Human CD4 T lymphocytes with increased intracellular ... - CiteSeerX

Human CD4⫹ T lymphocytes with increased intracellular cAMP levels exert regulatory functions by releasing extracellular cAMP Silvia Vendetti,*,1 Mario Patrizio,† Antonella Riccomi,* and Maria Teresa De Magistris* Departments of *Infectious, Parasitic and Immune-Mediated Diseases and †Drug Research and Evaluation, Istituto Superiore di Sanita`, Rome, Italy

Abstract: We have previously shown that cholera toxin (CT) and other cAMP-elevating agents induce up-regulation of the inhibitory molecule CTLA-4 on human resting T lymphocytes. In this study, we evaluated the function of these cells. We found that purified human CD4ⴙ T lymphocytes pretreated with CT were able to inhibit proliferation of autologous PBMC in a dose-dependent manner. It is interesting that this phenomenon was not mediated by inhibitory cytokines such as IL-10, IL-4, or TGF-␤ but was in part caused by the release of extracellular cAMP by the CD4ⴙ T lymphocytes. Purified CD4ⴙ T cells pretreated with forskolin, a transient cAMP inducer, or with dibutyryl cAMP, an analog of cAMP, did not exert suppressive functions, suggesting that a sustained production of cAMP, such as that induced by CT, was required to identify a novel regulatory function mediated by CD4ⴙ T cells. Our results show that CD4ⴙ T lymphocytes can exert regulatory functions through the release of extracellular cAMP and that the cyclic nucleotide acts as a primary messenger, which could play a biological role in the modulation of immune responses. J. Leukoc. Biol. 80: 880 – 888; 2006. Key Words: T cells 䡠 suppression 䡠 cholera toxin 䡠 immunoregulation

INTRODUCTION The role of cAMP as a second messenger in cells of the immune system has been widely described [1]. In particular, elevation of intracellular cAMP concentration in T lymphocytes has an inhibitory effect on proliferation and IL-2 production [2] and has been shown to affect early T cell activation events [3]. The engagement of the TCR induces an increase of intracellular cAMP [4, 5], and it has been demonstrated that costimulatory signals are needed to overcome its inhibitory effects during T cell activation [6]. In accordance with this, increased intracellular cAMP has been implicated in the induction of T cell anergy [7], a state of unresponsiveness that occurs when T cells are stimulated through CD3/TCR in the absence of costimulation. It has been widely reported that 880

Journal of Leukocyte Biology Volume 80, October 2006

anergic T lymphocytes exert suppressive functions in vitro and in vivo [8-10]. Cholera toxin (CT) is a bacterial toxin of 84.7 kDa, which ADP-ribosylates the ␣ subunit of G proteins constitutively activating adenylate cyclase and leading to an increase of cAMP synthesis [11]. We have previously demonstrated that CT and other cAMP-elevating agents induce up-regulation of soluble and membrane isoforms of the inhibitory molecule CTLA-4 in resting T lymphocytes [12]. The CTLA-4 molecule is constitutively expressed on T cells with regulatory activity (Treg); they do not proliferate or produce IL-2 and are able to inhibit the proliferative response and cytokine production of effector T cells [13–15]. Treg cells consist of heterogeneous T cell populations, which can be classified into two major categories, including the naturally occurring CD4⫹CD25⫹ Treg and those induced in the periphery [16]. The repeated stimulation with antigen or polyclonal activators in the presence of inhibitory cytokines such as IL-10 or TGF-␤ leads to the induction of IL-10-producing Treg [type 1 regulatory cell (Tr1)] and TGF-␤-producing Treg [T helper cell type 3 (Th3)], respectively [17, 18]. It has also been reported that immunosuppressive drugs induce human and mouse CD4⫹ T cells to differentiate in vitro into Treg producing IL-10 [19]. Further, Tr1 clones have been established in vivo in mice and humans infected with pathogens that cause persistent or chronic infections [20, 21]. Regardless of their origin, all the induced Treg populations described to date, except for anergic T cells, which suppress by cell-to-cell contact, have the capacity to suppress in a cytokine-dependent manner. However, whether T cells exert regulatory functions through the release of inhibitory factors other then cytokines remains to be determined. In the attempt to analyze the function of CD4⫹ T lymphocytes treated with CT or other cAMP-elevating agents, we evaluated their regulatory activity and asked whether cAMP played any role. We found that human-purified CD4⫹ T lymphocytes pretreated with CT were able to inhibit T cell proliferation, and the suppression was, at least in part, mediated by the release of

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Correspondence: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena 299, Rome 00161, Italy. E-mail: [email protected] Received January 31, 2006; revised April 28, 2006; accepted May 19, 2006; doi: 10.1189/jlb.0106072

0741-5400/06/0080-880 © Society for Leukocyte Biology

extracellular cAMP. This suggests that cAMP can play a role in the modulation of the immune response acting as a primary extracelluar messenger.

