Adenosine produced as a byproduct of metabolic activity is present in all tissues and produces dose-dependent suppression of TCR signaling. Naive T cell maintenance depends on inhibition of TCR signals by environmental sensors, which are yet to be fully defined. We produced mice with a floxed adenosine A2A receptor (A2AR) gene, Adora2a, and show that either global A2AR deletion or cre-mediated T cell deletion elicits a decline in the number of naive but not memory T cells. A2AR signaling maintains naive T cells in a quiescent state by inhibiting TCR-induced activation of the phosphatidylinositide 3-kinase (PI3K)–AKT pathway, thereby reducing IL-7Rα down-regulation and naive T cell apoptosis. Patterns of IL-7Rα expression on T cells in chimeric mice reconstituted with Adora2a+/+ and Adora2a−/− bone marrow cells suggest that decreased IL-7Rα in naive T cells is a cell-intrinsic consequence of Adora2a deletion. In addition, A2AR expression increases in early thymic T cell development and contributes to progression of double-negative thymic precursors to single-positive thymocytes with increased IL-7Rα expression. Therefore, A2AR signaling regulates T cell development and maintenance to sustain normal numbers of naive T cells in the periphery.
Adenosine is produced as a result of metabolic processes and has cell signaling roles that are mediated by four G-protein–coupled adenosine receptors: A1, A2A, A2B, and A3 (Fredholm et al., 2011). These receptors are antagonized by naturally occurring and widely consumed methylxanthines, caffeine and theophylline, as well as by more potent synthetic antagonists (Haskó et al., 2008; Fredholm et al., 2011; Linden and Cekic, 2012). Adenosine is constitutively produced and increases in response to cellular hypoxia and stress. It readily crosses cell membranes via nucleoside transporters to gain access to cell surface receptors (Yao et al., 2011). Extracellular adenosine is also produced from the degradation of adenine nucleotides by exonucleases. ATP and ADP are converted to AMP and adenosine after release to the extracellular space through membrane channels (Huang et al., 2007), from cell death, or as granular components of platelets, mast cells, or neurons. Therefore, adenosine is available in all tissues and organs and changes in concentration depending on the tissue type and physiological conditions.
Naive and memory T cells are maintained in the periphery to provide appropriate antigen-specific recognition to eliminate pathogens and tumors. IL-7 signaling and TCR engagement by self-peptide–MHC molecules provide signals needed for the development, survival, and homeostatic proliferation of naive T cells. Memory T cells also rely on IL-7 for survival but use IL-15 for homeostatic proliferation (Surh and Sprent, 2000, 2008). Recent evidence suggests that naive T cells are actively maintained in a quiescent state that requires integration of proliferative and survival signals with signals from environmental cues. However, the nature of these environmental cues is not fully understood. Here we identify one such cue as adenosine. The A2AR is the predominant adenosine receptor subtype expressed by T cells (Su et al., 2004) and is induced when these cells are activated (Lappas et al., 2005). A2AR activation increases cAMP to suppress TCR signaling (Ohta and Sitkovsky, 2001; Lappas et al., 2005; Ohta et al., 2009; Linden and Cekic, 2012). In tissues, basal adenosine concentrations are high enough to engage A2ARs (Su et al., 2004). We show that endogenous adenosine is sensed by A2ARs as an environmental cue that prevents IL-7R down-regulation after TCR stimulation. This signaling pathway increases naive T cell survival.
A2AR deficiency impairs peripheral T cell homeostasis
An analysis of the ImmGen database (Heng and Painter, 2008) confirms prior studies showing that A2AR mRNA is the predominant adenosine receptor transcript expressed by T cells (Fig. 1 A). Compared with wild-type animals, mice lacking the A2AR gene, Adora2a, have smaller spleens (wild type = 110 ± 6 mg vs. Adora2a deficient = 73.75 ± 6 mg) and LNs (not depicted), suggesting intrinsic signaling by A2ARs even in unstressed mice. We compared the frequencies and numbers of lymphoid cell populations in Adora2a+/+ and Adora2a−/− mice. Global Adora2a deletion significantly reduced the number of naive T cells (CD44loCD4+ and CD44loCD8+ T cells) in blood and peripheral LNs (Fig. 1, B and C) without affecting numbers of B, NK (Fig. 1 B), or myeloid cells (not depicted). To a lesser but still significant extent, numbers of A2AR-deficient CD4+CD44hi and CD4+Foxp3+ T cells were also reduced in LNs but not spleen (Fig. 1 D). This may be because of a reduction in the precursor naive CD4+ T cell population. Overall, these data demonstrate that basal A2AR signaling contributes to the maintenance of naive T cell numbers in the periphery.
