Potentiates TCR Signaling Phosphodiesterase 4 to Lipid Rafts

TCR- and CD28-Mediated Recruitment of Phosphodiesterase 4 to Lipid Rafts Potentiates TCR Signaling

This information is current as Hilde Abrahamsen, George Baillie, Jacob Ngai, Torkel of October 2, 2021. Vang, Konstantina Nika, Anja Ruppelt, Tomas Mustelin, Manuela Zaccolo, Miles Houslay and Kjetil Taskén J Immunol 2004; 173:4847-4858; ; doi: 10.4049/jimmunol.173.8.4847 http://www.jimmunol.org/content/173/8/4847 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TCR- and CD28-Mediated Recruitment of Phosphodiesterase 4 to Lipid Rafts Potentiates TCR Signaling1

Hilde Abrahamsen,* George Baillie,‡ Jacob Ngai,*† Torkel Vang,* Konstantina Nika,§ Anja Ruppelt,* Tomas Mustelin,§ Manuela Zaccolo,¶ Miles Houslay,‡ and Kjetil Taske´n2*

Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alone does not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMP was generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation, ␤-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) was recruited to lipid rafts which down-regulated cAMP levels. Whereas inhibition of protein kinase A increased TCR-induced immune responses, inhibition of PDE4 blunted T cell cytokine production. Conversely, overexpression of either PDE4 or ␤-arrestin augmented TCR/CD28-stimulated cytokine production. We show here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipid Downloaded from rafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus. The Journal of Immunology, 2004, 173: 4847–4858.

yclic AMP, generated by G-protein-mediated (Gs␣) acti- and, as a consequence, TCR-mediated cAMP production must be vation of adenylyl cyclase (AC),3 is a common and ver- regulated for T cell activation to occur. Since the only known way C satile second messenger controlling numerous cellular of reducing intracellular cAMP levels is through activation of the http://www.jimmunol.org/ processes including inhibition of mitogenic responses in fibro- large family of cAMP PDEs (10Ð12), they are poised to play a key blasts and T cells. Although cAMP is known to activate protein regulatory role in regulation of T cell function. The majority of the kinase A (PKA) (1), Epac (exchange protein directly activated by cAMP-hydrolyzing activity in T cells is known to be mediated by cAMP) (2) and cyclic nucleotide-gated ion channels (3), PKA is the PDE3 and PDE4 cAMP PDE families (13Ð15). Indeed, there is likely to be the major target of cAMP in T cells because neither currently considerable interest in the PDE4 family as selective Epac (our unpublished data and Ref. 4) nor cyclic nucleotide-gated PDE4 inhibitors that exert a profound anti-inflammatory action are ion channels are expressed in lymphocytes (3). Compartmental- being developed to treat asthma and chronic obstructive pulmo- ization of receptors, cyclases, and PKA by A-kinase-anchoring nary disease (16Ð18), where T cells are thought to provide one of by guest on October 2, 2021 proteins (5, 6) as well as generation of local pools of cAMP within the key cell targets for their action. Furthermore, there is now the cell by the action of anchored cAMP phosphodiesterase (PDE) considerable evidence demonstrating that individual PDE4 iso- isoforms (7) underpin the high degree of specificity of action in forms display distinct patterns of intracellular targeting, indicating PKA-mediated signaling. that they are likely to play a key role in compartmentalized cAMP It has previously been demonstrated that stimulation of the T signaling (10, 11). Indeed, PDE4 enzymes have very recently been cell Ag receptor (TCR) elevates cAMP (8). However, increased shown to interact with the signaling scaffold protein ␤-arrestin (19) cAMP levels in T cells inhibits T cell function and proliferation (9) that together serve to regulate signaling through ␤2-adrenergic receptors. However, the functional role of PDE4 in the proximal TCR signaling in primary T cells is not well known, although it *Biotechnology Centre and †School of Pharmacy, University of Oslo, Oslo, Norway; ‡Institute of Biomedical and Life Sciences, University of Glasgow, United Kingdom; was recently demonstrated that PDE4B2 stably transfected into ¤Infectious and Inflammatory Disease Center, and Program of Signal Transduction, Jurkat cells localizes to the immunological synapse upon activa- ¶ Cancer Research Center, Burnham Institute, La Jolla, CA 92037; and Dulbecco tion (20), suggesting a role for PDE4 in proximal T cell signaling. Telethon Institute and Venetian Institute of Molecular Medicine, Padova, Italy It is generally accepted that proximal TCR-mediated signaling is Received for publication March 5, 2004. Accepted for publication August 9, 2004. initiated in specialized sphingolipid- and cholesterol-enriched mi- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance crodomains in the cell membrane called lipid rafts (21). Lipid rafts with 18 U.S.C. Section 1734 solely to indicate this fact. function as signaling platforms that are comprised of or recruit 1 This work was supported by grants to K.T. from the Programme for Advanced protein complexes involved in the proximal signal transduction in Studies in Medicine, the Research Council of Norway, the Norwegian Cancer Society, T cells (22). The importance of such membrane microdomains has and Novo Nordic Foundation Committee; by grants to M.H. from the Medical Re- search Council (U.K.) G8604010 and European Union (QLG2-CT-2001-02278); by been demonstrated in T cells, as the integrity of lipid rafts is nec- grants to M.Z. from Telethon Italy (TCP00089), the Italian Cystic Fibrosis Research essary for propagation of TCR-induced signaling to occur (23). Foundation, and the Fondazione Compagnia di San Paolo; by a grant to K.T., M.H., and M.Z. from the European Union (RTD Grant QLK3-CT-2002-02149); and by One of the most proximal events taking place in T cells after en- grants to T.M. from the National Institutes of Health (AI48032 and AI53585). gagement of the TCR is activation of the Src family protein ty- 2 Address correspondence and reprint requests to Dr. Kjetil Taske«n, Biotechnology rosine kinases, in particular Lck, and phosphorylation of the Centre, University of Oslo, P.O. Box 1125, N-0317 Oslo, Norway. E-mail address: ITAMs present in the CD3 subunits (for review, see Ref. 24). This [email protected] process is inhibited by C-terminal Src kinase (Csk) (25, 26). The 3 Abbreviations used in this paper: AC, adenylyl cyclase; PKA, protein kinase A; PDE, phosphodiesterase; Csk, C-terminal Src kinase; IBMX, 3-isobutyl-1-methylx- molecular mechanism for the inhibitory effect of cAMP on prox- anthine; SH, Src homology. imal T cell signaling involves PKA-mediated phosphorylation of

