrate that WT CD4+ cells rapidly hydrolyze exogenous ADP to efficiently generate adenosine in vitro. Cd39-null CD4+ cells show markedly diminished rates of ADP phosphohydrolysis with no production of adenosine (). These data confirm that functional, classic Foxp3+/T reg cells constitutively express CD39 and CD73 and that the biochemical pathway leading to the synthesis of adenosine is operative.
Figure 3. Concomitant expression of CD39/CD73 and generation of adenosine distinguishes T reg cells from other T cells. (A) CD39/NTPDase activity in CD4+/CD25+ and CD4+/CD25− cells. (B) Hydrolysis of extracellular 14C-radiolabeled (more ...)
In contrast, the two other CD4+ subpopulations, namely Foxp3−/CD39+ and Foxp3−/CD39−, fail to generate adenosine from ADP (), which is in keeping with low CD73 expression levels. CD8+ T cells included as a CD39−/CD73+ control do not generate adenosine. Therefore, within the T cell compartment, only the Foxp3+(GFP+) T reg cell possesses the intact ectonucleotidase catalytic machinery, and these properties are maintained during cellular activation (unpublished data). Such cells might dictate the generation of pericellular adenosine (at extravascular sites) to potentially mediate immunoregulation at a cellular level.
Adenosine suppresses T cell responses
The hypothesis that adenosine may comprise, at least in part, a component of the suppressive machinery of T reg cells is supported by the independent demonstration of CD73 expression by T reg cells (26
). Adenosine exerts its effects by binding to adenosine type 1 purinergic G protein–coupled cell surface receptors, which are termed A1, A2a, A2B, and A3. Adenosine is a potent inhibitor of T cell responses (19
), and the A2A receptor has been identified as the major antiinflammatory adenosine receptor associated with T cells (21
The expression patterns of the A2A receptor were evaluated in T reg cells and CD4+/CD25− cell populations. Under resting conditions, the highest A2A mRNA levels are present in T reg cells, which, in the presence of adenosine generated from CD39, may account for the anergic state typical of this population. Minimal A2A mRNA expression could be detected in CD4+/CD25− cells (), but effector T cells acquire A2A receptors during activation, starting at 4 d after stimulation and with expression peaking at day 6 (). Little or no expression of A2B receptors is identified in T cells (unpublished data). To determine whether signaling through the A2A receptor has functional consequences in CD4+/CD25− cells, a panel of adenosine agonists with defined selectivity toward the full array of adenosine receptors was tested. The proliferation of CD4+/CD25− cells stimulated by allogeneic cells in culture was substantially inhibited in a dose-dependent fashion in the presence of A2A-specific agonists (ATL146e), confirming a potent pharmacological effect (). Much weaker effects were obtained using compounds with somewhat nonselective A1 and A3 receptor agonist activity ().
Figure 4. Adenosine generated by CD39 and CD73 suppresses T cell proliferation. (A) mRNA expression of adenosine A2A receptor. Cells were activated for 5 d in vitro. Data are means of three independent experiments. (B) ATL146e inhibits T cell proliferation in response (more ...)
The central role of adenosine generation in the control of CD4+
proliferation was then tested by using lymphocytes from mutant mice null for A2A receptors. We noted that A2A-null CD4+
proliferation rates in response to TCR stimulation in the presence of irradiated accessory cells are markedly increased by day 6 (). These observations are in agreement with the known kinetics of A2A receptor up-regulation and validate further previous observations regarding A2A receptor properties in T effector cells (21
). The timing of up-regulation of the A2A receptor during T cell activation suggests that this novel immunosuppressive loop is functional during the late phases of T effector cell activation and proliferation, likely coinciding with maximal nucleotide leak from increasingly hypoxic or activated cells (30
Effects of CD39 upon adenosinergic loops that comprise a component of the suppressive machinery of T reg cells
CD39 critically regulates levels of ADP (), a crucial feed-forward inhibitor of CD73 (31
). Therefore, we further studied Cd39
-null mice to determine how disruption of the ectonucleotidase cascade impacts T reg cell functions. Cd39
-null mice express CD4+
T cells in the expected numbers in peripheral blood and lymphoid organs. These cells express traditional markers of T reg cells, such as Foxp3 and GITR. However, T reg cells isolated from Cd39
-null animals display features consistent with an activated state, such as enhanced mRNA expression of CD25 and CTLA-4 (). These features possibly reflect aberrant purinergic signaling, resulting in the loss of any putative inhibitory adenosinergic autocrine loop. Moreover, Cd39
-null T reg cells are markedly dysfunctional in other ways: specifically, they are not anergic and proliferate excessively in response to anti-CD3 and anti-CD28 triggering and to alloantigens without exogenous IL-2 ().
Figure 5. T reg cells from Cd39-null mice are constitutively activated and fail to suppress CD4+/CD25+ cell proliferation. (A) CD25 and CTLA-4 mRNA expression in CD4+/CD25− and CD4+/CD25+ cells isolated from Cd39 (more ...)