MATERIALS AND METHODS Media and reagents RPMI 1640 supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin (Gibco, Grand Island, NY), and 10% FCS (Hyclone Laboratories, Logan, UT) was used as complete medium in all cultures. Anti-CD3 (Clone UCHT1) and anti-CD28 (Clone CD28.2) mAb were purchased from Immunotech (Westbrook, ME) and PharMingen (San Diego, CA), respectively. Neutralizing anti-IL-10 (23738), -IL-10R (37607.11), -IL-4 (3007.11) and TGF-␤ (9016.2) mAb were purchased from R&D Systems (Minneapolis, MN). CT and CT-B subunit (CT-B) were purchased from Calbiochem-Novabiochem (San Diego, CA) or List Biological Laboratories (Campbell, CA); forskolin (FSK), dibutyryl cAMP (dbcAMP), cAMP, and phosphodiesterase (PDE) were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell isolation and CD4⫹ lymphocyte purification PBMC were isolated from healthy donors by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density centrifugation. CD4⫹ T lymphocytes were purified by negative selection using an immunomagnetic cell sorter (Miltenyi Biotec, Germany). Briefly, PBMC were labeled using a cocktail of hapten-conjugated mAb anti-CD8, CD11b, CD16, CD19, CD36, and CD56 molecules and MACS MicroBeads coupled to an anti-hapten mAb. The magnetically labeled cells were depleted by retaining them on a column using a MidiMACS cell separator.

Proliferation assay Purified CD4⫹ T cells (5⫻106/ml) were incubated in the absence or in the presence of CT (3 ␮g/ml), CT-B (3 ␮g/ml), FSK (50 ␮M), or dbcAMP (0.5 mM). After 24 h, cells were harvested, irradiated (3000 rad), washed extensively, and incubated for 1 h in round-bottom, 96-well plates with autologous PBMC (6⫻104) at different ratios (3:1, 2:1, 1:1). Then, increasing concentrations of anti-CD3 mAb (Immunotech) or increasing concentrations of tetanus toxoid (TT) antigen were added to the cultures, and the proliferation was evaluated by 3 H-thymidine incorporation. Plates were incubated for 48 h at 37°C with 5% CO2, and 3H-thymidine (Amersham, Aylesbury, UK) was added (1 ␮Ci/well). After 18 h, cells were harvested, and incorporated radioactivity was measured by micro␤-counting. In some experiments, neutralizing mAb anti-IL-4, -IL-10, -IL-10 receptor (IL-10R), or –TGF-␤ (1 ␮g/ml) were added singularly or as a cocktail to the cultures. In others experiments, PDE was added at different concentrations (0.5–5 ␮g/ml) to the cultures 30 min before adding anti-CD3 mAb. To evaluate the effect of exogenously added cAMP and dbcAMP, PBMC were incubated in the presence or in the absence of cAMP or dbcAMP (0.1 mM), with or without PDE (5 ␮g/ml), and stimulated with anti-CD3 mAb (0.5 ␮g/ml). T cell proliferation was evaluated by 3H-thymidine incorporation after 66 h of culture.

Transwell experiments In transwell assays, cells were separated by a membrane (6.5 mm diameter, 0.4 mm pore size) in 24-well plates (Costar, Corning, NY). The lower compartments of the wells contained PBMC (8⫻105). The upper compartments contained medium alone, untreated or with CT-pretreated CD4⫹ T cells (2.4⫻106). In some experiments, untreated or CT-pretreated CD4⫹ T lymphocytes were placed in the lower chambers together with PBMC, which were stimulated with anti-CD3 mAb (0.5 ␮g/ml) for 48 h, and proliferation was evaluated by harvesting the cells from the lower compartments and by incubating them in the presence of 3H-thymidine for further 18 h.

Supernatant ultrafiltration Purified CD4⫹ T cells (5⫻106/ml) were incubated in the absence or in the presence of CT (3 ␮g/ml) for 24 h. Thereafter, the cells were harvested, washed

extensively, and incubated in 24-well plates (5⫻106/ml) for 48 h. Culture supernatants from untreated and CT-pretreated CD4⫹ T lymphocytes were collected and filtered using Amicon Ultra-4 centrifugal filters (Millipore, Bedford, MA) with membrane cut-off of 10 k, which allowed the separation of low molecular weight compounds. Filtered supernatants from untreated or CT-pretreated CD4⫹ T cells were supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin (Gibco), and 10% FCS (Hyclone Laboratories) and added (75% of total culture volume) to PBMC in round-bottom, 96-well plates. After 1 h, cells were stimulated with anti-CD3 mAb (0.5 ␮g/ml). The proliferation was evaluated by 3H-thymidine after 66 h.

