Adenosine-Induced Intracellular cAMP Accumulation Impairs T Cell-Mediated Antitumor Responses
It is now understood that T cells play a major role in tumor control (118–120). As will be discussed however, elevated levels of adenosine in the TME can potently impair T-cell function by inducing accumulation of intracellular cAMP.

Levels of Adenosine Receptors on the T Cell Surface
Murine (121–127) and human (128–132) T cells express all four ARs, and levels of A2AR (122, 124–127, 129), A2BR (126, 127, 130), and A3R (127, 131) increase upon T cell activation. However, the biology of T cells is primarily affected by the predominantly expressed A2AR (122, 123, 128, 132). Of note, similarly to CD39 and CD73, A2AR, and A2BR are upregulated due to hypoxia-induced HIF1α (133) transcriptional activity. Moreover, mRNA levels of both A2AR and A2BR are upregulated in T cells specifically upon provision of anergic stimulus (134). Validating these findings, adoptively transferred tumor-specific T cells isolated from tumors contained twice the A2AR mRNA levels than counterpart T cells isolated from spleens of tumor-bearing mice (135). Since triggering of the different ARs initiates diverse and even antagonistic signaling pathways, the net cellular effects of adenosine are determined by the relative surface expression of its receptors. It is clear, however, that treatment of human (136, 137) or murine (38, 126, 138, 139) T cells with adenosine or adenosine analogs induces A2AR- (38, 126, 137–139) as well as A2BR- (38, 136) mediated intracellular cAMP build-up.

The Mechanics of cAMP-Mediated T Cell Suppression
The secondary messenger of adenosine cAMP, also a derivative of ATP, is involved in a diverse range of cellular functions including metabolism, transcription, and growth, while oscillations of its levels within distinct cell populations are paramount for the regulation of multiple bodily functions, such as endocrine, cardiovascular, neuronal, and immune processes (140). The intracellular concentration of cAMP is determined by the antagonistic activities of ACs, and of cAMP-specific phosphodiesterases (PDEs), proteins that hydrolyze cAMP to 5′-AMP. Although cAMP can diffuse within the cytosol, the co-localization of the highly-targeted AC and PDE activities in particular subcellular regions results in the formation of distinct cAMP microdomains within which co-localized cAMP effectors are activated by in-situ generated cAMP before its swift degradation (141, 142). The formation of such microdomains is mediated by AKAPs, scaffold proteins shown to bind ACs, PDEs as well as effectors of the cAMP-signaling pathway (143, 144). Of the 10 currently identified AC isoforms, T cells express AC3, AC6, AC7 and AC9 (145, 146) with most cAMP production catalyzed by AC7 (146). As previously described, A2AR and A2BR are coupled to Gαs which stimulates the activity of ACs. Of the 11 PDE families characterized to date, isoforms belonging to the relatively strong-affinity (147) cAMP-binding families of PDE1 (145, 148), PDE3 (145, 149), PDE4 (145, 149), PDE7 (145, 149–151), PDE8 (145, 151, 152), and PDE11 (145) have been observed within T cells, with most cAMP hydrolysis carried out by PDE3 and PDE4 isoforms (148, 149, 153). Of note, cAMP levels in T cells can also be augmented by additional factors in the TME including prostaglandin E2 (PGE2) (154), norepinephrine (155), histamine (156), the neuropeptides VIP and PACAP (157, 158), and low pH (159). Additional phenomena contributing toward cAMP build-up within effector T cells include TCR triggering (160, 161) as well as direct cAMP transfer by tumor cells (162) or Tregs (163) via gap junctions.
Accumulation of cAMP within the T cell cytosol induces the activity of protein kinase A (PKA) and of exchange protein directly activated by cAMP (EPAC). PKA, the dominant effector of the cAMP signaling pathway (164) is an heterotetramer comprising two catalytic (C) subunits, maintained in an inactive state by tethering to two regulatory (R) subunits (165). Binding of cAMP to the R-subunits induces a conformational change resulting in the release of the C-subunits (166). As a result, liberated PKA C-subunits within T cells phosphorylate a wide variety of substrates affecting multiple signaling pathways (167). It is well established that sustained PKA activity disrupts signaling induced by triggering of the TCR, of the co-stimulatory receptor CD28 (168, 169) as well as by the IL-2 receptor (IL-2R) (170). Negative regulators of these signaling pathways, whose activity is bolstered by PKA, include Csk (171), SHP-1 (172), SHIP1 (173), HPK1 (174), and PP2A (175). Conversely, PLCγ1 (176, 177), Raf-1 (178, 179), JAK3 (170), RhoA (180, 181), VASP (182) as well as the transcription factors NFAT (183, 184) and NFkB (185, 186) constitute mediators or endpoint effectors of the aforementioned axes whose activity is dampened by PKA.
PKA activity also significantly affects cytoplasmic potassium concentration within T cells by inhibiting the activity of Kv1.3 (187) and KCa3.1 (188, 189), channels which are responsible for the bulk of potassium efflux by T cells (190). In a negative-feedback fashion, PKA induces reduction of the cytosolic cAMP concentration by directly phosphorylating AC6 in an inhibitory fashion (191) as well as isoforms of PDE3 (192), PDE4 (193, 194), PDE8A (195) in a stimulatory manner. At the transcriptional level, PKA augments the activity of CREB cAMP responsive element binding (CREB), cAMP responsive element modulator (CREM) and activating transcription factor-1 (ATF-1) (196), which induce or counteract the transcription of multiple inflammation-relevant genes such as IL-2 (197–199), IFNγ (200–202), IL-4 and IL-13 (203, 204), IL-17 (205–208), and FoxP3 (209, 210). Specifically, PKA promotes the transcriptional activity of CREB by phosphorylating it thus increasing its affinity for its co-activators CBP and p300 (211), and by promoting the nuclear localization of CRTC (212), another family of CREB co-activators. Finally, PKA directly phosphorylates and activates ATF-1 (213) as well as distinct CREM isoforms (214) in a way similar to CREB.
The guanine nucleotide exchange factor EPAC1 is another effector of cAMP in T cells (215, 216). cAMP binds to the cAMP-responsive N-terminal region of EPAC1 and induces an open conformation rendering its catalytic core accessible to its effectors (217, 218). The most heavily characterized EPAC1 effector in T cells is the anergy-associated GTPase Rap1 (219, 220) which in its GTP-bound form is targeted to the plasma membrane (221) where it inhibits TCR-induced MEK-ERK activation by sequestering Raf-1 (220, 222).