serine 364 in Csk, resulting in Csk activation and subsequent in- with LAT Abs revealed that fractions 2Ð5 normally contained the majority of LAT and were used as a measurement for successful separation. Peak hibition of Lck (27). Furthermore, PGE2 and other inputs leading to elevated levels of cAMP before stimulation of the TCR inhibit raft fractions were made by mixing fractions 2Ð5. For all cAMP measurements, fractions were prepared with Brij 98 as detergent. All other sepa- signaling through the TCR due to increased association between rations were done in the presence of Triton X-100. After Brij 98 cell sep- Csk and Csk-binding protein/protein associated with glycosphin- aration, all fractions were stimulated with anti-CD3 (5 ␮g/ml) alone or with golipid-enriched microdomains residing in lipid rafts (28). How- anti-CD3 in combination with anti-CD28 (1 ␮g/ml) in the presence of ATP Ј ever, it is not known to what extent cAMP and PKA may inhibit (1 mM) and MgCl2 (15 mM). After 1 min of Ab incubation F(ab )2 (20 ␮ Ј ␮ g/ml) were added for Ab cross-ligation. F(ab )2 (20 g/ml) were also T cell activation during TCR triggering in the absence of concom- added to controls. Reactions were stopped in reaction termination buffer itant ligand-operated stimuli through G protein-coupled receptors supplied with the cAMP radioimmunoassay. Activity was normalized for such as, for example, PGE2 that drive the generation of cAMP. protein content. In this study, we investigate TCR-mediated cAMP production Stimulation of T cells for time course studies and subsequent PKA activation in primary T cells and the role of PDE4 in the propagation of T cell signaling in the absence and PBL were incubated at 37¡C for 10 min and then stimulated with anti-CD3 ␮ ␮ Ј presence of CD28 coreceptor stimuli. We show that the TCR-me- alone (5 g/ml) or anti-CD3/anti-CD28 (1 g/ml). Thereafter, F(ab )2 (20 ␮g/ml) were added for Ab cross-ligation and incubation proceeded for diated increase in cAMP levels in rafts is generated by recruitment 1Ð10 min as described in relevant figures. Reactions were ended by adding and dissociation of G proteins and that PKA activation serves to ice-cold RPMI 1640 followed by cell pelleting and lysis in standard lysis down-modulate signal transduction through the TCR. We show buffer (30) containing n-octyl-␤-D-glucoside (50 mM). Equal cell numbers here for the first time that concurrent TCR and CD28 stimulation were used at each time point for lipid raft purification after stimulation. acts to increase raft-localized PDE4 enzymes, enhancing the deg- Immunoprecipitations were as before (30). Downloaded from radation of cAMP and, thereby, potentiating T cell activation. Densitometric scanning analysis Thus, recruitment of PDE4 isoforms to lipid rafts is required and necessary for a maximal T cell immune response to occur. Scanned x-ray films were subjected to densitometric analysis with the program Scion image (www. scioncorp.com). Materials and Methods PDE activity assay Cells and transfections After ultracentrifugation and lipid raft separation, the collected fractions http://www.jimmunol.org/ Human peripheral blood T cells were purified by negative selection (95Ð were analyzed for PDE activity (36). Assays were conducted in duplicate 98% pure) as described previously (29) and transfected in accordance with with the presence and absence of the PDE4 selective inhibitor rolipram (10 ␮ the manufacturer’s instructions (Amaxa, Cologne, Germany) nucleofector M) to determine the rolipram-dependent PDE4 activity. PDE3 activity kit for peripheral T cells, catalogue no. VPA-1002). The human leukemic was similarly assessed but in this instance through chemical ablation in the ␮ T cell line Jurkat TAg was cultured and transfected by electroporation as presence of the PDE3 selective inhibitor cilostimide (1 M, C 79781; previously described (30) (10Ð40 ␮g DNA for each transfection). Unless Sigma-Aldrich). Protein concentrations were quantified according to the otherwise indicated, all experiments were conducted in normal primary method of Bradford using BSA as a standard. Activity was normalized for peripheral blood T cells. protein content. Reagents and Abs IL-2 assay and cAMP assay by guest on October 2, 2021 Rolipram (R-6520), Brij 98 (P-5641), PMA (P-8139), ionomycin (I-0634), For IL-2 production, T cells were stimulated with beads coated with anti- mFLAG M2 Ab (F-3165), and pFLAG Ab (F-7425) were purchased from CD3 and anti-CD28 Abs for 18 h (two beads per cell). For inhibition of ␮ ␮ Sigma-Aldrich (St. Louis, MO), n-octyl-␤-D-glucoside (19775) was ob- PKA or PDE4, cells were treated with 10 M H-89 or 10 M rolipram, tained from USB (Cleveland, OH). 