Cytokine assay Purified CD4⫹ T cells (5⫻106/ml) were incubated in the absence or in the presence of CT (3 ␮g/ml) and CT-B (3 ␮g/ml) in round-bottom, 96-well plates and stimulated with plate-coated anti-CD3 mAb (5 ␮g/ml) and soluble antiCD28 mAb (1 ␮g/ml). Culture supernatants were collected after 48 h and frozen at – 80°C until assayed for IL-10, IFN-␥, IL-4, and IL-5 content by sandwich ELISA. The levels of these cytokines were assayed by using antibody pairs of mouse anti-human IL-10, IFN-␥, IL-4, and IL-5 (2 ␮g/ml) and biotin-conjugated rat anti-human IL-10, IFN-␥, IL-4, and IL-5 (0.5 ␮g/ml) from Pierce Endogen (Rockford, IL).

cAMP measurement CD4⫹-purified T cells (5⫻106/ml) were incubated in the absence or in the presence of CT (3 ␮g/ml) or FSK (50 ␮M), and culture supernatants were collected after 24 h to measure the accumulation of extracellular cAMP. In same experiments, cells were harvested, irradiated (3000 rad), washed extensively, and cultured at 2 ⫻ 105/well in round-bottom, 96-well plates in the presence of increasing concentrations of PDE (0.5–5 ␮g/ml). In this case, supernatants were collected at different time-points after 1, 4, and 24 h. Culture supernatants (90 ␮l) were then added with 10 ␮l 0.1 M HCl and processed for cAMP determination by a RIA following sample acetylation as described [22]. The sensitivity of the test was 1 ftmol.

Statistical analysis Microsoft Excel (Microsoft Corp., Redmond, WA) was used for statistical analysis. Data were expressed as mean ⫾ SD, and statistical significance was determined by Student’s t-test. P ⬍ 0.05 was considered statistically significant.

RESULTS CD4⫹ T lymphocytes pretreated with CT inhibit the proliferation of bystander, autologous PBMC First, we asked whether human T lymphocytes treated with CT show regulatory activity. Purified CD4⫹ T lymphocytes isolated from PBMC of healthy donors were incubated in the presence of CT; CT-B, the B subunit of CT, which lacks the ADP-rybosyl transferase activity; FSK, a transient cAMP inducer; or dbcAMP, an analog of cAMP, for 24 h. Thereafter, the cells were irradiated (3000 rad), washed extensively, and cocultured with autologous PBMC stimulated in the presence of anti-CD3 mAb. T cell proliferation was evaluated after 48 h of culture. Figure 1, A and B, shows that human-purified CD4⫹ T lymphocytes pretreated with CT were able to inhibit the proliferation of autologous PBMC in a dose-dependent manner (P⬍0.05 for all the concentrations of anti-CD3 mAb used), whereas the untreated T cells did not. The same results were obtained using non-irradiated CD4⫹ T lymphocytes pretreated with CT (data not shown). Treatment with CT-B did not affect the proliferation of bystander PBMC (Fig. 1C), suggesting that the enzy-

Vendetti et al. Suppression by cAMP released from CD4ⴙ T cells

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ing doses of TT. We found that CT pretreated CD4⫹ T cells were able to inhibit the TT-specific proliferation as compared with untreated CD4⫹ T cells (P⬍0.05 for all doses of TT used; Fig. 2). These data show that CT-pretreated CD4⫹ T lymphocytes have regulatory functions also in an antigen-specific system.

The inhibition of proliferation by CT-pretreated CD4⫹ T cells also occurs in the absence of cellto-cell contact

Fig. 1. CT-pretreated CD4⫹ T lymphocytes inhibit the proliferation of bystander, autologous PBMC. Purified CD4⫹ T lymphocytes (5⫻106/ml) were cultured in the presence of medium alone (A), CT (3 ␮g/ml; B), CT-B (3 ␮g/ml; C), FSK (50 ␮M; D), and dbcAMP (0.5 mM; E). After overnight incubation, CD4⫹ T cells were washed three times, irradiated (3000 rad), and cultured with autologous PBMC (6⫻104) at different CD4⫹/PBMC ratios (3:1, 2:1, and 1:1) in the presence of increasing doses of anti-CD3 mAb (from 0.005 to 0.5 ␮g/ml). T cell proliferation was evaluated after 66 h by 3H-thymidine incorporation. The data shown are from one representative experiment of six performed.

