Cyclic AMP underpins suppression by regulatory T cells

Authors


Full correspondence Dr. Josef Bodor, Institute of Immunology, University Medical Center JGU Mainz, D-55131 Mainz, Germany

Fax: +49-6131-17-6202

e-mail: bodor@uni-mainz.de

Abstract

Elevated levels of intracellular cyclic adenosine monophosphate (cAMP) in naturally occurring T regulatory (nTreg) cells play a key role in nTreg-cell-mediated suppression. Upon contact with nTreg cells, cAMP is transferred from nTreg cells into activated target CD4+ T cells and/or antigen-presenting cells (APCs) via gap junctions to suppress CD4+ T-cell function. cAMP facilitates the expression and nuclear function of a potent transcriptional inhibitor, inducible cAMP early repressor (ICER), resulting in ICER-mediated suppression of interleukin-2 (IL-2). Furthermore, ICER inhibits transcription of nuclear factor of activated T cell c1/α (NFATc1/α) and forms inhibitory complexes with preexisting NFATc1/c2, thereby inhibiting NFAT-driven transcription, including that of IL-2. In addition to its suppressive effects mediated via ICER, cAMP can also modulate the levels of surface-expressed cytotoxic T lymphocyte antigen-4 (CTLA-4) and its cognate B7 ligands on conventional CD4+ T cells and/or APCs, fine-tuning suppression. These cAMP-driven nTreg-cell suppression mechanisms are the focus of this review.

Introduction

Naturally occurring CD4+ T regulatory (nTreg)cells are essential for maintaining peripheral tolerance; they prevent autoimmunity and limit chronic inflammatory diseases [[1]]. Immune responses, both protective and harmful, are principally mediated by T and B cells, which possess enormous diversity in antigen recognition, potent effector functions, and long-lasting immunologic memory. Every adaptive immune response involves the recruitment and activation of not only effector T and B cells but also nTreg cells, and the balance between effector and regulatory lymphocytes is critical for the proper control of adaptive immune responses. This balance is also critical for establishing or breaching tolerance against self- and nonself-antigens.

Aside from peripheral conversion, the majority of nTreg cells are generated in the thymus with their frequency increasing from the late CD4+CD8+ double positive to the CD4+CD8 single positive stage [[2, 3]]. Remarkably, this coincides with the stage of thymic development during which human medullary thymocytes acquire cyclic AMP (cAMP) mediated expression of inducible cAMP early repressor (ICER) [[4]]. As ICER is a mediator of nTreg-cell suppression (see below and the section cAMP and Foxp3 direct ICER-mediated suppression), this suggests that competence to suppress develops in nTreg cells simultaneously with the ability of CD4+ T cells to be suppressed via upregulation of ICER.

nTreg cells can have both beneficial effects, for example, preventing autoimmune diseases, and deleterious effects, for example, impairing effective antitumor responses. Understanding the mechanisms of immunological self-tolerance, including those regulated by nTreg cells, will provide insight into how insufficient immune responses, such as those against tumor antigens, can be augmented or, conversely, how exaggerated immune responses such as graft rejection or autoimmunity can be restrained.

nTreg-cell function cannot be attributed to a single dominant pathway or molecule and several mechanisms acting either directly or indirectly at the site of antigen presentation create a regulatory environment that promotes bystander suppression and infectious tolerance. Details of these mechanisms are emerging, including the role that cAMP plays in the expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) and its cognate B7 ligands [[5, 6]]. Tang and Bluestone [[7]] have described a three-tiered model of the function of nTreg cells in which “homeostatic” control forms the first tier, damage control the second, and infectious tolerance the third. With reference to this model, it seems that cAMP and ICER are likely to play a significant role in the steady (homeostatic) state, when nTreg cells exert control over the immune system in lymphoid organs. When this steady state is breached, for example, in autoimmune and transplantation settings, nTreg cells are further activated to engage the second tier of “damage control.” Activated nTreg cells contain enhanced levels of intracellular cAMP, secrete immunosuppressive lymphokines, such as IL-10, IL-35, and TGF-β, and upregulate the expression of CTLA-4 and the ectoenzymes CD39 and CD73. Infectious tolerance seems to be established by contact-dependent mechanisms involving both cAMP and CTLA-4.