8-Bromoadenosine-3Ј,5Ј-cyclic mono- respectively, for 20 min at 37¡C before stimulation. Standard IL-2 assay phosphorothioate (Rp-8-Br-cAMPs) was obtained from BIOLOG Life Sci- (S2050; R&D Systems, Minneapolis, MN) was used to measure secreted Ј IL-2. cAMP radioimmunoassays (cAMP kit from NEN, Boston, MA, cat- ence Institute (Bremen, Germany). Anti-Fc F(ab )2 for cross-ligation of bound Ab and peroxidase-conjugated secondary reagents were obtained alogue no. SP004) were performed in accordance with the manufacturer’s from Jackson ImmunoResearch Laboratories (West Grove, PA). H-89 instructions. (371963) was obtained from Calbiochem (San Diego, CA). Protein A/G- ␨ NFAT-AP-1 luciferase assay PLUS agarose and Abs toward Lck (sc-433), CD3- (sc-1239), Gs␣ (sc- 823), Gq␣ (sc-393), and Gi-2a (sc-7276) were purchased from Santa Cruz Jurkat Tag cells were transfected with NFAT-AP-1 luciferase reporter con- ⑀ ⌷⌲⌻ Biotechnology (Santa Cruz, CA). Anti-CD3 ( 3) Ab was affinity- struct (10 ␮g) and Renilla-TK luciferase (1 ␮g) in combination with vec- purified from a hybridoma cell line from American Type Culture Collec- tors encoding ␤-arrestin 2, PDE4B2, PDE4A4, PDE4D1, or empty vector tion (CRL-8001; Manassas, VA). Magnetic beads coated with anti-CD14 (20 ␮g) as indicated in Fig. 5D. Sixteen hours after transfection cells were (Dynabeads M-450 CD14, 111.12), anti-CD19 (Dynabeads M-450 CD19, either left unstimulated, stimulated with anti-CD3 (2.5 ␮g/ml) alone, stim- 111.04), and anti-CD3/anti-CD28 Abs (Dynabeads CD3/CD28 T cell ex- ulated with a combination of anti-CD3/anti-CD28 (2.5 ␮g/ml/0.5 ␮g/ml), pander, 111.31) were obtained from Dynal Biotech (Oslo, Norway). mAb or stimulated with PMA (40 nM) and ionomycin (10 ␮M). Six hours after toward CD28 (clone CD28.2) was purchased from Immunotech (Marseille, stimulation, cells were harvested and lysed. Thereafter, luciferase activity France). Anti-linker for activation of T cells (LAT; 06H807), anti-p44/42 was measured by a dual luciferase reporter assay system from Promega MAPK (06-182), and anti-Tyr(P) mAb (4G10) were purchased from Up- (E1960; Madison, WI). NFAT-AP-1 luciferase activity in any sample was state Biotechnology (Lake Placid, NY). Abs recognizing phospho-416 Src T normalized against Renilla luciferase activity in the same sample. Mea- family (phospho-394 Lck/phospho-417 Fyn ) (2101), p42/p44 MAPK surements were done in triplicates. NFAT-AP-1 activity in each series was (9106), and PKA substrate phospho-specific Ab (abbreviated anti-RXXPS/ calculated as percentage of control (PMA/ionomycin) that was found to be PT, 9621) were purchased from Cell Signaling Technology (Berverly, unaffected by increased cAMP levels (see Fig. 5C). MA). Abs toward PDE4A, PDE4B, PDE4D, and ␤-arrestin were as previously described (19, 31Ð33). Intracellular staining and FACS analysis Purification and stimulation of lipid rafts Three hours after transfection, primary T cells were stimulated with anti- CD3 (5 ␮g/ml)/anti-CD28 (1 ␮g/ml) Abs (or not) and cross-linked with Isolation of Triton X-100 (0.7%)- and Brij 98 (1%)-insoluble rafts were Ј ␮ ␮ F(ab )2 (10 g/ml), or a mix of PMA (25 nM) and ionomycin (10 M). performed as described in detail elsewhere (34, 35) except that cells were Stimulation proceeded for6hina96-well plate. Pelleted cells were fixed, lysed in standard lysis buffer (30). Brij 98 lysis was performed at 37¡C for stained and analyzed as previously described (37). 7 min. After lysis both Triton X-100 lysates and Brij 98 lysates were completely homogenized in a Dounce homogenizer, mixed with 80% su- Constructs crose, loaded at the bottom of a 40Ð5% sucrose gradient (in 25 mM MES (pH 6.5), 5 mM EDTA, and 150 mM NaCl), centrifuged at 200,000 ϫ g for FLAG ␤-arrestin 2 (19), PDE4A4 (38), PDE4B2 (32), PDE4D1 (39), 20hat4¡C, and fractionated into 12 fractions from the top. Western blot Csk-wt and Csk-SH3-SH2 (30) were as previously described. The Journal of Immunology 4849