Cell-intrinsic A2AR signaling regulates IL-7Rα expression
Maintenance of naive T cells is mediated by homing signals to peripheral lymphoid organs through CCR7 and CD62L and survival signals from growth factor receptors. Deletion of Adora2a did not cause significant reductions in the expression of homing receptors CCR7 (Fig. 2 A) or CD62L (Fig. 2 B), suggesting that the decrease in naive T cell numbers in Adora2a−/− mice is not caused by a homing defect. IL-7 is the major survival factor for naive T cells. We observed a substantial reduction in the cell surface expression of IL-7Rα (CD127) on naive T cells from Adora2a−/− mice as compared with wild-type controls (Fig. 2 C). CD127 is not changed on Adora2a−/− CD44hiCD4+ T cells and only slightly decreased on Adora2a−/− CD44hiCD8+ T cells. Hence the number of cells with a memory phenotype (CD44hi) is minimally changed in Adora2a−/− mice (Fig. 1 B) despite a substantial decrease in the naive T cell population (Fig. 1, B and C).
Global A2AR deletion in mice produces a constellation of effects mediated by deletion of receptors on multiple cell types. The consequences vary among mouse strains but may include aggressiveness, hypoalgesia, high blood pressure, and increased basal levels of inflammatory mediators derived in part from APCs (Ledent et al., 1997; Ohta and Sitkovsky, 2001). To determine whether Adora2a deletion has cell-intrinsic effects to influence T cell number or IL-7R expression, we performed mixed bone marrow reconstitution experiments. We transferred 1:1 mixtures of bone marrow cells derived from Adora2a+/+ (CD45.1+) and Adora2a−/− (CD45.2+UBC-EGFP) mice to irradiated wild-type (CD45.2) recipients and measured the ratio of transferred T cells after 8 wk. Similar experiments used EGFP+ hosts and bone marrow from Adora2a+/+ (CD45.1+) and Adora2a−/− (CD45.2+) to exclude the possibility that a decrease in T cell numbers might be caused by EGFP expression. Analogous experiments were conducted using mixtures of Adora2b+/+ (CD45.2+) and Adora2b−/− (CD45.1+) bone marrow. Compared with wild-type cells, low numbers of Adora2a-deficient but not Adora2b-deficient T cells were detected in reconstituted recipients, consistent with the conclusion that T cell A2AR (Fig. 2 D) but not A2BR signaling (Fig. 2 E) facilitates T cell homeostasis.
We observed different patterns of IL-7Rα expression on T cells in chimeric mice reconstituted with Adora2a+/+ and Adora2a−/− bone marrow cells, suggesting that decreased IL-7Rα in naive T cells is a cell-intrinsic consequence of Adora2a deletion (Fig. 2 F). Accordingly, transfer of a 1:1 mixture of Adora2a+/+ (CD45.1+) and Adora2a−/− (CD45.2+UBC-EGFP) spleen and LN cells to wild-type recipients (CD45.2+) for 0–2 wk resulted in a reduced ratio of Adora2a−/− to Adora2a+/+ in naive but not memory phenotype T cells over time (Fig. 2 G). To determine whether the decrease in IL-7Rα expression in Adora2a−/− T cells influences their sensitivity to IL-7, we cultured Adora2a−/− or Adora2a+/+ cells from LNs in the absence or presence of IL-7. A2AR deficiency significantly decreased the maximum response to IL-7 (Fig. 2 H) without affecting the receptor affinity for IL-7 (see EC50 values for IL-7), suggesting that a decrease in the number of IL-7Rs reduces survival. These data demonstrate that cell-intrinsic A2AR signaling favors peripheral T cell accumulation by regulating IL-7R expression.
Protein kinase A (PKA) stimulation by A2AR activation prevents TCR-induced down-regulation of IL-7Rα
IL-7R and TCR signaling are both known to inhibit IL-7Rα expression. Therefore, we measured the effects of A2AR activation on TCR- or IL-7–induced decreases in cell surface IL-7Rα expression. Fig. 3 A shows that the addition of the selective A2AR agonist CGS 21680 to T cells in vitro significantly inhibits TCR- but not IL-7–induced down-regulation of cell surface IL-7Rα (CD127), suggesting that A2AR signaling interferes with TCR but not IL-7R signaling. CGS 21680 reduced the potency but not the maximal effect of αCD3 to down-regulate CD127. These results are consistent with the concept that A2AR signaling attenuates submaximal TCR signaling. Unlike naive T cells, maintenance of memory phenotype T cells in general does not require self-peptide–MHC and TCR interactions in vivo (Surh and Sprent, 2008). Therefore, differential TCR stimulation may explain why IL-7Rα expression is strongly reduced in naive but not memory phenotype T cells despite similar levels of Adora2a expression in both T cell types (Fig. 1 A).