The transcriptional repressor ICER was originally described as a “master regulator” of the cAMP response [[8]]. ICER was shown to be a dominant negative regulator of cAMP-responsive transcription in the hypothalamic–pituitary–gonadal axis [[8]] and later this function was also detected in lymphocytes [[4]]. The suppressive potency of nTreg cells has been shown to depend on cAMP [[9]] (as predicted by Rudensky and colleagues [[10]]), which induces ICER that, due to alternative promoter usage, is generated from the 3′ region of the gene-encoding CREM (cAMP-response modulator) [[8]]. Since ICER is initiated from an intronic cAMP-responsive promoter located downstream of the CREM transactivation domain, the consequent lack of a transactivation domain renders ICER the only known cAMP-inducible repressor among the cAMP-responsive element binding (CREB)/CREM transcription factors (reviewed in [[11]]). Among other roles, ICER preferentially inhibits the production of IL-2, an essential growth factor for autoaggressive T-effector cells [[4, 12]]. Importantly, a dominant negative form of CREB (DN-CREB) acts in a fashion analogous to ICER and, when ICER or DN-CREB is transgenically overexpressed in the T lymphocytes of mice, a profound T-cell proliferative defect characterized by markedly decreased IL-2 production ensues [[13, 14]]. This defect in IL-2 production parallels observations in conventional CD4+ T cells cultured with nTreg cells [[12, 15-17]]. Together these data suggest that ICER plays an important role in nTreg-cell-mediated suppression of IL-2 synthesis.

Here, we discuss in detail how cAMP-mediated transcriptional mechanisms leading to attenuation of IL-2 could direct both basal and activated nTreg-cell contact-dependent suppression of conventional CD4+ T cells. We present the hypothesis that cAMP underpins suppression by nTreg cells through the inhibitory function of ICER in the nucleus, which, in addition to cAMP transfer via gap junction intercellular communications (GJICs), is controlled, at least in part, by upstream interactions between CTLA-4 and B7.

“Supraphysiological” levels of intracellular cAMP in nTreg cells

A connection between cAMP and nTreg-cell function was described first by Bopp et al. who showed that the high intracellular cAMP levels in murine nTreg cells are critically involved in contact-dependent suppression of conventional CD4+ T cells [[9]]. nTreg cells harbor high “supraphysiological” levels of intracellular cAMP that are transferred to conventional CD4+ T cells via cell-contact-dependent GJICs. These findings correspond with the data of Rudensky and colleagues showing that expression of the cAMP-degrading phosphodiesterase 3b (PDE3b) is reduced in murine nTreg cells [[18]] by direct binding of Foxp3 to the Pde3b gene [[19]]. Thus, Foxp3 is, at least in part, responsible for the elevated levels of cAMP in nTreg cells. In addition, Foxp3 downregulates miR-142-3p, which silences adenylyl cyclase (ADCY9) mRNA, also leading to upregulated cAMP production in nTreg cells [[20]] (Fig. 1A).

Figure 1.

nTreg cells direct ICER into the nucleus of activated conventional CD4+T cells via cAMP (A) “Supraphysiologically” high intracellular cAMP levels are generated in nTreg cells, at least in part, by Foxp3-mediated downregulation of the Pde3b gene [[19]]. Furthermore, Foxp3 also downregulates miR-142-3p targeting adenylyl cyclase (ADCY9) mRNA resulting in upregulated cAMP production [[20]]. cAMP is then transferred from nTreg cells to the activated conventional CD4+T cells via gap junction intercellular communications (GJICs) [[9]]. There cAMP has at least two effects: first it induces ICER expression and second it enables the nuclear localization of ICER leading to transcriptional attenuation of IL-2 synthesis by suppression of NFATc1/α gene expression and/or formation of inhibitory NFAT/ICER protein complexes [[28, 44]]. In addition, cAMP may upregulate surface expression of CTLA-4 in suppressed conventional CD4+T cells [[5]], thus conferring a B7 inhibitory signal to target cell populations as depicted in Fig. 2. (B) In the absence of nTreg cells, for example, after ablation of nTreg cells in DEREG mice, TCR triggering and CD28 costimulation via CD28 superagonist (CD28SA) mAb results in cytosolic localization of ICER, which disables its function as a transcriptional repressor leading to unopposed NFAT-driven transcription [[28]]. When ICER is ousted to the cytosol, NFAT is translocated into nucleus and drives vigorous IL-2 expression in conventional CD4+T cell upon CD28 costimulation (activated T cell). (C) GJICs transferring cAMP from nTreg to conventional CD4+T cells lead to the maintenance of ICER in the nucleus of both cell populations during nTreg-cell-mediated suppression. In the presence of a CD28 signal (either CD3/CD28 in vitro or CD28A in vivo), ICER and NFAT colocalize in the nucleus of activated conventional CD4+T but not in nTreg cells; the latter failing to translocate NFAT to the nucleus upon CD28 costimulation [[28, 40]]. In the suppressed conventional CD4+T cells, nuclear colocalization of ICER and NFAT leads to the inhibition of NFATc1/α gene induction and/or the formation of inhibitory NFAT/ICER protein complexes, thus inhibiting NFAT-driven transcription [[12, 28]].