Results the isolated lipid raft fractions with these inhibitory Gs␣ Abs be- TCR-mediated cAMP production is increased upon fore stimulation reduced the in vitro anti-CD3/anti-CD28-induced 3-isobutyl-1-methylxanthine (IBMX) treatment and cAMP production almost back to control levels (Fig. 1G). Con- may occur in lipid rafts versely, incubation with Ab blocking Gi␣ effector coupling increased both the basal and stimulated cAMP levels to 1.6 and 2 The TCR-induced elevation of cAMP levels has been observed pmol cAMP/106 cells, respectively, demonstrating the specificity previously in T cells (8). We demonstrate here that TCR stimula- of action of these reagents (data not shown). Together these data tion in the absence of PDE inhibitors resulted in a modest but indicate that in vitro TCR stimulation induces cAMP production in consistent cAMP production (Fig. 1A). However, in the presence lipid rafts through a Gs-mediated process. Additionally, in intact of the nonselective PDE inhibitor IBMX, both basal and TCR- cells, generation of cAMP seems to be increased by concurrent induced cAMP levels were further increased (Fig. 1A). In contrast, recruitment of additional G ␣ to rafts and dissociation of inhibitory in T cells activated by TCR and CD28 cross-ligation, cAMP levels s Gi␣ from rafts. Furthermore, by blocking PDE activity using the actually decreased (Fig. 1B). An increase in cAMP levels was only PDE inhibitor IBMX, TCR-induced cAMP production is increased observed in the presence of IBMX (Fig. 1B). Since the cAMP identifying a key role of PDEs in regulation of cAMP levels upon levels in total cell lysates were low, we next explored the possi- T cell activation. bility that the TCR-stimulated accumulation of cAMP might be localized to lipid rafts since these membrane domains have been T cell stimulation increases PDE4 activity in lipid rafts shown to provide a key focus for initiation of TCR-mediated sig- Localization of PDE isoforms to specific cellular compartments naling in T cells (23). To evaluate this, we purified lipid rafts from (10) has been shown to provide a pivotal contribution to the gen- primary T cells using Brij 98 as a detergent. As previously shown, eration of local intracellular cAMP gradients in various cell types Downloaded from lysis of cells with Brij 98 at 37¡C before sucrose gradient ultra- (7, 45). A number of PDE isoforms are expressed in T cells centrifugation prevents the TCR-CD3 complex from being dis- (PDE3B, 4A, 4B, 4D, 7A1, 7A3, 8A), of which PDE3 and PDE4 solved from rafts (35). Upon lipid raft purification with Brij 98, we provide the majority of cAMP PDE activity (13, 15, 46). The fact ␨ identified CD3 ,Gs␣ (Fig. 1C), and low levels of the coreceptor that the nonselective PDE inhibitor IBMX augmented cAMP pro- CD28 (data not shown) in the fractions corresponding to lipid rafts. duction upon T cell stimulation indicates a role for PDEs in reg- In comparison, lipid raft purification with Triton X-100 dissolves ulating cAMP levels in T cells (Fig. 1, A and B). Given that PDE4 http://www.jimmunol.org/ ␨ the TCR-CD3 from the rafts (data not shown). The fact that CD3 selective inhibitors are considered to mediate part of their thera- and Gs␣ copurifies with these purified cell-free fractions indicates peutic potential by acting on T cells and that a key feature of PDE4 that at least part of the cAMP production machinery may already isoforms is their intracellular targeting (10, 11), we set out to de- be present in lipid rafts, without the need for recruitment of other termine whether PDE4 activity was localized to lipid rafts where T components. Indeed, when lipid raft fractions isolated by sucrose cell signaling is initiated (47) and where the majority of cAMP 2ϩ gradient centrifugation were reconstituted with Mg /ATP and seems to be produced. Doing this we see that although anti-CD3 subsequently stimulated with anti-CD3/anti-CD28, high levels of stimulation induces PDE4 activity in fractions corresponding to cAMP production were detected in lipid raft fractions (up to 3-fold lipid rafts (Fig. 2, A and AЈ), CD28 costimulation leads to a pro- by guest on October 2, 2021 above control in 1 min) (Fig. 1D). This reflects increased AC ac- found raft-associated increase in PDE4 activity (Fig. 2, B and BЈ). tivity occurring in isolated lipid rafts in response to TCR stimu- In marked contrast to this, lipid raft fractions even from T cells lation in purified raft fractions. Comparable results were obtained subjected to anti-CD3/anti-CD28 stimulation contained little or no by in vitro TCR stimulation by anti-CD3 treatment alone (data not PDE3 activity (Fig. 2, C and CЈ). shown). Furthermore, PKA-mediated phosphorylation of putative raft-localized PKA substrates was detected immediately after TCR Enhanced recruitment of PDE4 and ␤-arrestin to lipid rafts stimulation of primary T cells (Fig. 1E). Densitometric analysis after TCR and CD28 costimulation revealed a 4- to 5-fold increase in PKA-mediated phosphorylation The specific increase in PDE4 activity in lipid raft fractions upon of a protein of ϳ80 kDa. Phosphorylation of the same protein as TCR/CD28 engagement might suggest that temporal changes in shown in Fig. 1E was reduced upon H-89 pretreatment (data not PDE4 activity can play a key role in tuning intracellular activation- shown). This provides further evidence of the activation of the induced gradients of cAMP in T cell lipid rafts and thereby in- cAMP signaling pathway upon T cell activation. crease signal propagation upon costimulation. Since a key feature Gi,Gs, and AC have been reported to segregate into lipid rafts of T cell activation is to induce the redistribution of components (40, 41). Therefore, we next examined whether these G proteins both to and from lipid rafts, we next explored whether there was played a role in the raft-associated cAMP increase. In resting T any specific recruitment of PDE4 enzymes to lipid raft fractions