To investigate whether the suppression mediated by CT-pretreated CD4⫹ T lymphocytes was dependent on cell-to-cell contact or on secretion of soluble factors, we performed transwell experiments. Untreated or CT-pretreated CD4⫹ T lymphocytes were placed in the upper chambers of transwells, whereas autologous PBMC were placed in the lower chambers in the presence or absence of anti-CD3 mAb. In parallel, untreated or CT-pretreated CD4⫹ T lymphocytes were placed in the lower chambers together with PBMC. We found that CT-pretreated CD4⫹ T cells inhibited the proliferation of autologous, antiCD3-stimulated PBMC, even in the absence of cell-to-cell contact, although the inhibition was slightly lower than that observed when PBMC and CT-pretreated CD4⫹ T lymphocytes were placed in the lower chambers together (P⬍0.05; Fig. 3A). These results suggest that soluble factors are involved in the suppression induced by CT-pretreated CD4⫹ T cells, although cell-to-cell contact plays also a role in the inhibition. To rule out the possibility that a low amount of CT could be released by the CT-pretreated CD4⫹ T cells and could exert inhibitory effect on target cells, we filtered the supernatants from untreated or CT-pretreated CD4⫹ T cells using filters with

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matic activity of CT is required for the induction of CD4⫹ T lymphocytes with regulatory activity. On the contrary, purified CD4⫹ T cells pretreated with FSK or with dbcAMP did not suppress the proliferation of bystander anti-CD3-stimulated PBMC (Fig. 1, D and E). This suggests that a sustained production of cAMP, such as that obtained by CT treatment, is required for the induction of CD4⫹ Treg cells. Furthermore, we analyzed the phenotype and the functional state of the CD4⫹ T cells pretreated with CT. As we observed previously [12], CT-pretreated CD4⫹ T cells up-regulated the inhibitory molecule CTLA-4 as compared with untreated cells (data not shown). Conversely, the expression levels of the activation markers CD25 and CD69 were not affected by the treatment (data not shown). Moreover, CT-pretreated CD4⫹ T cells were strongly inhibited in their capacity to proliferate and to produce IL-2 upon anti-CD3 stimulation (data not shown), suggesting that CT induces an anergic-like, functional state in T cells. To investigate whether CT-pretreated CD4⫹ T lymphocytes were able to inhibit antigen-specific T cell responses, we cultured untreated or CT-pretreated CD4⫹ T cells from donors immune to TT with autologous PBMC stimulated with increas-

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TT (μg/ml) PBMC + Untreated CD4+ PBMC + CT-pretreated CD4+ Fig. 2. CT-pretreated CD4⫹ T lymphocytes inhibit the proliferation of autologous TT-specific PBMC. Untreated or CT-pretreated CD4⫹ T lymphocytes from donors immune to TT antigen were cultured with autologous PBMC (6⫻104) at a CD4⫹:PBMC ratio of 3:1 in the presence of increasing doses of TT antigen. T cell proliferation was evaluated after 66 h by 3H-thymidine incorporation. The data shown are from one representative experiment of two performed.

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sion. It cannot be excluded, however, that soluble factors with higher molecular weight may contribute to the inhibitory effect.

The inhibitory cytokines IL-10, IL-4, and TGF-␤ are not involved in the suppression induced by CT-pretreated CD4⫹ T cells

Fig. 3. Inhibition of T cell proliferation by CT-pretreated CD4⫹ T lymphocytes also occurs in the absence of cell-to-cell contact. (A) The effect of CT-pretreated CD4⫹ T lymphocytes on PBMC proliferation was investigated using a transwell system. PBMC (8⫻105) were placed in the lower wells, and irradiated untreated or CT-pretreated CD4⫹ T lymphocytes (2.4⫻106) were cultured in the upper wells. In parallel, untreated or CT-pretreated CD4⫹ T lymphocytes were placed in the lower chambers together with PBMC. Cells were stimulated with anti-CD3 mAb (0.5 ␮g/ml), and the PBMC proliferation was evaluated after 66 h by 3H-thymidine incorporation. (B) The inhibitory effect of filtered supernatants (membrane cut-off 10 k) from cultures of untreated or CT-pretreated CD4⫹ T cells was evaluated on PBMC (1⫻105/well) stimulated with anti-CD3 mAb (0.5 ␮g/ml), and the proliferation was analyzed after 66 h by 3H-thymidine incorporation. The data shown are from one representative experiment of three performed.