Importantly, human nTreg cells also harbor high levels of intracellular cAMP, which are increased even further after T-cell receptor (TCR) stimulation [[21]] and/or CD4 engagement [[22, 23]]. High levels of intracellular cAMP have been observed in oocytes that maintain prophase arrest using the orphan Gs-linked receptor GPR3, which activates adenylyl cyclase mediated conversion of ATP to cAMP [[24]]. Remarkably, another G protein-coupled receptor GPR83, overexpressed in conventional CD4+ T cells, has been shown to lead to the induction of Foxp3+ nTreg cells under specific inflammatory conditions in vivo [[25]]. The data on GPR83 [[25]] were further corroborated by the study of Kim and Leonard [[26]] implying a causal link between cAMP and Foxp3 expression.

cAMP and Foxp3 direct ICER-mediated suppression

Forced expression of Foxp3 allows for the acquisition of a regulatory phenotype in naïve CD25 T cells [[27]]. In analogy with the situation in nTreg cells, forced expression of Foxp3 in conventional CD4+ T cells induces ICER expression, presumably through elevated levels of intracellular cAMP [[12, 28]]. Similarly, forced Foxp3 expression in conventional CD4+ T cells increases CTLA-4 expression [[27]]. Based on genome-wide analysis of Foxp3 target genes in the nucleus of nTreg cells, ICER/CREM was identified as a direct target of Foxp3 binding [[29]]; however, CTLA-4 was not among the Foxp3 targets, suggesting that CTLA-4 expression was regulated by Foxp3 indirectly [[30]]. It is noteworthy that signaling through cAMP induces upregulation of CTLA-4 in the absence of TCR stimulation in resting human CD4+ T cells [[5]]. Therefore, cAMP-dependent signaling alone can trigger CTLA-4 expression in resting (and probably also in suppressed) conventional CD4+ T cells using a pathway distinct from that of costimulation. Moreover, forced retroviral expression of Foxp3 facilitates ICER expression in Foxp3 transductants and allows such transductants to suppress activated conventional CD4+ T cells in a CTLA-4-dependent fashion [[12, 28]]. Thus, in conventional CD4+ T cells, ICER-driven transcriptional attenuation of IL-2 could be conveyed by cAMP transfer via GJICs conferred by direct contact with nTreg cells, which may proceed, together with contact-dependent CTLA-4-mediated termination of T-cell proliferation, even though these conventional CD4+ T cells lack endogenous Foxp3 expression [[31]].

A new form of immunoregulation has recently been described that implicates ICER in the switch toward a regulatory phenotype in T helper 1 (Th1) cells [[32]]. When ICER induction is triggered in Th1 cells via the complement regulator CD46, IL-2 synthesis is strongly attenuated in the absence of Foxp3 suggesting an autonomous suppressive role for ICER. This is in agreement with a previous study implying ICER-transgenic splenocytes in suppressive function in the mixed lymphocyte reaction [[33]]. Therefore, ICER may play an important role in the induced suppressive potency of conventional CD4+ T cells leading to an inducible (i)Treg cell phenotype and/or “infectious” tolerance of target cell populations.

The relationship between Foxp3 and cAMP has been further explored in scurfy (sf) mice. Sf mice do not harbor functional nTreg cells since the mice have a mutation in the Foxp3 gene resulting in a lack of functional Foxp3 protein; the mice display an autoimmune phenotype [[34]]. The nonfunctional nTreg cells may carry potentially self-reactive TCRs that trigger the pathogenesis. However, such nonfunctional nTreg cells could not be identified via the nTreg-cell marker Foxp3 because of the lack of this protein. Crossing sf mice with DEREG (depletion of Treg cells, a conditional depletion system) mice meant that this limitation could be overcome since DEREG mice express GFP under the control of an additional Foxp3 promoter [[34, 35]]. The expression of GFP in cells is therefore indicative of so-called “would-be” nTreg cells (sf nTreg cells). By this measure, it was determined in vitro that sf nTreg cells had lost their suppressive capacity and that this coincided with a substantial reduction in their intracellular cAMP levels. Importantly, the surface expression of CTLA-4 on sf nTreg cells was unaffected, suggesting that in “would-be” nTreg cells CTLA-4 could be induced independently of cAMP. However, the expression of CTLA-4 in the absence of cAMP did not lead to effective suppression [[34]], suggesting that cAMP is instrumental for nTreg-cell-mediated suppression in vivo. The proposed role of Foxp3, cAMP, ICER, and CTLA-4 in the mechanism of nTreg-cell-mediated suppression is summarized in Fig. 1A.

nTreg-cell-mediated suppression of IL-2: A role for cAMP and ICER in vivo

In order to further understand the role of cAMP in nTreg-cell function, the effect of cAMP on ICER was investigated in two independent mouse models, using different techniques in each [[28]]. In the first model, the cAMP concentration, the subcellular (nuclear versus cytoplasmic) localization of ICER, and IL-2 gene expression levels were monitored in activated conventional CD4+ T cells of DEREG mice upon ablation of nTreg cells. In the second model, the vital dye calcein that spreads from donor to recipient cells via GJICs was used to identify conventional CD4+ T cells after transfer of calcein-loaded (OVA323-339) TCR-specific nTreg cells into OVA-specific TCR-transgenic mice lacking endogenous nTreg cells [[28]].