cells, Gs␣ was detected both in lipid rafts as well as in nonraft upon concomitant TCR and CD28 engagement. The PDE4 family

fractions (Fig. 1, C and F). However, the level of Gs␣ in rafts was consists of four subfamilies (A, B, C, and D), each of which gen- rapidly and transiently increased upon anti-CD3/anti-CD28 cross- erates a series of isoforms by the use of distinct promoters and ligation of whole cells, with a decline to basal levels occurring alternative mRNA splicing (10, 11). However, within any one sub-

after 2 min (Fig. 1F, first panel). Conversely, the G protein Gi␣, family, the C-terminal regions of all active isoforms are identical. which acts to inhibit AC, was present at high levels in rafts from This has been effectively exploited to generate antisera that are not resting T cells but dissociated rapidly from rafts following TCR only specific for each subfamily but thereby identify all isoforms stimulation (Fig. 1F, second panel). As shown previously by oth- within a particular subfamily (39). Using antisera specific for the

ers (42), the G protein Gq␣ is recruited to rafts upon engagement four PDE4 subfamilies, we found that the long PDE4A4 isoform, of the TCR (Fig. 1F, third panel). However, this G protein is not the short PDE4B2 isoform, and the short PDE4D1/2 isoforms were considered to be directly involved in regulating cAMP signaling all rapidly and concomitantly recruited to lipid rafts upon TCR and

(43). Since both AC and its activator, Gs␣, are present in lipid rafts, CD28 costimulation (Fig. 3A). This suggests that the increased we next set out to hamper the anti-CD3/anti-CD28-induced cAMP PDE4 activity seen in lipid rafts upon TCR/CD28 stimulation at

production in vitro by using speciﬁc blocking Abs that inhibit Gs␣ least in part is a result of increased levels of PDE4 in membrane function by interfering with effector coupling (44). Pretreatment of microdomains. 4850 PDE4 RECRUITMENT TO RAFTS INCREASES T CELL ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. cAMP levels increase upon T cell activation. A, Increase in total cellular cAMP levels upon TCR stimulation. Puriﬁed primary T cells were Ј Ј stimulated with either F(ab )2 as a control or with anti-CD3 and F(ab )2 in the absence and presence of IBMX. Subsequently, cAMP levels were measured by radioimmunoassay. The experiment is representative of three similar experiments. B, TCR-induced cAMP levels in T cells are reduced upon cross- Ј Ј ligation of the TCR with CD28. Primary T cells were stimulated with F(ab )2 or with anti-CD3/anti-CD28 and F(ab )2 in the absence and presence of IBMX. ␨ cAMP levels were measured as in A. C, CD3 and Gs␣ are localized in T cell lipid rafts. Insoluble membranes from T cells were prepared using Brij 98 as detergent. Lysates from the 11 ﬁrst fractions were analyzed with the indicated Abs. D, Anti-CD3/anti-CD28-mediated cAMP increase occurs in lipid rafts. Insoluble membranes from T cells were prepared as in C (Brij 98), whereupon the TCR is not dissolved from rafts. Each fraction harvested from the Ј sucrose density gradient centrifugation was reconstituted with Mg/ATP and stimulated with anti-CD3, anti-CD28, and F(ab )2 at 37¡C for 1 min. Subse- quently, cAMP production was measured by radioimmunoassay. Protein content in each fraction was measured. The data shown are representative of three Ј representative experiments. E, T cell activation increases PKA activity in rafts. Primary T cells were stimulated with anti-CD3 and F(ab )2 and subsequently subjected to sucrose gradient separation with Triton X-100. Peak raft fractions were analyzed for proteins phosphorylated by PKA (Anti-RXXPS/PT). The blot shown is representative of four experiments. Densitometric scanning revealed about a 4-fold induction in PKA activity (average, 4.2 Ϯ 1.2, n ϭ 4).

F, Concomitant reduction of Gi␣ levels and increased Gs␣ levels in lipid rafts upon anti-CD3/anti-CD28 stimulation. T cells were stimulated and separated as in E. Peak raft fractions were analyzed for the presence of Gs␣,Gi␣,Gq␣ as well as LAT. G, Preincubation of raft fractions with inhibitory Abs toward Gs␣ reduces anti-CD3/anti-CD28-induced cAMP production. T cell lysates were subjected to sucrose gradient fractionation with Brij 98 as in A. Raft fractions were reconstituted with Mg/ATP, not pretreated or pretreated with Abs inhibiting Gs␣, stimulated as indicated, and cAMP production compared with control was calculated. The Journal of Immunology 4851 Downloaded from

FIGURE 2. Recruitment of PDE4 activity to T cell lipid rafts after T cell activation. A and B, T cell activation induces PDE4 activity in lipid raft http://www.jimmunol.org/ Ј fractions. Peripheral T lymphocytes were left unstimulated or stimulated with either anti-CD3 and F(ab )2 (A) or a combination of anti-CD3/anti-CD28 and Ј F(ab )2 (B) for 2 min. Lysates were subjected to sucrose gradient centrifugation and each of the harvested fractions was subsequently analyzed for PDE4 activity and protein content (AЈ and BЈ). Time-dependent increase in PDE4 activity in T cell rafts after stimulation. Cells were stimulated for 0, 2, 3, and 5 min with either anti-CD3 alone (AЈ) or anti-CD3/anti-CD28 in combination (BЈ), separated, and analyzed as in A. Data are presented as induction of PDE4 activity at different time points (average Ϯ SEM) compared with control (set to 1). Induction of PDE4 activity at 2 min upon anti-CD3 stimulation was 2.0 Ϯ 0.8-fold (average Ϯ SEM, n ϭ 3). Induction of PDE4 activity at 2 min upon anti-CD3/anti-CD28 stimulation was 4.5 Ϯ 0.8-fold (average Ϯ SEM, n ϭ 4; C and CЈ). No signiﬁcant increase in PDE3 activity in rafts upon anti-CD3/anti-CD28 stimulation. Cells were stimulated with anti-CD3/anti-CD28 for 3 min, separated as in A, and analyzed for PDE3 activity. PDE3 activity following anti-CD3/anti-CD28 treatment was 1.1 Ϯ 0.1-fold (average Ϯ SEM, n ϭ 3) compared with untreated cells. by guest on October 2, 2021