Overview of the Inhibitory Effects of cAMP on T-Cell Biology
A variety of molecules, including cAMP analogs, direct AC activators (e.g., forskolin and cholera toxin) and PDE inhibitors have been used to elucidate the diverse effects of intracellular cAMP accumulation on T-cell biology. In the presence of such molecules (223–228) as well as by A2AR triggering (125, 126, 229) the capacity of previously unstimulated T cells, CD4+ or unfractionated, to differentiate post-activation toward cells that produce Th1 (125, 126, 223–225, 229) or Th2 (226–229)-signature cytokines is drastically diminished. This occurs in a PKA-dependent fashion (230, 231) through multi-level disruption of TCR- or CD28-induced signaling (122, 232). Intriguingly, A2AR agonist-induced impairment of IFNγ production remains evident even when A2AR agonist-pretreated T cells are re-stimulated in the absence of this agent (139). Furthermore, agents that directly activate the cAMP pathway (233–235), as well as adenosine (122, 138, 232, 236, 237), have been shown to restrict stimulation-induced AKT activation (122, 232, 233, 238) and to induce stabilization of β-catenin, which restricts maturation toward terminally differentiated effector cells (239). Moreover, such agents can prevent FasL upregulation, thus averting FasL-mediated activation-induced cell death (AICD) (127, 138, 235, 237). Finally, such molecules abolish mitogenic-stimulus-induced T cell proliferation, in a PKA-dependent manner (240), by downmodulating the transmission of TCR/ CD28- and IL-2 (241)-initiated signaling, as well as IL-2 production (126, 229, 231) and IL-2Ra expression (242).
Forskolin, cAMP analogs, PDE inhibitors (152, 243–245) and adenosine (188, 246–248) also diminish T cell adherence (152, 243, 246, 248) by down-modulating the expression levels of ICAM-1 (249, 250) as well as of the integrins α4 (251, 252) and β2 (251, 253), components of VLA-4 and LFA-1, respectively. Such agents also impair T-cell migration (188, 244, 245, 247) by inducing KCa3.1 inhibition (188, 189). In addition, cAMP-mediated signaling (230, 254, 255) or the presence of A2AR agonists (139, 168, 230, 231) diminishes T cell cytotoxicity, in a PKA-dependent manner (168, 230, 231), probably as a result of impaired TCR signaling, motility/adhesion, granule exocytosis (138), as well as due to decreased expression of FasL, Granzyme B (GzB), and perforin (127).
Lastly, cholera toxin (256), PDE inhibitors (257–259), forskolin (157) and A2AR agonists (126, 260) not only skew T cells toward the Treg lineage via induction of FoxP3 expression (126, 256–258, 260), but also enhance the capacity of Treg cells to suppress responder T cells (258–260), at least in part by upregulating CTLA-4 levels (157, 260). Thus, cAMP can potently diminish the differentiation and effector activities of CD4+ and CD8+ T cells, while promoting the differentiation toward Tregs, as well as their suppressive capacity.