In the first model, cAMP concentrations and ICER localization were examined in T cells of DEREG mice under conditions of polyclonal activation using a stimulatory CD28-specific mAb [[36]]. The CD28 “superagonistic” antibody (CD28SA), which triggers both TCR and CD28, was found to preferentially activate nTreg cells in vivo, but this activation was dependent on the concomitant stimulation of conventional CD4+ T cells, which initially provided IL-2 but then were rapidly prevented from doing so by the activated nTreg cells. Subsequently, the activated nTreg cells further suppressed proliferation of, and IL-2 release by, the conventional CD4+ T cells. Attenuation of IL-2 transcription was found to occur following colocalization of ICER and nuclear factor of activated T cell (NFAT) in the nuclei of activated conventional CD4+ T cells [[28]] (Fig. 1B and C). Ablation of nTreg cells from the T-cell compartment of DEREG mice (by diphteria toxin [[35]]) led to the cytosolic localization of ICER in activated conventional CD4+ T cells upon CD28SA administration (Fig. 1B) and, under these conditions, cytosolic ICER failed to suppress IL-2 synthesis in activated conventional CD4+ T cells in vivo [[28]]. Importantly, recent reports on the subcellular localization of ICER have revealed that phosphorylation of discrete serine residue(s) can target ICER for ubiquitination [[37]] and lead to its subsequent translocation from the nucleus to the cytosol in proliferating cancer cells [[38]]. The cytosolic localization of ICER observed in both proliferating conventional CD4+ T cells and cancer cells suggests that nuclear ICER may play an important role in proliferation arrest.

In the absence of nTreg cells in the DEREG model, conventional CD4+ T cells, following activation with CD28SA, not only display cytosolic ICER and IL-2 production but also PDE4 is recruited to the lipid rafts. Upon CD28 costimulation, degradation of cAMP by the recruited PDE4 counteracts the local TCR-induced production of cAMP [[39]]. In the presence of nTreg cells, direct contact between the nTreg and conventional CD4+ T cells leads to the transfer of cAMP through GJICs and the nuclear localization of ICER in conventional CD4+ T cells, even upon CD28SA activation (Fig. 1B and C) [[28]]. Thus, despite delivery of a strong CD28 costimulatory signal, nTreg cells may reinstate suppression via cAMP transfer and enable ICER suppressive functions in activated conventional CD4+ T cells in vivo.

nTreg cells induce cAMP/ICER-dependent inhibition of IL-2 synthesis in vivo

In the second model mentioned above, cAMP transferred by nTreg cells via GJICs was directly measured by ELISA in calcein-positive target cell populations [[28]]. Mice that bear CD4+ T cells expressing a transgenic OVA323-339-specific T-cell receptor but lacking nTreg cells (on a RAG1−/− background) were immunized with OVA peptide in complete Freund's adjuvant in one footpad. CD3/CD28-expanded calcein-loaded OVA TCR-transgenic nTreg cells were then adoptively transferred and CD4+ T cells were isolated and their calcein content determined by flow cytometry. ICER was found in the nucleus of calcein-positive conventional CD4+ T cells isolated from the draining lymph nodes, that is, those conventional CD4+ T cells that had interacted with calcein-loaded nTreg cells. In contrast, ICER was cytosolic in calcein-negative CD4+ T cells isolated from, for example, the nondraining lymph nodes, that is, those conventional CD4+ T cells that had not interacted and formed GJICs with nTreg cells. Importantly, cAMP transfer from nTreg to conventional CD4+ T cells was dependent on OVA stimulation since nonactivated target CD4+ T cells (no OVA immunization) did not show any cAMP transfer despite being isolated from the draining lymph nodes, that is, in the presence of nTreg cells [[28]]. The calcein-positive conventional CD4+ T cells showed a significant increase in intracellular cAMP levels, which was tightly correlated with the nuclear localization of ICER and reduced IL-2 production. A direct role for ICER in the attenuation of IL-2 synthesis was demonstrated by hyperproduction of IL-2 in ICER-deficient conventional CD4+ T cells upon CD3/CD28 costimulation (T. Bopp, unpublished observations). This is consistent with the inhibition of IL-2 synthesis by nuclear (but not cytosolic) ICER observed in vivo [[28]]. Together these findings demonstrate that during nTreg-cell-mediated suppression nTreg cells transfer cAMP through GJICs, which leads to the retention of ICER in the nucleus of conventional CD4+ T cells resulting in the attenuation of IL-2 synthesis in vivo (Fig. 1).