membrane cut-off of 10 k. The inhibitory effect of filtered supernatants was then tested on PBMC stimulated in the presence of anti-CD3 mAb. Figure 3B shows that the supernatant from CT-pretreated CD4⫹ T cells was still able to inhibit the proliferation of anti-CD3-stimulated PBMC. The inhibition was 45% when compared with anti-CD3-stimulated cells and 31% when compared with the inhibition exerted by the supernatant of untreated CD4⫹ T cells. The slight inhibition observed with the supernatant of untreated CD4⫹ T cells may be a result of the withdrawal of factors important for T cell growth caused by filtration. Indeed, although filtered supernatants from untreated and CT-pretreated CD4⫹ T cells were supplemented with several growth factors after the filtration, we cannot exclude that additional factors, which are normally present in the complete medium, are missing in the filtered supernatants. These data suggest that the suppression mediated by CT-pretreated CD4⫹ T cells is not a result of the CT, which may be released by the cells, and they show that soluble factors with low molecular weight are involved in the suppres-

Next, the possible involvement of inhibitory cytokines was evaluated. In particular, the production of IL-10 by purified CD4⫹ T lymphocytes cultured in the presence or in the absence of CT and stimulated by coated anti-CD3 and soluble anti-CD28 mAb was analyzed. The results reported in Figure 4A show that IL-10 production induced by anti-CD3/CD28 stimulation was strongly inhibited in purified CD4⫹ T lymphocytes treated with CT. Furthermore, to test whether the production of IL-10 could be induced by the presence of mononuclear cell types, we measured the levels of IL-10 in the supernatants of untreated and CT-pretreated CD4⫹ T cells co-cultured with autologous PBMC and stimulated with antiCD3 mAb. The amount of IL-10 in the culture containing CT-pretreated CD4⫹ T cells was lower than that found in the culture containing untreated CD4⫹ T cells (P⬍0.05; Fig. 4B), which as reported above, did not mediate the suppression. The level of IL-10 in the culture containing untreated CD4⫹ T cells was higher than that found in the culture containing PBMC alone, and this likely reflects the production of IL-10 by PBMC and untreated CD4⫹ T cells. Altogether, these data show that IL-10 production by CD4⫹ T cells is inhibited by CT. To evaluate the effect of CT-pretreated CD4⫹ T cells on the production of Th1 and Th2 cytokines, we measured the levels of IFN-␥, IL-4, and IL-5 in the supernatants of untreated and CT-pretreated CD4⫹ T cells co-cultured with autologous PBMC and stimulated with anti-CD3 mAb. The amount of these cytokines in the cultures containing CT-pretreated CD4⫹ T cells was lower than that found in the culture containing untreated CD4⫹ T cells (P⬍0.05; data not shown), suggesting that CT-pretreated CD4⫹ T cells inhibit Th1 and Th2 cytokine production. To investigate the role of IL-10 in the suppression mediated by CT-pretreated CD4⫹ T lymphocytes, neutralizing anti-IL-10 and anti-IL-10R mAb were added to the co-cultures containing untreated or CT-pretreated CD4⫹ T cells and autologous PBMC. As shown in Figure 5A, the addition of anti-IL-10 and anti-IL-10R did not prevent the inhibition mediated by CTpretreated CD4⫹ T lymphocytes. To test whether the secretion of TGF-␤ or IL-4 was involved in the suppression, neutralizing anti-TGF-␤ or a cocktail of anti-IL-4, -IL-10, and -TGF-␤ mAb (blocking mAb) was added to the cultures, and their capacity to prevent the inhibition of T cell proliferation was evaluated. The results in Figure 5, B and C, demonstrate that none of these cytokines was involved in the inhibitory effects mediated by CT-pretreated CD4⫹ T lymphocytes, suggesting that other inhibitory factors might be released by CT-pretreated CD4⫹ T lymphocytes.

Extracellular release of cAMP by purified CD4⫹ T lymphocytes treated with CT It has been reported that CT and other cAMP-elevating agents can induce the release of cAMP in the extracellular compart-


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Fig. 4. The suppression exerted by CT-pretreated CD4⫹ T lymphocytes is not mediated by the secretion of IL-10. (A) Purified CD4⫹ T lymphocytes (2⫻106/ml) were stimulated with plate-coated anti-CD3 (5 ␮g/ml) and soluble antiCD28 (1 ␮g/ml) mAb in the presence or in the absence of CT (3 ␮g/ml), and the amount of IL-10 in culture supernatants was evaluated by ELISA after 48 h. (B) IL-10 level was evaluated in culture supernatants of PBMC (6⫻104) stimulated for 48 h with anti-CD3 mAb (0.5 ␮g/ml) in the presence of untreated or CT-pretreated CD4⫹ T lymphocytes (18⫻104) at a CD4⫹:PBMC ratio of 3:1. The data shown are from one representative experiment of three performed.