TCR stimulation causes activation of the phosphatidylinositide 3-kinase (PI3K)–AKT pathway, which negatively regulates IL-7Rα expression (Pallard et al., 1999; Barata et al., 2004; Riou et al., 2007; Kerdiles et al., 2009; Hand et al., 2010). Therefore, unrestrained AKT activity reduces naive T cell numbers by decreasing T cell survival and accumulation of memory phenotype T cells. The PI3K–AKT pathway is inhibited by PKA in response to elevated cAMP (Kim et al., 2001; Lou et al., 2002). Because A2AR signaling regulates T cell signaling events primarily through PKA, we hypothesized that A2AR signaling reduces AKT activation and down-regulates IL-7Rα in a PKA-dependent manner. A2AR stimulation significantly reduced AKT phosphorylation (Fig. 3 B) after TCR signaling, and this effect was blocked by the competitive A2AR antagonist SCH 58621. Accordingly, basal AKT phosphorylation, which was measured by flow cytometry analysis (because immunoblotting was not sufficiently sensitive), was higher in A2AR-deficient naive T cells than in wild-type naive T cells (Fig. 3 C). The effect of A2AR stimulation to increase IL-7Rα expression after TCR stimulation was completely reversed by the highly selective PKA inhibitor KT 5720 (Fig. 3 D). To determine whether PI3K inhibition and A2AR stimulation are additive, we examined the effects of strong TCR stimulation in the absence or presence of the PI3K inhibitor LY 294002 and the A2AR agonist CGS 21680. We chose this strategy because IL-7R down-regulation after weak TCR stimulation can be completely reversed by CGS 21680 (Fig. 3 C) and a possible additive effect of PI3K inhibition and A2AR stimulation may not be observed. LY 294002 alone was sufficient to inhibit TCR-dependent IL-7Rα down-regulation, and A2AR stimulation and PI3K inhibition together were not additive (Fig. 3 E). Interestingly, IL-7 is also known to stimulate the PI3K–AKT signaling pathway. However, CGS 21680 had no effect on IL-7–induced down-regulation of the IL-7R. Recent studies have shown that IL-7–induced down-regulation of IL-7R is regulated primarily by the JAK3–STAT5 pathway (Henriques et al., 2010; Ghazawi et al., 2013). In CD8 T cells, inhibition of PI3K did not prevent IL-7–induced IL-7R down-regulation (Ghazawi et al., 2013). Furthermore, addition of the PI3K inhibitor LY 294002 had no effect on IL-7–induced IL-7R down-regulation in cultured CD4 T cells (Fig. 3 F), indicating that IL-7 and TCR signaling use different pathways to regulate IL-7 receptor expression. Memory phenotype T cells do not require endogenous TCR signals to be maintained. However, they still may receive these TCR signals, which can be regulated by A2AR signaling. Therefore, we stimulated naive and memory T cells and tested the effect of A2AR stimulation on CD127 expression. A2AR signaling had a larger impact on CD127 expression in naive T cells yet still significantly inhibited CD127 down-regulation in memory phenotype T cells (Fig. 3 G), indicating that (a) A2AR signaling is more effective at preventing CD127 down-regulation during tonic TCR signaling in naive than in memory T cells, (b) memory phenotype T cells may express different factors that prevent them from down-regulating CD127 expression during tonic TCR signals, or (c) memory phenotype T cells may not respond to tonic TCR signals as naive T cells do. Overall, these data suggest that stimulation of the PKA pathway by A2AR activation controls IL-7Rα expression by inhibiting TCR-induced AKT activation more in naive than memory T cells.
A2AR signaling regulates proliferation and survival of naive T cells
Homeostatic proliferation of naive T cells is driven by TCR interactions with MHC complexes presenting self-peptides (Ernst et al., 1999; Goldrath and Bevan, 1999; Muranski et al., 2000). Deletion of negative regulators of TCR activation or unrestrained AKT activity leads to loss of quiescence and increased homeostatic proliferation (Yang et al., 2011). To investigate the impact of A2AR signaling on T cell proliferation, we performed BrdU incorporation assays. Fig. 4 A shows that a greater percentage of Adora2a−/− than Adora2a+/+ naive T cells but not memory T cells (Fig. 4 B) incorporate BrdU, consistent with decreased TCR signaling activity in naive T cells in response to endogenous A2AR signaling. Similar BrdU incorporation in Adora2a−/− and Adora2a+/+ memory T cells suggests that lymphopenia in Adora2a−/− mice is not responsible for increased proliferation of naive Adora2a−/− T cells.