␤ ␤ It has recently been shown that upon 2-adrenergic stimulation more PDE4/ -arrestin was recruited upon costimulation with of HEK293 cells a cytoplasmic complex consisting of ␤-arrestin CD28 (Fig. 3C). This led us to evaluate the impact of costimula- and PDE4 is recruited to membranes where the phosphorylated tion on recruitment of the PDE4/␤-arrestin complex to lipid rafts. ␤ 2-adrenergic receptor is localized (19, 48). Intriguingly, we found Indeed, cross-linking of CD3 alone was far less effective in re- here that anti-CD3/anti-CD28 stimulation caused a clear recruit- cruiting both ␤-arrestin and ERK to lipid rafts compared with co- ment of ␤-arrestin to T cell lipid raft fractions concurrently with stimulation with anti-CD3/anti-CD28 (Fig. 3C). In the same ex- PDE4 (Fig. 3A). ␤-Arrestin serves as a cytosolic scaffold protein periment, PDE4 activity was lower upon CD3 stimulation that can bind a variety of signaling molecules and recruit them to compared with CD3/CD28 stimulation (data not shown) as de- the plasma membrane upon appropriate receptor stimulation (49). scribed in Fig. 2. One such species is the MAPK/ERK (50Ð52) and we demonstrate The diterpene forskolin interacts directly with the catalytic unit here (Fig. 3A) that anti-CD3/anti-CD28 stimulation also serves to of AC to cause its activation. Treating T cells with this agent concomitantly recruit ERK along with ␤-arrestin to lipid rafts in T clearly fails to show any recruitment of PDE4 to rafts (Fig. 3D), cells. Furthermore, the fraction of ERK present in lipid rafts was although forskolin always gave a robust cellular increase in cAMP evidently active, as determined by phospho-specific Abs toward compared with CD3 stimulation (Fig. 3E). This demonstrates that Thr(P)202 and Tyr(P)204 in ERK (Fig. 3A). PDE4 recruitment is not elicited through any increase in intracel- To examine whether PDE4 and ␤-arrestin exist in a complex lular cAMP levels and argues toward a specific role for the TCR- both before and after T cell stimulation, we transfected T cells with and CD28-mediated signaling in recruitment of PDE4 to rafts. An- a vector encoding a FLAG-tagged ␤-arrestin, and immunoprecipi- ti-CD3 stimulation was used as a control in these experiments as tated ␤-arrestin from both unstimulated cells and cells stimulated only low levels of PDE4 are recruited upon anti-CD3 stimulation, with anti-CD3/anti-CD28. Interestingly, ␤-arrestin and PDE4D co- allowing detection of potentially low PDE4 recruitment upon for- immunoprecipitated from both unstimulated and stimulated cells skolin treatment. (Fig. 3B), indicating that they exist in a preassembled complex in Only a few TCRs are concomitantly ligated under normal cir- T cells as has been shown to occur in other cell types (19). Other cumstances in vivo, generating incomplete activation events that PDE4 isoforms were not tested here, as they have been described eventually lead to anergy or cell death (53). However, CD28 co- to coimmunoprecipitate with ␤-arrestin before. stimulation amplifies the weak TCR-induced signals leading to full Stimulation using anti-CD3 alone was not as effective as CD28 T cell activation and clonal expansion in vivo. When T cells were costimulation in increasing PDE4 activity in lipid raft fractions stimulated with anti-CD28 Abs alone here, we found that this was (Fig. 2). Indeed, compared with anti-CD3 stimulated cells, 2.5-fold sufficient to induce recruitment of ␤-arrestin to lipid rafts (Fig. 3F). 4852 PDE4 RECRUITMENT TO RAFTS INCREASES T CELL ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. A PDE4/␤-arrestin complex is recruited to T cell lipid rafts following T cell activation. A, The levels of PDE4, ␤-arrestin, and ERK in lipid Ј rafts are increased upon anti-CD3/anti-CD28 stimulation. After stimulation with anti-CD3, anti-CD28 and F(ab )2 human peripheral T cells were lysed and subjected to sucrose gradient ultracentrifugation with Triton X-100. Peak raft fractions (fractions 2Ð5) from cells activated for various periods of time were analyzed with the indicated Abs. B, Jurkat TAg cells were transfected with empty vector or with a construct coding for FLAG-␤-arrestin, stimulated as indicated, and lysed in standard lysis buffer. Immune complexes were precipitated with Abs toward FLAG and analyzed for the presence of PDE4D and FLAG ␤-arrestin. C, Enhanced recruitment of PDE4 and ␤-arrestin to T cell lipid rafts after anti-CD28 costimulation. T cells were stimulated either with Ј ␤ anti-CD3 and F(ab )2 alone or in combination with anti-CD28, peak raft fractions were analyzed for the recruitment of -arrestin and ERK at the indicated times of activation. Recruitment of ␤-arrestin to rafts with anti-CD3/anti-CD28 compared with anti-CD3 alone was 2.5 Ϯ 0.2-fold (average Ϯ SEM, n ϭ 3). D, Increased cAMP induced by forskolin is not sufﬁcient for PDE4 recruitment to rafts. T cells were stimulated either with forskolin (3 min) or anti-CD3 Ј and F(ab )2 (3 min) that give low levels of PDE4 in rafts and thereafter subjected to sucrose gradient ultracentrifugation. Peak raft fractions (fractions 2Ð5) ␮ Ј were analyzed with the indicated Abs. E, Forskolin induces a robust cAMP increase. T cells were stimulated with forskolin (10 M) or anti-CD3 and F(ab )2 for 3 min. Thereafter, cAMP was measured. F, Anti-CD28 stimulation of T cells is sufﬁcient for ␤-arrestin recruitment to lipid rafts. T cells were stimulated with indicated combinations of anti-CD3, anti-CD28, and anti-CD4 and analyzed with Abs detecting ␤-arrestin and LAT. The Journal of Immunology 4853 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. PKA is activated following T cell activation and regulates proximal T cell signaling events. A, TCR stimulation induced phosphorylation of the phosphatase HePTP on Ser23, a known PKA phosphorylation site. Jurkat TAg cells were transfected with hemagglutinin-tagged HePTP and stimulated with C305 (IgM), that cross-ligates the TCR on Jurkat cells. Phosphorylation of HePTP, a good readout for PKA activation in lymphocytes, was analyzed with an Ab with specificity toward phospho-Ser23 in HePTP. B, PKA inhibition by H-89 or the cAMP antagonist Rp-8-Br-cAMPs decreases TCR-induced CREB phosphorylation. Purified human T lymphocytes were pretreated with H-89 (upper panel) or Rp-8-Br-cAMPs (lower panel) and Ј subsequently stimulated with anti-CD3 and F(ab )2 for the indicated time periods and thereafter analyzed for CREB phosphorylation by immunoblotting. C, PKA is activated following TCR stimulation and is involved in down-regulation of the TCR-induced signal. Purified human T lymphocytes were left untreated or pretreated with H-89 and subsequently stimulated with anti-CD3 for the indicated time periods. TCR-induced T cell activation upon PKA inhibition was assessed by detection of ␨-chain phosphorylation (4G10) and autophosphorylation of Lck and Fyn (Anti-PY394Lck/anti-PY417FynT). D, PKA inhibition increases ␨-chain phosphorylation 2- to 4-fold. Densitometric scanning analysis of ␨-chain phosphorylation (normalized for the amount of ␨) for three similar experiments as shown in A is presented as fold induction over control (average Ϯ SEM, n ϭ 3). E, ␤-arrestin and PDE4A4 potentiates proximal T cell signaling. T cells were transfected with 2 ␮g vector, ␤-arrestin, or PDE4A4. Sixteen hours after transfection, cells were stimulated with anti-CD3 ␮ Ј ␮ ␨ (2.5 g/ml) and F(ab )2 (20 g/ml) for the indicated times. Lysates were analyzed for -chain phosphorylation.