ment of different tissues and cells [23, 24]. Thus, to investigate whether cAMP was involved in the suppression mediated by CT-pretreated CD4⫹ T lymphocytes, we evaluated if CD4⫹ T cells cultured in the presence of CT or FSK were able to release cAMP into the extracellular compartment. Purified CD4⫹ T lymphocytes were cultured in the presence and in the absence of CT or FSK for 24 h, and the level of cAMP in the supernatants was evaluated by RIA assay. We found that untreated CD4⫹ T cells constitutively released cAMP at a basal level (1.35⫾0.6 pmoles/107 cells) and that the levels of cAMP in the supernatants were increased by the presence of CT and FSK (20.2⫾4.5 and 15.4⫾1.5 pmoles/107 cells, respectively; Fig. 6A). By comparing the amount of cAMP secreted in the supernatants with the total cAMP produced by the cells (calculated as the cAMP measured in the cell extracts plus the amount of cAMP estimated in the supernatants), we calculated that CT-pretreated CD4⫹ T lymphocytes released 55.5 ⫾ 9% of the total cAMP produced after 24 h incubation with CT. The levels of extracellular cAMP released by CD4⫹ T cells, treated or untreated with CT or FSK, were evaluated at different time-points after the removal of the stimulus. We found that cAMP was released in a time-dependent manner only by the cells that had been treated with CT (Fig. 6B). Indeed, FSKpretreated CD4⫹ T cells, which did not inhibit the proliferation of bystander PBMC, released low levels of cAMP after the removal of FSK. These levels were lower than the levels found in the supernatants of untreated cells (Fig. 6B). This phenomenon may be a result of a mechanism of feedback regulation of the adenylate cyclase activity. To rule out the possibility that the accumulation of extracellular cAMP in the supernatants was the result of a passive release by dying cells, the vitality of untreated and CT-pretreated CD4⫹ T cells was evaluated at different time-points 884


after the removal of CT, by propidium iodide (PI) staining. We found that more then 97% of untreated and CT-pretreated CD4⫹ T populations were alive (PI-negative) after 6 h and more then 93% after 24 h of culture (data not shown). These data show that cAMP can be released by CD4⫹ T lymphocytes in the extracellular compartments and that CD4⫹ T cells pretreated with CT continue to release cAMP after the removal of CT.

The inhibition of proliferation by CT-pretreated CD4⫹ T lymphocytes is partially prevented by treatment with PDE To test whether extracellular cAMP was involved in the suppressive effects mediated by CT-pretreated CD4⫹ T lymphocytes, PDE, an enzyme capable of metabolizing cAMP to AMP, was added to the cocultures containing irradiated CT-pretreated or untreated CD4⫹ T cells and autologous PBMC. After 1 h of incubation, the cells were stimulated with anti-CD3, and their ability to proliferate was evaluated. Figure 7A shows that the inhibition of T cell proliferation by CT-pretreated CD4⫹ T lymphocytes was partially prevented by treatment with 5 ␮g/ml PDE (P⬍0.05 for all concentrations of anti-CD3 mAb used), suggesting that extracellular cAMP plays a role in the suppression. To confirm that PDE treatment was able to metabolize the extracellular cAMP, the level of cAMP in the supernatants of CT-pretreated CD4⫹ T lymphocytes was evaluated at different time-points in the presence of an increasing concentration of PDE (0.5–5 ␮g/ml). We found that PDE was able to metabolize cAMP in a dose-dependent manner (Fig. 7B). These results suggest that extracellular cAMP plays a role in the inhibition mediated by CT-pretreated CD4⫹ T cells. In this context, cAMP could be regarded as a primary messenger with inhibitory activity. http://www.jleukbio.org

agents, had regulatory activity. We found that human-purified CD4⫹ T lymphocytes pretreated with CT were able to inhibit proliferation of TT-specific or anti-CD3-stimulated, autologous PBMC in a dose-dependent manner. It is interesting that this phenomenon was, at least in part, a result of the release of extracellular cAMP by the purified CD4⫹ T lymphocytes. Conversely, purified CD4⫹ T cells pretreated with FSK, a transient cAMP inducer, or with dbcAMP, an analog of cAMP, did not suppress the proliferation of bystander anti-CD3-stimulated PBMC, suggesting that a sustained production of cAMP is required to identify a novel, regulatory function mediated by CD4⫹ T cells. The T cells with regulatory functions, which we describe here, are new in the method of generation and in their mode of suppression. Indeed, the in vitro-induced Treg described so far are generated by culturing T lymphocytes with antigen or polyclonal activators in the presence of inhibitory cytokines such as IL-10 or TGF-␤ [18, 25, 26]. Furthermore, these cells are able to suppress the activity of effector T lymphocytes through the release of IL-10 and/or TGF-␤. It is interesting that