IL-7 signaling prevents apoptosis and keeps naive T cells alive (Surh and Sprent, 2008). We observed that Adora2a−/− T cells are hyporesponsive to IL-7 (Fig. 2 E). Accordingly, decreased IL-7Rα expression was associated with decreased expression of antiapoptotic Bcl-2 (Fig. 5, A and B), which is a downstream target of IL-7 signaling, whereas the total cell expression of proapoptotic Bax was not affected (Fig. 5 A). Adora2a deletion increased the number of apoptotic naive T cells (Annexin V+Live-Dead−) in blood and increased the number of dead T cells (Annexin V+Live-Dead+) in peripheral LNs (Fig. 5, C and D). (Note that some cell death occurs during tissue processing. Therefore, the actual differences in apoptosis are likely larger than illustrated.) These data indicate that A2AR-deficient T cells proliferate more than wild-type cells but fail to accumulate as a result of increased apoptosis.
A2AR signaling affects thymic T cell development
Adenosine concentrations are higher in the thymus than in other organs, possibly because of rapid cell turnover (Resta et al., 1997; Cekic et al., 2011). Adora2a mRNA transiently increases during early thymic T cell development and peaks during the DN2B phase (ImmGen database consortium [Heng and Painter, 2008]; Fig. 6 A). Therefore, we next sought to determine whether A2AR signaling influences thymic T cell development. In Adora2a−/− mice, numbers of CD4 and CD8 double-negative (DN phase) populations (early thymic precursors) are unchanged, whereas CD4 or CD8 double-positive (DP) and single-positive (SP) cells decrease significantly (Fig. 6 B). To determine whether this is caused by cell-intrinsic signaling, we measured the ratios of thymic precursors in chimeric mice reconstituted with 1:1 mixtures of bone marrow from Adora2a+/+ (CD45.1) and Adora2a−/− (CD45.2) mice transferred to recipient mice (CD45.2+UBC-EGFP+) 8–12 wk after irradiation. Compared with wild-type cells, large reductions in the relative proportions of Adora2a−/− DP and SP but not DN thymic precursors were detected in reconstituted recipients (Fig. 6 C). To determine whether A2AR expression during early thymic T cell development is important for subsequent progression to DP and SP precursors, we produced Adora2af/f mice and crossed these to syngeneic C57BL/6J mice carrying the Cre recombinase transgene under control of the Lck promoter (Fig. 6 D, top). As illustrated in Fig. 6 A, Lck is first activated toward the end of the DN phase (Sprent and Surh, 2011). A2AR mRNA expression isolated from Adora2af/f-LckCre mice was reduced by 77% in DP, 72% in CD8SP, and almost 100% in CD4SP thymocytes, whereas no significant reduction was observed in DN precursors as compared with mRNA expression in thymocytes from Adora2af/f littermates not expressing Cre recombinase, indicating efficient deletion of the Adora2a gene from T cells (Fig. 6 D, bottom). As can be seen in Fig. 6 E, lck-mediated deletion of the A2AR does not change the proportions and numbers of thymic precursors. This suggests that increased Adora2a transcription during early thymic development (Fig. 6 A) has important effects on thymic progression of T cell precursors toward a mature T cell phenotype. The surge in A2AR transcription that occurs during the DP stage may influence the survival of cells as they enter the SP stage. Thymic progression is not affected by lck-dependent deletion that occurs after the pulse of A2AR transcription (Fig. 6, A and D).
A2AR signaling is required for naive T cell maintenance in the periphery
We considered the possibility that naive T cells are reduced in numbers in Adora2a−/− mice only as a result of decreased thymic output. Because Lck-mediated deletion of Adora2a did not affect thymic progression, we compared the numbers of naive T cells in Cre+ mice with Cre− littermates. We still observed a significant reduction in naive T cell numbers in the periphery without changes in B or NK cells or other T cell subtypes (Fig. 7, A–C). We also observed significant reductions in IL-7Rα expression and IL-7 responsiveness among naive T cells in Cre+ mice (Fig. 7, D and E). Overall, these data suggest that A2AR signaling helps to maintain normal numbers of naive T cells by regulating both thymic output and survival in the periphery.
IL-7R expression starts decreasing in Adora2a−/− T cells during thymic development and completely recovers in the absence TCR signaling
Besides engaging self-peptide–MHC complexes in the periphery, T cells also go through a selection process during their development when thymocytes with newly formed rearranged TCRs are selected based on their ability to interact with self-peptide–MHC complexes. IL-7R expression temporarily decreases during this process because of TCR signals and increases when newly formed T cells become SP and mature. DP thymocytes receiving too weak or too strong TCR stimuli are deleted at this stage. Because we hypothesize that A2AR signaling can fine tune TCR signaling to maintain IL-7R expression, we measured IL-7R expression on thymic precursors. Fig. 8 A shows that global deletion of Adora2a reduces IL-7Rα (CD127) expression in SP precursors. This effect is cell intrinsic because we observed reduced CD127 expression in Adora2a−/− thymocytes developing in the same thymus with Adora2a+/+ thymocytes after 1:1 bone marrow reconstitution (Fig. 8 B), suggesting that Adora2a−/− cells may receive stronger TCR signals during positive selection and either die or fail to fully up-regulate their IL-7Rs. Lck-dependent deletion of Adora2a did not affect IL-7Rα expression in thymic precursors (Fig. 8 C), suggesting that A2AR receptors produced during early thymic development are retained and help to maintain normal numbers of thymic precursors. Decreased IL-7Rα expression after DP to SP transition suggests an incomplete maturation of SP Adora2a−/− thymocytes. The proportions of Qa2hiHSAlo cells were significantly lower among SP thymocytes isolated from Adora2a−/− mice as compared with wild-type controls (Fig. 8 D and Fig. S1), suggesting incomplete maturation rather than accumulation in thymus is the contributing factor in reduced naive T cell numbers in the periphery of Adora2a−/− mice.