In contrast, upon cross-ligation of the TCR or CD28 with CD4, a tion of the recruitment of PDE4 isoforms to lipid raft fractions T cell surface marker to which the Src kinase Lck binds, no ad- upon T cell activation and regulation of raft-associated cAMP sig- ditional effect on recruitment of ␤-arrestin was found (Fig. 3F). naling has particular functional signiﬁcance. We thus set out to Altogether, these results suggest that signals generated upon CD28 gain insight into the possible physiological consequences of TCR- stimulation are critical for enhancing the recruitment of the PDE4 induced cAMP production by analyzing the effect of PKA- and ␤-arrestin to lipid rafts in a cAMP-independent fashion. mediated signaling on proximal TCR activation in T cells. As a start, we began to analyze whether TCR stimulation increases PKA is activated upon TCR stimulation and inhibits proximal T PKA-mediated signaling. It has previously been demonstrated that cell signaling the phosphatase HePTP is directly phosphorylated at serine 23 by The fact that PDE4 selective inhibitors inhibit T cell functioning PKA in T cells (55), making serine 23 phosphorylation of HePTP therapeutically (17, 54) might suggest that our novel demonstra- a good readout for PKA activity. Stimulation of Jurkat cells with 4854 PDE4 RECRUITMENT TO RAFTS INCREASES T CELL ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. PKA and PDE4 are activated following T cell activation and play opposite regulatory roles in titrating T cell immune functions. A, IL-2 secretion is impaired upon rolipram and H-89 pretreatment. Primary T cells pretreated with rolipram or H-89 were left unstimulated, stimulated with beads coated with anti-CD3 and anti-CD28, or with a mix of PMA and ionomycin. Twenty hours after stimulation IL-2 secreted from the cells was measured with ELISA (average Ϯ SEM, n ϭ 3). B, Expression of PDE4B2 and ␤-arrestin increases IL-2 production in T cells. Primary T cells were transfected with indicated constructs. At 3 h after transfection, cells were stimulated with anti-CD3/anti-CD28-coated beads and 18 h after stimulation cells were pelleted and IL-2 secreted was analyzed by ELISA (average Ϯ half range, n ϭ 2). C, Increased cAMP does not affect PMA/ionomycin-induced proximal IL-2 promoter (NFAT-AP-1-luc) activity. Jurkat cells were transfected with NFAT-AP-1 luciferase and with Renilla luciferase. Eighteen hours after transfection, cells were pretreated with forskolin (15 min) and subsequently stimulated with PMA and ionomycin for 6 h and then analyzed for luciferase activity. D, Increased IFN-␥ production in PDE4-expressing and ␤-arrestin-expressing T cells. Primary T cells were transfected with indicated constructs. Cells were Ј stimulated at 3 h after transfection with anti-CD3/anti-CD28 and F(ab )2, harvested 6 h after stimulation (5 h in the presence of brefeldin A), ﬁxed, and permeabilized, then stained with Abs detecting intracellular IFN-␥. FACS analysis was subsequently performed to determine (Figure legend continues) The Journal of Immunology 4855