Fig. 5. Neutralizing mAb against IL-10, IL-10R, TGF-␤, and IL-4 do not prevent the suppression mediated by CT-pretreated CD4⫹ T lymphocytes. Untreated or CT-pretreated CD4⫹ T lymphocytes (18⫻104) were cultured with autologous PBMC (6⫻104) at the ratio of 3:1 in the presence or in the absence of neutralizing mAb against IL-10 (1 ␮g/ml) and IL-10R (1 ␮g/ml; A), TGF-␤ (1 ␮g/ml; B), or a cocktail of anti-IL-4, -IL-10, and –TGF-␤ (1 ␮g/ml; C). Cells were stimulated by increasing doses of anti-CD3 mAb (from 0.005 to 0.5 ␮g/ml), and the proliferation was evaluated 66 h later by 3H-thymidine incorporation. The data shown are from one representative experiment of three performed.

Exogenous cAMP inhibits T cell proliferation To evaluate if exogenously added cAMP is able to inhibit T cell proliferation, we measured the proliferation of anti-CD3-stimulated PBMC (10⫻104/well) in the presence or in the absence of cAMP. We found that cAMP inhibited T cell proliferation (54% of inhibition; Fig. 8) and that this inhibition was partially reversed by adding PDE to the cultures (P⬍0.05). In this context, we also studied the specificity of the cAMP-inhibitory effect by using dbcAMP, an analog of cAMP, which is not hydrolyzed by cAMP-specific PDE. We found that dbcAMP strongly inhibited anti-CD3-induced T cell proliferation (83% of inhibition) and that the treatment with PDE did not reverse the inhibitory effect (Fig. 8). These data suggest that exogenous cAMP can be up-taken by the cells and is able to exert inhibitory functions.

DISCUSSION In this study, we evaluated whether human T lymphocytes, which had been exposed to CT or to other cAMP-elevating

Fig. 6. Sustained release of extracellular cAMP by purified CD4⫹ T lymphocytes treated with CT. (A) Purified CD4⫹ T lymphocytes (5⫻106/ml) were cultured with medium alone, CT (3 ␮g/ml), or FSK (50 ␮M) for 24 h, and the accumulation of cAMP in the supernatants was evaluated by RIA assay. (B) The amount of cAMP released by CD4⫹ T lymphocytes, treated or untreated with FSK or CT, was evaluated at different time-points (1, 4, and 24 h) after the removal of stimuli. The data shown are from one representative experiment of three performed.


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Time (hours)

Fig. 7. Inhibition of proliferation by CT-pretreated CD4⫹ T lymphocytes is partially prevented by PDE treatment. (A) Untreated or CT-pretreated CD4⫹ T lymphocytes (18⫻104) were cultured with autologous PBMC (6⫻104) at ratio of 3:1 in the presence or in the absence of PDE (5 ␮g/ml) for 1 h. Then, cells were stimulated by increasing doses of anti-CD3 mAb (from 0.005 to 0.5 ␮g/ml), and the proliferation was evaluated 66 h later by 3H-thymidine incorporation. (B) Evaluation of extracellular cAMP at different time-points in cultures containing CT-pretreated CD4⫹ T lymphocytes in the presence of increasing concentrations of PDE by RIA assay. The data shown are from one representative experiment of three performed.

our findings show that the inhibition mediated by CT-induced CD4⫹ T cells was not mediated by these cytokines. Indeed, IL-10 production induced by anti-CD3/CD28 stimulation was strongly inhibited in purified CD4⫹ T lymphocytes treated with CT. Furthermore, neutralizing mAb specific for IL-10 and IL-10R did not prevent the suppression mediated by CTpretreated CD4⫹ T cells. In addition, neutralizing mAb specific for TGF-␤ and IL-4, two cytokines involved in driving the differentiation of T cells with regulatory activity [27, 28], also failed to prevent the inhibition. CT and adenylate cyclase toxin from Bordetella pertussis have been described to promote the induction of IL-10-producing Treg cells to coadministered antigen by enhancing the production of IL-10 by dendritic cells, which in turn, induce the differentiation of T cells with regulatory phenotype [29, 30]. Here, we show that CT also has the capacity to induce a new class of T cells with regulatory activity by interacting directly with T lymphocytes. This and other findings [31] suggest that following microbial infections, different classes of T cells with regulatory functions may be generated. Furthermore, several bacteria produce cAMP-inducing toxins or they release cAMP themselves [32, 33]; thus, our results imply that immunosuppressive effects may be generated in conditions of high, local cAMP production. 886