Because our data suggest that it is the lack of regulation of TCR signals during thymic selection or peripheral maintenance in the absence of A2AR signaling that reduces IL-7Rα expression, we hypothesized that reduced IL-7Rα expression is not a permanent developmental defect; therefore, the cessation of TCR signaling should restore IL-7R expression in Adora2a−/− T cells. To test this, we measured the expression of IL-7Rα on CD4+ T cells in freshly isolated single cell suspensions or after sorting and incubation overnight at 37°C in medium supplemented with 5% fetal bovine serum. As can be seen in Fig. 8 E, IL-7Rα expression in naive Adora2a−/− CD4+ T cells increased to the levels of wild-type T cells after overnight incubation in vitro at 37°C. We observed a more modest but similar pattern for IL-4Ra expression (not depicted). Unlike IL-4 and IL-7R, IL-2R expression does not decrease with TCR stimulation. Accordingly, expression of IL-2R was similar between freshly isolated or incubated T cells from both A2AR-deficient and wild-type animals (not depicted). To show that absence of TCR signaling increases CD127 expression in Adora2a−/− T cells in vivo, we adoptively transferred GFP+ Adora2a−/− naive CD4+ T cells to wild-type or MHCII−/− mice. CD127 expression in adoptively transferred naive Adora2a−/− T cells remained low in wild-type animals but increased to the level in naive host cells in MHCII−/− mice (please note CD127 expression in wild-type and MHCII−/− T cells is similar). These data suggest that A2AR signaling by endogenous adenosine is important for the homeostatic balance of CD127 expression.
We show here that deletion of A2ARs strongly impacts naive T cell development and survival in C57BL/6 mice. A2ARs couple to the heterotrimeric G protein, primarily Gs and partially to Golf in the central nervous system (Schwindinger et al., 2010). These G proteins activate adenylyl cyclase and increase cAMP and PKA activity, which in turn inhibits AKT (Kim et al., 2001; Lou et al., 2002). AKT is a master regulator that stimulates T cell proliferation and reduces IL-7Rα expression downstream of TCR stimulation (Pallard et al., 1999; Rathmell et al., 2003; Barata et al., 2004; Riou et al., 2007; Kerdiles et al., 2009; Hand et al., 2010). IL-7 signaling is required for naive T cell survival. The current study shows that A2AR/PKA activation limits IL-7R down-regulation by reducing TCR-mediated activation of the PI3K–AKT pathway. The results imply that tissue levels of endogenous adenosine are high enough to activate A2ARs. Interestingly, adenosine concentrations are higher in the thymus than other organs (Resta et al., 1997; Cekic et al., 2011). The current study also shows that Adora2a expression, which increases in early thymic precursors, helps these cells to progress through normal thymic development and to up-regulate IL-7Rα along with thymocyte maturation markers Qa2 and HSA after positive selection. The decrease in IL-7Rα in naive A2AR−/− cells is completely reversed by culturing cells in the absence of TCR stimulation in vitro, suggesting that reduced IL-7Rα expression after thymic selection or during peripheral maintenance is not a permanent developmental defect. Therefore, although adenosine limits homeostatic T cell proliferation, it supports naive T cell development and survival.
TCR stimulation with self-peptide–MHC complexes drives homeostatic proliferation of naive T cells (Surh and Sprent, 2000, 2008). However, unlike memory T cells, only a small fraction of naive T cells proliferate during their lifetime, whereas most stay quiescent (Surh and Sprent, 2000). This suggests that survival plays a major role in maintaining naive T cell numbers. This explains why increased T cell proliferation in response to A2AR deletion fails to maintain normal T cell numbers. The data implicating the IL-7R as a key target of A2AR signaling agree with the proposal by Kerdiles et al. (2009) that IL-7 is a limiting factor for T cell survival. Although PI3K–AKT signaling supports survival and proliferation of T cells, unrestrained AKT activity significantly inhibits IL-7Rα expression, Stat5 phosphorylation, and Bcl-2 expression, thereby decreasing the ability of cells to compete for limited IL-7 and to survive, despite increased homeostatic proliferation (Rathmell et al., 2003; Kerdiles et al., 2009; Hand et al., 2010).