nTreg cells: NFAT does not translocate efficiently to the nucleus

The transcriptional landscape in murine nTreg cells is influenced by their high intracellular levels of cAMP. For example, murine nTreg cells constitutively express high levels of ICER which is not significantly expressed in conventional CD4+ T cells until cAMP is elevated, for example, upon cAMP transfer via GJICs mediated by nTreg cells [[11, 28]]. nTreg cells also show markedly increased ICER RNA and protein levels in vivo, in comparison with conventional CD4+ T cells, with the ICER typically being nuclear in nTreg cells irrespective of CD28 costimulation [[28]]. This is most likely due to the elevated levels of intracellular cAMP. Furthermore, upon CD28 costimulation, nTreg cells fail to efficiently translocate NFATc1 to the nucleus and are unable to produce the IL-2 that can be readily induced in conventional CD4+ T cells (Fig. 1B and C). This inability to translocate NFATc1 to the nucleus upon activation has also been confirmed in human nTreg cells isolated from peripheral blood lymphocytes (PBLs) [[40]]. In murine nTreg cells, the failure to translocate NFATc1 is associated with reduced calcium flux, diminished calcineurin activation, and increased activity of glycogen synthase kinase-3β, a negative regulator of NFATc1 [[41]]. This inability to efficiently translocate NFATc1 to the nucleus of nTreg cells [[28]] casts doubt on the NFAT/Foxp3 complex proposed to play a critical role in the suppressive function of nTreg cells [[42]].

NFAT/ICER in conventional CD4+T cells: A critical inhibitory checkpoint?

While NFATc1 has been found to be essential for IL-2 expression [[28]], a complex of NFAT and ICER can strongly suppress IL-2 expression in conventional CD4+ T cells [[43, 44]]. Such inhibitory NFAT/ICER complexes bind to multiple composite NFAT/AP-1 DNA sites in vitro, and are likely to play an important role in the suppression of numerous NFAT-driven cytokines and chemokines, such as TNF-α, IL-4, IL-13, GM-CSF, MIP-1α, and MIP-1β [[13, 43, 44]]. NFAT/ICER complexes are also presumably involved in transcriptional attenuation of NFAT-driven cytokines and chemokines during nTreg-cell-mediated suppression of conventional CD4+ T cells (e.g. following CD28SA administration in DEREG mice [[28]]). A critical role for NFAT factors forming inhibitory complexes with ICER in conventional CD4+ T cells is further strengthened by observations indicating that conventional CD4+ T cells from NFATc2/c3 double-deficient mice are unresponsive to suppression, even though nTreg-cell development and function are normal [[45]]. When intracellular cAMP levels are elevated in conventional CD4+ T cells, ICER and/or CREMα inhibits the induction of c-fos and prevents subsequent formation of the AP-1 complex [[13, 44, 46]]. The reduced levels of the AP-1 complex allow ICER to outcompete AP-1 at NFAT/AP-1 DNA-binding motifs and NFAT/ICER complexes to be preferentially formed. Hence, upon contact with nTreg cells, inhibitory NFAT/ICER complexes are likely to play an important role in transcriptional attenuation of NFAT-driven cytokine and chemokine expression in suppressed conventional CD4+ T cells [[13, 28, 44]].

cAMP and CTLA-4: Concerted signaling through B7

CTLA-4 is a dominant negative receptor of the CD28 superfamily of immune regulatory molecules. Several groups have investigated the involvement of constitutive CTLA-4 expression in the suppressive mechanisms of nTreg cells [[47-50]]. Two initial studies performed independently (in Shimon Sakaguchi's and Fiona Powrie's laboratories) indicated that nTreg-cell-mediated suppression could be abrogated by CTLA-4 blockade using anti-CTLA-4 antibodies in vitro [[48, 49]]. Furthermore, Sakaguchi's research group showed that an nTreg-cell-specific CTLA-4 deficiency impaired the suppressive function of nTreg cells in vivo [[47]]. In particular, Sakaguchi's data demonstrated that nTreg cells could downregulate B7-1 and B7-2 expression on dendritic cells (DCs). Moreover, CTLA-4 blockade leading to abolition of nTreg-cell function has been shown in vitro to disrupt nTreg-cell-mediated expression of ICER [[12]]. Thus, nTreg cells interacting through their CTLA-4 with B7 expressed on activated conventional CD4+ T cells and/or APCs could potentiate suppression via induction of ICER. Based on these data we propose that, in addition to cAMP-mediated activation of ICER function, nTreg-cell suppression of conventional CD4+ T cells may act synergistically with signals conveyed by CTLA-4/B7 interactions [[28]]. In particular, CTLA-4/B7 interactions between nTreg and conventional CD4+ T cells may confer a B7-mediated inhibitory signal into the conventional CD4+ T cells [[51, 52]], which is mediated, at least in part, via ICER induction and/or protection of ICER from degradation [[12, 28]]. Therefore, B7 engagement may strengthen the cAMP-driven function of ICER (induction and nuclear localization) in suppressed conventional CD4+ T cells and thus attenuate IL-2 synthesis either by direct transcriptional repression through DNA binding of ICER and/or via protein–protein interactions of ICER with NFAT within NFAT/ICER inhibitory complexes [[28, 44]]. This view is consistent with recent observations by Allison and colleagues [[50]] that indicate that blockade of CTLA-4 on both conventional and nTreg cells decreases suppression and facilitates the antitumor activity of anti-CTLA-4 antibodies in vivo.