Concordant with data derived from A2A-null CD4+/CD25− (), proliferation responses of CD4+/CD25− T cells obtained from Cd39-null mice are exaggerated after 6 d of stimulation, which is coincident with the up-regulation of adenosine receptor A2A in CD4+/CD25− cells (). As a further confirmation of direct connections between CD39 expression and adenosine generation, effects of the addition of soluble exogenous NTPDases (apyrase grade VII) to Cd39-null T cell cultures were tested. This reconstitution effectively suppresses the abnormal proliferation of CD4+/CD25− cells () and restores anergy within the T reg cell pool (not depicted).
We next examined the suppressive ability of WT and Cd39-null T reg cells to inhibit the proliferation of WT or Cd39-null effector cells, respectively. An in vitro system was chosen whereby effector cells were activated with anti-CD3 and anti-CD28. This system was chosen to eliminate the expression of CD39 on stimulating cells, which confounds analyses. We found that Cd39-null T reg cells were 50–60% less effective at inhibiting Cd39-null effector cell proliferation compared with WT counterparts at all ratios tested (unpublished data).
To further determine the importance of the CD39-adenosinergic axis upon T cell function, we examined the ability of T reg cells from WT and Cd39-null animals to suppress the proliferation of CD4+/CD25− cells from WT and A2A-null mice. Although the elimination of Cd39 substantially retards adenosine generation, it does not account for local adenosine release. Therefore, we generated an in vitro system in which the cellular machinery that generates adenosine or the dominant receptors that bind adenosine are intact or disrupted, respectively. Given the kinetics of A2A receptor up-regulation (), this system was analyzed at day 5, a time point when, in the absence of T reg cells, almost all effector T cells have entered into proliferation. Under such conditions, Cd39-null T reg cells are ~50% less effective at suppressing A2A-null T cell proliferation when compared with the ability of WT T reg cells to suppress WT CD4+/CD25− proliferation when mixed at a ratio of 1:1 (). This effect is shown by the increased numbers of A2A-null CD4+/CD25− T cells that have entered into proliferation. The disparity between the suppressive capacities of WT and Cd39-null T reg cells holds at decreased numbers relative to effectors, with greater numbers of effector T cells entering into proliferation at each reduced ratio (unpublished data). Interestingly, analysis of cultures at earlier time points failed to demonstrate a difference in the suppressive ability between WT and Cd39-null T reg cells (unpublished data), once again indicating that Cd39 and the subsequent generation of adenosine are effective late in the proliferation of T cells.
These data further imply that adenosine generated from the hydrolysis of nucleotides exerts substantive inhibitory effects primarily through the A2A receptor in vitro, which is additive to any other cell–cell contact putative mechanisms that dictate T reg cell function. CD4+
T cells isolated from Cd39
-null animals also exhibit attenuated suppressive capabilities in vivo, as assessed in a transplant rejection model in immune reconstituted mice. Here, WT and mutant T reg cells are transferred in excess with CD4+
T cells, and the capacity to prevent the rejection of skin allografts was examined. As expected (32
), adoptive transfer of WT T reg cells with WT CD4+
cells at the well-defined 4:1 ratio results in long-term allograft survival (>40 d). In contrast, when Cd39
-null T reg cells are transferred together with Cd39
-null T effectors at the same cellular ratios, four out of eight grafts are rejected at a median of 24.5 d (P = 0.02). Transfer of CD4+
T cells alone from either WT or Cd39
-null animals does not induce rejection (not depicted); no statistically significant differences are observed in the rejection times induced by the transfer of WT or Cd39
cells alone (). These results are the first demonstration that the CD39-adenosinergic axis plays a nonredundant role in CD4+
cell suppression mechanisms in vivo, giving credence to our phenotypic studies and other expression analyses (8
). Our data strongly suggest that adenosine generation is an important component of the T reg cell armamentarium and is essential for full suppressive function.
CD39 and CD73 are components of a larger family of ectoenzymes that degrade nucleotides and include molecules for which a clear-cut role in lymphocyte activation and migration has been extensively proven, such as CD26 (34
) and CD38 (35
). All of these enzymes evolved from highly conserved ancestral proteins, usually acquiring membrane anchorage and developing parallel, albeit independent, functions. The acquisition of receptor functions such as signal transduction, localization in membrane lipid microdomains, and physical and functional association with partners specialized in signal transduction are particularly relevant. Similar properties also have been shown, albeit in a more limited manner for CD39 (36
Our data demonstrate that CD39 is a novel cell surface marker of Foxp3 T reg cells. Functionally, the coexpression of CD39 and CD73 with the pericellular generation of adenosine dictates a substantial component of the suppressive capabilities of T reg cells. CD39 might regulate immune T cell suppression by the downstream production of adenosine acting via the A2A receptor. Advances in the understanding of how CD39 impacts T reg cell suppressive functions may help develop novel therapeutic avenues to target common inflammatory and immune disorders.