Interestingly, IL-7R signaling itself causes the activation of the PI3K–AKT pathway and down-regulation of IL-7Rα. However, our findings and others suggest that the PI3K pathway is not the primary regulator of IL-7–induced IL-7R down-regulation (Henriques et al., 2010; Ghazawi et al., 2013). In addition to activating the PI3K–AKT pathway, IL-7R signaling also activates the Jak3–Stat5 pathway. It has recently been shown that IL-7–induced down-regulation of IL-7R is regulated by two independent mechanism through Jak3–Stat5 signaling (Henriques et al., 2010; Ghazawi et al., 2013). The fact that A2AR activation selectively prevents TCR- but not IL-7R–induced down-regulation of IL-7Rα suggests that A2AR signaling selectively interferes with TCR-associated signaling events. Consistent with our observations, constitutive activation of AKT or removal of FoxO1 and Tsc1 (which are negatively regulated by AKT) also decreases naive T cell numbers without affecting numbers of memory T cells (Rathmell et al., 2003; Kerdiles et al., 2009; Yang et al., 2011). Recent studies show that unlike unrestrained AKT activation, constitutive activation of Stat5 significantly increases T cell survival without causing a large decrease in cell surface IL-7Rα expression (Hand et al., 2010). Therefore, it appears that naive T cell quiescence and survival can be achieved through a balanced activation of AKT and Stat5 signaling.
Our study demonstrates a greater effect of Adora2a deletion on naive T cells than memory T cells. Unlike naive T cells, most memory T cells lose the need for TCR interactions and rely on IL-7 and IL-15 for survival and homeostatic proliferation (Surh and Sprent, 2000, 2008). Accordingly, our study shows that A2AR signaling selectively prevents TCR-induced IL-7Rα down-regulation (Sprent and Surh, 2011). In Adora2a−/− mice, naive T cells have increased basal AKT activation and homeostatic proliferation. These events not only evoke loss of quiescence and reduced naive T cell survival, but also enhance accumulation of memory phenotype T cells. This may explain why the numbers of T cells with a memory phenotype in Adora2a−/− mice and wild-type mice are similar despite the fact that Adora2a−/− mice have lower numbers of precursor naive T cells.
The decrease in the numbers of naive T cells and CD127 expression were less dramatic after Lck-mediated deletion as compared with global deletion of Adora2a, suggesting that reduction in thymic precursors contributes to decreased numbers of naive T cells in the periphery. It is also possible that the remaining (23%) A2AR transcript expression in DP thymocytes after cre-mediated deletion is sufficient to partially rescue T cells from losing CD127 expression. Therefore, our data suggest that early expression of A2ARs in the thymus contributes to the up-regulation of CD127 during positive selection that may contribute to the differential effects of global versus Lck-mediated Adora2a deletion.
Although adenosine accumulates in stressed or inflamed tissues, it also contributes to baseline homeostasis of several physiological processes, including cardiovascular (tissue oxygen delivery), neuronal (sleep cycle), and renal (glomerular filtration). In this study, we propose a new role for adenosine in the regulation of T cell homeostasis through A2AR signaling. It will be of interest in future studies to examine the effects of A2AR deletion during stressful conditions when adenosine is elevated. In the immune system, adenosine dampens excessive inflammation after tissue injury and activates tissue remodeling responses such as angiogenesis and fibrosis, thereby helping to establish long-term homeostasis after local or systemic disturbances. Ongoing studies indicate that A2AR signaling helps to maintain T cells in adenosine-rich hypoxic solid tumor microenvironments (unpublished data) parallel to our observation that adenosine may regulate CD127 expression even in effector/memory phenotype T cells. Therefore, determining how to extract therapeutic benefits by targeting adenosine receptors without disrupting homeostatic activities will be an important goal of future research.
MATERIALS AND METHODS
Cell lines, animals, and reagents.
Animal experiments were approved by the Animal Care and Use Committee of the La Jolla Institute for Allergy and Immunology. 6-wk-old C57BL/6 and MHCII-deficient mice were purchased from the Jackson Laboratory and used for experiments after being acclimated for 2–6 wk. Generation of the Adora2af/f mice is described below. LckCre mice (Lee et al., 2001) were obtained from Taconic. Adora2af/f-LckCre−/−, Adora2af/f-LckCre+/−, Adora2a−/−, Adora2b−/− (gift from K. Ravid, Boston University, Boston, MA), and RAG1−/− mice were bred in the La Jolla Institute for Allergy and Immunology. Yellow fluorescent reactive dye was purchased from Invitrogen. Antibodies recognizing total Akt, phosphorylated Akt (Ser473), and β-actin for immunoblotting were obtained from Cell Signaling Technology. Fluorescent antibodies used in this study, their sources, and dilutions used are listed in Table S1.