B7 expressed on activated CD4+T cells: A target molecule for suppression

B7-1 (CD80) and B7-2 (CD86) encode type I transmembrane proteins that interact with CD28 and CTLA-4 expressed on T cells. Based on observations by Cantor and colleagues [[51, 52]], the transmission of a suppressive signal by nTreg cells requires the engagement of the B7 expressed on activated conventional CD4+ T cells. Indeed activated conventional CD4+ T cells lacking the B7 receptor (i.e. cells isolated from B7-1 and B7-2 double knockout (B7-DKO) mice) showed increased resistance to nTreg-cell-mediated suppression. Moreover, adoptively transferred activated B7-DKO conventional CD4+ T cells provoked a lethal wasting disease in lymphopenic mice, even when transferred together with nTreg cells [[51]]. Suppression of B7-DKO conventional CD4+ T cells could be restored by T-cell-specific lentiviral expression of full length but not truncated forms lacking the transmembrane/cytoplasmic domain of the B7 receptor [[51]]. It has also been shown that triggering with an anti-B7-1 mAb induced ICER expression in activated conventional CD4+ T cells [[12]]. Furthermore, coculture of B7-DKO conventional CD4+ T cells with wild-type nTreg cells did not efficiently suppress IL-2 production nor induce ICER significantly [[12]]. Thus it seems that, in B7-DKO conventional CD4+ T cells, the increased resistance to nTreg-cell-mediated suppression results from diminished ICER expression, accompanied by increased IL-2 synthesis.

APCs: Direct targets of nTreg cells

The suppression of APCs by nTreg cells represents a very effective approach to dampen T-cell-dependent immune responses. It has been previously shown that nTreg cells can interact directly with DCs immediately after cell transfer into mice [[53]]. nTreg cells either promote the secretion of suppressive factors by the target DC population or abrogate the activity of DCs. Engagement of CTLA-4 on nTreg cells with B7 expressed on APCs results in the induction of indoleamine 2,3-dioxygenase (IDO), which in turn leads to immune suppression as a consequence of tryptophan depletion and production of pro-apoptotic metabolites [[54]]. Importantly, coculture of murine DCs and nTreg cells can increase DC cAMP levels and IL-10 synthesis, which leads to rapid downregulation of the costimulatory molecules B7-1 and B7-2 on the DCs [[55]]. This is consistent with the ICER-mediated downregulation of B7-1 (and to a lesser extent of B7-2), triggered by the nuclear localization of ICER following nTreg-cell-mediated cAMP transfer into conventional CD4+ T cells and/or APCs [[28, 55, 56]]. In the absence of B7 on APCs, the consequent lack of a CD28 signal in conventional CD4+ T cells may prevent activation of cAMP-degrading PDEs [[39]] and thus strengthen the cAMP/ICER-mediated suppression in vivo [[28]]. Therefore, transendocytosis by CTLA-4 on nTreg cells leading to the removal of B7 from APCs and the subsequent absence of a CD28 signal in conventional CD4+ T cells [[57]] may substantially contribute to the elevated intracellular cAMP levels in conventional CD4+ T cells after TCR triggering [[28]]. This protective effect of cAMP may lead to nuclear accumulation of ICER and repression of NFAT-driven transcription including that of IL-2 [[12, 28]]. It is assumed that suppression of cytokine and chemokine expression, along with the cAMP/ICER-mediated transcriptional attenuation of B7 in DCs, plays an important role in nTreg-cell-mediated suppression [[12, 28, 56, 57]]. A suppressive role for cAMP in conjunction with CTLA-4/B7 interactions is supported by observations suggesting that functional GJICs accumulate at the immuno-logical synapse during T-cell activation [[58]]. Therefore, transfer of cAMP via gap junction is enhanced at the immunological synapse and may further contribute to the cAMP-mediated CTLA-4 expression in suppressed conventional CD4+ T cells as summarized in Fig. 2.

Figure 2.