After the discovery of the role of cAMP as an intracellular second messenger, several studies reported the presence of the cyclic nucleotide in plasma and urine, suggesting that cAMP could be exported out of the cells [34 –36]. The release of cAMP from different cell types is an active transport against a concentration gradient, and it seems to be strictly regulated according to the stimulus and to the tissue involved [37–39]. However, the release of cAMP by T lymphocytes has not been reported so far. Here, we show that the stimulation of purified CD4⫹ T lymphocytes with CT or FSK induces the release of cAMP. According to the constitutive activation of the host adenylate cyclases by CT, only CT-pretreated CD4⫹ T cells sustained the release of cAMP after the removal of the stimulus. This allowed us to demonstrate that extracellular cAMP plays a biological role as a soluble mediator of T cell suppression. Indeed, we found that the inhibition of proliferation by CT-pretreated CD4⫹ T lymphocytes was, at least in part, prevented by treatment with PDE, an enzyme capable of metabolizing cAMP to AMP, suggesting that extracellular cAMP plays a role in the suppression. Furthermore, we observed that exogenous cAMP inhibited the anti-CD3-induced T cell proliferation, and this effect was partially reversed by the presence of PDE. These data further indicate that extracellular cAMP is able to exert inhibitory functions. The cellular mechanism by which cAMP released by CD4⫹ T lymphocytes influences the function of responding T cells needs further investigation. The inhibition could be a result of an influx of extracellular cAMP directly into cells, and this may account for the inhibition of proliferation, as an increased level of intracellular cAMP in T lymphocytes is known to have inhibitory effects [2, 40]. Alternatively, the binding of cAMP to specific membrane receptors could deliver inhibitory signals to the cells. In support of the first hypothesis, it has been shown that the influx of cAMP into smooth muscle cells has been found to be mediated by a system, which involves a transporter [41]. The amount of cAMP we measured in the supernatants of CT-pretreated CD4⫹ T lymphocytes, which ranged between 69 and 216 pM, could be delivered directly into the target cells. Conversely, although cAMP receptors have not yet been identified in mammals, they have been well-characterized in lower

Fig. 8. Exogenous cAMP inhibits T cell proliferation. PBMC (10⫻104/well) were stimulated with anti-CD3 mAb (0.5 ␮g/ml) in the presence of cAMP or dbcAMP (0.1 mM), with or without PDE (5 ␮g/ml), and T cell proliferation was evaluated after 66 h of culture by 3H-thymidine incorporation. The data shown are from one representative experiment of two performed.


eukaryotes. Four different cAMP receptors have been described in the amoeba Dictyostelium discoideum [42, 43]. They belong to the superfamily of seven transmembrane domain G protein-coupled receptors, which modulate the level of intracellular cAMP. It is interesting that these receptors exhibit a certain homology with the secretin receptor family [24, 44], and it is tempting to speculate that similar cAMP receptors may be present in mammalian cells, although this remains to be demonstrated. Another possible mechanism by which the efflux of cAMP could mediate inhibitory effects in target cells is the extracellular cAMP-adenosine pathway [45]. Extracellular cAMP can be converted into adenosine, which activates adenylate cyclase via A2 receptors, leading to an increase of intracellular cAMP [46]. However, in our experiments, adenosine does not seem to be the soluble factor responsible for the suppression, as extracellular PDE, which usually leads to an increase of AMP, a precursor of adenosine, prevented the inhibition of T cell proliferation. Furthermore, by using a nonselective adenosine receptor antagonist, we did not observe prevention of the inhibition (data not shown). The stimulation of different membrane receptors on T lymphocytes by hormones or neurotransmitters such as catecholamines, PGE2, and histamine modulates intracellular levels of cAMP, and cAMP as second messenger is responsible for the regulation of many cellular events [47]. We have shown that by treating purified CD4⫹ T lymphocytes with cAMPelevating agents, the cyclic nucleotide can be released by these cells and acts as an extracelluar messenger, conferring suppressive functions to the cells. A physiological role of the release of cAMP from T cells could be associated to a novel mechanism involved in the maintenance of the immune homeostasis or in the modulation of the inflammatory reactions against invading microbes. Although more in vivo studies are needed to clarify whether CT-treated CD4⫹ T lymphocytes play a role in vivo, in this study by using CT in vitro in a human system, we identified an unrecognized regulatory function mediated by CD4⫹ T cells. Altogether, these findings suggest that cAMP should be regarded not only as an intracellular second messenger but also as a primary messenger, which could play a biological role in the modulation of the immune response, enabling cell-to-cell communication.

ACKNOWLEDGMENTS This work was supported by a grant from “Ricerca Finalizzata” by the Ministry of Health (No. 4AMF). We thank Dr. Ragnar Lindstedt and Dr. Tommaso Costa for helpful discussion and critical comments about the manuscript.

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