Generation of floxed A2AR mice.
We obtained a targeting construct containing the A2AR gene (Adora2a) previously used to generate Adora2a−/− mice from J.F. Chen as a gift (Boston University). LoxP sites were inserted 300 bp upstream and 1.4 kb downstream 3′ of exon 2, which contains the ATG transcription initiation site. Deletion of exon 2 can effectively eliminate A2AR expression (Ledent et al., 1997). Diphtheria toxin A was placed downstream of the targeting construct to eliminate random integration. We also included positive and negative selection markers (loxP-neo-tk-loxP) to facilitate monitoring homologous recombination and subsequent marker deletion. To achieve this, we designed a targeting vector with some rare enzyme sites in the MCS (multiple cloning site) and three loxP sites in the order: MCS1–loxP–MCS2–loxP–neo–tk–loxP-MCS3-DTA. Exon 2 was inserted into MCS2, the 5′ homologous genome (4.3 kb) into MCS1, and the 3′ homologous genome (4.2 kb) into MCS3. The ultimate goal was to produce embryonic stem (ES) cells such that the only modification to the adora2a locus is the insertion of the two 34-bp loxP recognition sequences surrounding exon 2. After transfection of a targeting vector by electroporation, colonies that survived positive and negative selection were clonally isolated and screened by both PCR and Southern blot analysis for specific adora2a recombination. We used a PCR primer set to identify clones with complete homologous recombination. We deleted the selection marker cassette by transient transfection of Cre into the homologous recombination–positive ES cells in vitro. Correctly modified ES cells were enriched and injected into C57BL/6 blastocysts and then implanted into pseudo-pregnant foster mothers. ES cells were injected into C57BL/6 blastocysts. 64 blastocysts were each injected with 10–15 ES cells. Injected blastocysts were implanted into six pseudo-pregnant foster mothers. 32 pups were born and 13 chimeric mice were identified by coat color. The gender distribution of these chimeras was eight male and five female. Male chimeric mice were test-bred to ascertain the contribution of the injected 129sv ES cells to the germline. We crossed each male chimera mouse with two C57BL/6 female mice. Only two agouti mice were found in the first test breed, and one of them was an (129 x C57BL/6) N1F1 adora2af/w mouse as confirmed by PCR and Southern blotting. Many N1F1 floxed adora2a heterozygous mice of both genders were found in the later litters. To get more heterozygous floxed females to mate with tissue-specific Cre mice, we first used all the N1F1 mice for backcrosses with C57BL/6 mice until we had enough N2F1 and N3F1 mice to generate tissue-specific knockouts. We successfully crossed heterozygous floxed adora2a mice to generate homozygous floxed adora2a mice (Adora2af/f) by crossing male and female N3F1 heterozygous mice. Adora2af/f mouse generation formally started from N3F2 and were designated with the official name B6;129P-adora2atm1Ydj. We maintained the colony by homozygous inbreeding onto C57BL/6J and BALB/c backgrounds. For the current study, floxed mice were crossed with mice expressing Cre recombinase gene under Lck promoter as described in Lee et al. (2001) to obtain lymphoid-specific deletion of Adora2a.
Flow cytometry and cell sorting.
Single cell suspensions from the indicated tissues were prepared by sequential pressing through 100- and 40-µm cell strainers. After RBC lysis (BioLegend), cells were washed and resuspended in RPMI medium supplied with 10% fetal bovine serum and counted in a Z2-Coulter particle counter (Beckman Coulter). 3–5 × 106 cells were preincubated for 10 min in 100 µl FACS buffer with antibody to block Fc receptors. Each sample tube received 100 µl of fluorescently labeled antibody cocktail and was incubated for 30 min at 4°C in the dark. Cells were analyzed using an LSRII equipped with four lasers and FACS Diva software (BD). CD4 and CD8 T cells were enriched by negative selection (STEMCELL Technologies) or for some experiments positively selected by magnetic cell sorting (Miltenyi Biotech) and then stained for CD44. Thymocytes, naive T cells (CD44dim), and memory T cells (CD44hi) were sorted by FACS Aria (BD). Live-Dead fixable yellow (Invitrogen) was used to exclude dead cells before analysis or during cell sorting. Flow cytometry data were analyzed using FlowJo software (version 9.0.1; Tree Star).
Quantitative real-time PCR and immunoblotting.