Model of nTreg-cell-mediated suppression: cAMP transfer via GJICs facilitated by CTLA-4/B7 interactions. (A) nTreg cells constitutively express CTLA-4 which then interacts with B7 induced on APCs upon activation leading to accumulation of GJICs at the immunological synapse [[58]] facilitating cAMP transfer by nTreg cells and strengthening the suppressive functions of ICER in the nucleus of APCs. (B) The interaction of CTLA-4 with B7 expressed on activated conventional CD4+T facilitates transfer of cAMP through GJICs into conventional CD4+T cells (suppressed Tcells). This further amplifies the cAMP-driven CTLA-4 expression in suppressed conventional CD4+T cells [[5]]. According to this model, CTLA-4/B7 engagement allows B7 to confer inhibitory signals via ICER into APCs leading eventually to transcriptional downregulation of B7 at the immunological synapse [[28, 57]]. (C) In suppressed CD4+T cells, cAMP enables the nuclear functions of ICER, which attenuate IL-2 synthesis by suppression of NFATc1/α induction and/or formation of inhibitory NFAT/ICER complexes in suppressed conventional CD4+T cells [[28, 44]].

CD28: Trigger of a critical costimulatory pathway for the control of nTreg-cell homeostasis

Costimulation is key for the development and function of nTreg cells (reviewed in [[59]]). CD28 is expressed constitutively on nTreg cells and, besides playing an essential role in nTreg-cell development in the thymus [[60]], it is also the major costimulatory molecule for conventional CD4+ T cells. CD28 signaling and IL-2 production is of critical importance for the generation and maintenance of nTreg cells [[61]]. On the one hand, nTreg cells themselves need to receive a signal through CD28 to suppress conventional CD4+ T cells and, on the other, CD28 signaling in conventional CD4+ T cells induces them to produce IL-2, which in turn stimulates nTreg cells through the IL-2 receptor. In animal models of autoimmunity, both prophylactic and therapeutic administration of CD28SA, which synergistically activates CD28 and TCR signaling, prevented or at least greatly reduced the clinical symptoms of experimental autoimmune encephalitis [[62]]. Adoptive transfer of CD28SA-treated nTreg cells has shown that CD28SA mediates long-term protection against autoaggressive immune reactions by inducing the expansion and activation of nTreg cells. Therefore, CD28SA seemed to offer a promising novel treatment modality for human autoimmune diseases. However, in contrast to the benign and anti-inflammatory behavior of the rat- or mouse-specific CD28SA in animal models [[62]], the fully humanized human-CD28SA mAb designated TGN1412 not only expanded nTreg cell numbers but also induced a life-threatening cytokine release syndrome in conventional CD4+ T cells during a phase one clinical trial, despite having been shown to be without side effects in nonhuman primates [[63, 64]]. Strikingly, human effector memory CD4+ T cells expressing CD28, which are missing in nonhuman primates, were identified as the subset responsible for the life-threatening cytokine release syndrome observed during the CD28SA clinical trial [[65]]. Thus, species differences in CD28 expression observed between primate and human cells seem to explain fundamental differences in immune response upon administration of CD28SA.

One way to ameliorate the adverse side effects of CD28SA observed in the clinical trial would be to increase intracellular cAMP levels in conventional CD4+ T cells. For instance, treatment with the pan-PDE inhibitor 3-isobututyl-1-methyl-xantine (IBMX) leads to elevated levels of intracellular cAMP and can restore nTreg-cell-mediated suppression upon CD28 costimulation in vitro [[28, 66]]. Further exploration of cAMP/ICER-driven suppression in human conventional CD4+ T cells in the context of CD28SA-mediated nTreg-cell expansion could shed more light on the clinical utility of PDE inhibitors such as Rolipram, and a combination of treatment with cAMP-elevating agonists and TGN1412 may generate new therapeutic options for TGN1412. This aim is further justified by findings from recent clinical studies indicating that adoptive transfer of ex vivo expanded CD3/CD28-activated nTreg cells confers a reduced incidence of acute graft versus host disease [[67]].