RNAs from resulting sorted cells were isolated by RNA isolation kit (QIAGEN), and cDNAs from isolated RNAs were synthesized by 4× qScript cDNA Super Mix (Quanta Biosciences). Quantitative real-time PCR was performed by using TaqMan primers and TaqMan PCR master mix obtained from Applied Biosystems. All isolation procedures and reaction assays followed recommended manufacturer’s instructions. For immunoblotting experiments, a negative T cell enrichment kit was used (STEMCELL Technologies). Treated T cells were lysed in RIPA and sample buffer, and immunoblotting was performed as described previously (Cekic et al., 2011), modified to use the semidry iBlot transfer system from Invitrogen. Fluorescently labeled anti–rabbit secondary antibodies and the Odyssey imaging system from LI-COR Biosciences were used for the detection of proteins.
Adoptive cell transfer.
Spleen and peripheral LNs from age-matched Adora2a+/+ and Adora2a−/− mice were collected, and single cell suspensions were prepared by passing tissues through 40-µM mesh filters. Cells were counted, and mixtures of cell suspensions were injected into WT or MHCII mice i.v. by the retroorbital route. Single cell suspensions from spleens and LNs of recipient mice were analyzed by flow cytometry to determine proportions of T cells 1 or 2 wk after adoptive cell transfer into WT recipients or to measure the CD127 expression 1 d after the transfer into WT or MHCII−/− recipients.
Bone marrow transplantation.
Mice 6–12 wk of age were fasted for 24 h and then lethally irradiated (2 × 450 Rads for RAG1−/− and 2 × 500 Rads for C57BL/6 recipients). After the second radiation exposure, a 1:1 mixture of 5–10 × 106 bone marrow cells from donor mice (WT and A2AAR−/−) were injected i.v. Mice were treated with antibiotics from 3 d before until 2 wk after radiation. Single cell suspensions from spleens and LNs were analyzed by cytofluorometry to determine the proportion of T and B cells 7–8 wk after bone marrow transplantation.
In vitro test for IL-7 responsiveness and IL-7Rα expression.
For TCR stimulation or stimulation with recombinant mouse IL-7 (R&D Systems) T cells were enriched by CD4+ T cell enrichment kits (STEMCELL Technologies) and stimulated with different concentrations of IL-7 or carrier-free plate-bound anti-CD3 antibody (clone 145-2C11; BioLegend). Biotinylated CD44 was used during enrichment to specifically isolate the CD44dim (naive) T cell population. To evaluate the effects of A2AR signaling on TCR-induced IL-7Rα down-regulation, we stimulated isolated T cells with plate-bound anti-CD3 antibody in the presence or absence of 1 µM of the selective A2AR agonist CGS 21680, 100 nM of the PKA inhibitor KT 5720, or 2 µM of the selective PI3K inhibitor LY 294002 (Invitrogen). After 24 h of culture at 37°C (5% CO2) in RPMI medium supplied with 5% fetal bovine serum, cell surface staining and flow cytometry were used to detect cell surface expression of IL-7Rα (CD127). Doses of KT 5720 and LY 294002 were selected based on their Ki values (50 nM and 1.4 µM, respectively), and dose response experiments were performed using CD127 as readout.
106 LN cells were isolated from Adora2a+/+ and Adora2a−/− or Adora2af/f-LckCre−/− and Adora2af/f-LckCre+/− mice cultured in the absence or presence of the indicated concentrations of recombinant mouse IL-7 (BioLegend) for 3 d at 37°C (5% CO2) using RPMI medium supplied with 5% fetal bovine serum. Percentages of live T cells were analyzed by flow cytometry using Live-Dead yellow and/or Annexin V staining.
BrdU incorporation assay for proliferation.
Adora2a+/+ or Adora2a−/− mice received 0.3 mg/mouse BrdU 40, 37, 34, and 24 h before the harvest of tissues. Therefore, each mouse received a total of 1.2 mg BrdU. Cells were surface stained and fixed according to the manufacturer’s instructions with a BrdU staining kit (BD). APC-conjugated anti-BrdU antibody was added for 20 min before treatment with wash and permeabilization buffer, and cells were resuspended in FACS buffer.
Online supplemental material.
Fig. S1 shows the gating strategy used for Fig. 8 D. Table S1 lists antibody sources, clones, and dilutions.
We gratefully acknowledge Dr. Katya Ravid for her gift of Adora2b−/− mice, Dr. Jiang Fan Chen for his gift of a targeting construct containing the A2AR gene (Adora2a), Dr. Heba Nowyhed for her help and suggestions for BrdU proliferation assays, Ruey Ken for real-time PCR, Dennis Huynh for mouse husbandry and genotyping, and the Immunological Genome Project.
This work was supported by National Institutes of Health grant P01 HL073361 and by an American Heart Association postdoctoral fellowship (to C. Cekic).
The authors declare no competing financial interests.
Author contributions: C. Cekic conceived the research, conducted the experiments, analyzed the data, and wrote the manuscript; D. Sag assisted with some experiments; Y.-J. Day generated Adora2af/f mice; and J. Linden oversaw study deign and edited the manuscript.