cAMP and CD28: The hypoxic governance of nTreg cells in vitro and in vivo

There is an important discrepancy in the ability of murine nTreg cells to suppress murine conventional CD4+ T cells after CD3/CD28 costimulation in vitro and in vivo. In vitro, conventional anti-CD3 and anti-CD28 antibodies abrogate nTreg-cell-mediated suppression and promote IL-2 synthesis in nTreg cell assays and have been assumed to “break suppression” [[15]]. In vivo, however, administration of a CD28SA (triggering both the TCR and CD28) in mice resulted in enhanced nTreg-cell-mediated suppression and inhibition of IL-2 synthesis [[28, 36]]. A plausible explanation for these conflicting observations relates to the hypoxic governance of conventional CD4+ T cells [[68]], a regulatory mechanism that is underestimated because the majority of the current in vitro cellular immunology studies are performed at nonphysiologically high oxygen tensions that weaken hypoxia-adenosinergic signaling [[69]]. The hypoxia-adenosinergic tissue-protecting mechanism is triggered by inflammatory damage and by the hypoxia-driven accumulation of extracellular adenosine that signals into conventional CD4+ T cells via immunosuppressive, cAMP-elevating A2A adenosine receptors [[68-70]]. Extracellular adenosine is produced by the local tissue hypoxia-upregulated ectoenzymes ATPase/ADPase CD39 and the 5′-ectonucleotidase CD73, which are expressed on several cell types including primed uncommitted conventional CD4+ T cells and nTreg cells [[71, 72]] (Fig. 3).

Figure 3.

Hypoxia-adenosinergic signaling: An additional model for the cAMP-enabled and nTreg-cell-mediated suppressive function of ICER. CD39 and CD73 ectoenzymes on nTreg cells generate extracellular immunosuppressive adenosine from ATP, which adds to the suppressive effects of inflamed-tissue hypoxic adenosinergic signaling on conventional CD4+T cells [[71, 72]] acting via the A2AR expressed on CD4+T cells (both nTreg and conventional CD4+ T cells). A2AR signaling enhances the levels of intracellular cAMP and, presumably in synergy with the models described in Fig. 1 and 2, enforces nuclear localization of ICER leading to transcriptional attenuation of IL-2 production in suppressed conventional CD4+T cells.

It is well established that the extracellular adenosine-A2AR-cAMP pathway inhibits the effector functions of T cells depending on differences in oxygen tensions in different tissues and that A2A adenosine receptors expressed on nTreg cells are critically involved in suppressive functions under hypoxic conditions [[71]]. Nevertheless, data from DEREG mice demonstrating direct cAMP transfer from nTreg cells to conventional CD4+ T cells enabling ICER function in vivo, argue in favor of autonomous nTreg-cell function acting through cell-to-cell communication via GJICs independent of the A2AR-cAMP pathway [[9, 28, 36]]. This notion is further supported by recent observations that mice deficient in connexin 43, which is involved in GJIC formation, produce only a few nTreg cells and have an increased proportion of activated conventional CD4+ T cells [[73]]. Moreover, treatment of human conventional CD4+ T cells with the irreversible adenylyl cyclase inhibitor MDL-12 (which inhibits the adenylyl cyclase conversion of ATP to cAMP) do not prevent the suppression of such conventional CD4+ T cells by human nTreg cells [[40]]. In contrast, the treatment of human nTreg cells with MDL-12 completely abrogated their suppressive capacity [[22, 40]]. Hence, these findings strongly suggest that nTreg-cell-mediated suppression is independent of the endogenous cAMP induced in conventional CD4+ T cells in a receptor-mediated fashion and instead is primarily driven by the transfer of cAMP generated in nTreg cells. Since both scenarios propose elevated levels of intracellular cAMP in the target conventional CD4+ T cells, it is difficult, at the present time, to distinguish in vivo between these two scenarios. However, it is conceivable that GJICs facilitated in the immunological synapse by CTLA/B7 interactions could, in a synergistic fashion with adenosine, elevate intracellular cAMP levels and enable the nuclear function of ICER in target populations of conventional CD4+ T cells and/or APCs during CD28 costimulation.

Concluding remarks

In this article, we have proposed that upon contact with nTreg cells, cAMP is transferred from nTreg cells into activated target CD4+ T cells and/or APCs via GJICs to suppress their function. As a consequence, intercellular transfer of cAMP from nTreg cells into activated conventional CD4+ T cells in vivo results in ICER-mediated suppression of IL-2 upon delivery of CD28 signals. Moreover, ICER binds to the cAMP-responsive elements (CREs) in the promoter of NFATc1/α inhibiting its induction. Upon activation, ICER may form inhibitory complexes with preexisting NFATc1/c2 thus dampening NFAT-driven transcription, especially that of IL-2. In addition, during nTreg-cell-mediated suppression, cAMP modulates surface-expressed CTLA-4 and its cognate B7 ligands expressed on conventional CD4+ T cells and/or APCs. Hence, nTreg cells may control the immune regulatory network via cAMP that underpins – as a common denominator – two crucial immune suppressive mechanisms directed by ICER and CTLA-4.

Acknowledgements

We are indebted to members of Schmitt, Hünig, and Serfling laboratories for fruitful discussions during writing this review.

Conflict of interest

The authors declare no financial or commercial conflict of interests.

Abbreviations
CREM

cAMP-response modulator

GJIC

gap junction intercellular communication

ICER

inducible cAMP early repressor

IDO

indoleamine 2,3-dioxygenase. sf: scurfy

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