FIGURE 6. PKA and PDE4 have opposing functions in proximal T cell signaling and titrates the activation-induced response. We suggest a model whereby TCR triggering through G protein coupling to the AC generates cAMP and prevents full T cell activation response (upper panel). However, CD28 Downloaded from costimulation serves to amplify TCR-induced signals by recruiting the PDE4/␤-arrestin complex leading to cAMP degradation, down-modulation of inhibitory signals, and full T cell activation (lower panel). http://www.jimmunol.org/ by guest on October 2, 2021

anti-CD3-IgM (C305) resulted in rapid phosphorylation of serine ber ZAP-70 (58), which upon Lck phosphorylation mediates the 23 in this protein (Fig. 4A), clearly demonstrating TCR-induced phosphorylation of the important adapter molecule linker for ac- PKA activation. Next, we studied phosphorylation of CREB (56), tivated T cells (LAT) (for review, see Ref. 24). Phosphotyrosines another direct target of PKA, through a pathway that was largely on LAT in turn attract complexes of adapters and signaling mol- inhibited by pretreatment with H-89 (Fig. 4B, upper panel). Like- ecules like SH2 domain-containing leukocyte protein of 76 kDa wise, pretreatment of primary T cells with the PKA antagonist (SLP-76) necessary to mediate a functional T cell response (59). Rp-8-Br-cAMPS reduced the activation-induced CREB phos- Indeed, upon pretreatment of T cells with the PKA inhibitor H-89, phorylation (Fig. 4B, lower panel). Altogether, this demonstrates early T cell activation markers such as ␨-chain phosphorylation TCR-induced PKA activation in primary T cells. and phosphorylation in the catalytic domain of Lck/FynT were Next, we explored what functional role TCR-induced PKA ac- increased (Fig. 4C). Densitometric analysis revealed that in the tivity plays in the proximal TCR signaling. One of the most prox- presence of H-89, TCR-induced ␨-chain phosphorylation was in- imal events taking place upon TCR stimulation is activation of the creased 2- to 4-fold compared with control cells (Fig. 4D). Fur- Src family kinases Lck and FynT and phosphorylation of the CD3 thermore, phosphorylation of other molecules important for T cell ␨-chains (57). The phosphorylated ␨-chains function as docking activation such as LAT and SLP-76 (24) were also increased under sites for the Src homology (SH) 2 domain of the Syk family mem- conditions of low PKA activity (data not shown). Lastly, we

percent IFN-␥-producing cells in the transfected GFP-positive population. Two hundred thousand GFP-positive cells were analyzed for each transfection. The experiment shown is representative of two independent experiments from two different blood donors. E, IFN-␥ production in PDE4-expressing and ␤-arrestin-expressing T cells. Graphic presentation from the experiment shown in Fig. 5D. Viable, GFP-positive cells were gated, and the IFN-␥ production was calculated as percentage of PMA/ionomycin induced for each transfection. F, Increased transcription from the proximal IL-2 promoter (NFAT-AP- 1-luc) in Jurkat TAg cells expressing PDE4 isoforms and ␤-arrestin. Jurkat TAg cells were transfected with the constructs indicated, Renilla luciferase and NFAT-AP-1-luciferase reporter constructs. Eighteen hours after transfection, cells were stimulated with anti-CD3, anti-CD3/anti-CD28, or PMA/ionomycin for 6 h and then analyzed for luciferase activity. NFAT-AP-1 luciferase activity was normalized against Renilla luciferase in the same sample. The experiment shown has triplicate measurements (average Ϯ SD, n ϭ 3) and is representative of three independent experiments. 4856 PDE4 RECRUITMENT TO RAFTS INCREASES T CELL ACTIVATION wanted to investigate whether expression of ␤-arrestin or PDE4A4 IBMX was reported Ͼ15 years ago (8, 60). Despite this, the sig- influenced the proximal signaling events as well. As shown in Fig. nificance of activation-induced cAMP production has been ill-un- 4E, ␤-arrestin clearly potentiated proximal events like ␨-chain derstood to date, as has the location in the cell where cAMP is phosphorylation (Fig. 4E). Overexpression of PDE4A4 also po- generated. In this study, we show that T cell activation rapidly tentiated ␨-chain phosphorylation although the induction was not induces the production of the immunoinhibitory signaling mole- as evident as when ␤-arrestin was expressed (Fig. 4E). cule cAMP in lipid rafts, resulting in raft-associated PKA activation. Our data indicate that the concomitant recruitment to lipid PKA and PDE4 control T cell function rafts of the stimulatory G protein, Gs, and dissociation of the in- ␤ Since -arrestin and PDE4 expression potentiate proximal signal- hibitory G protein, GI, play a key role in the activation of AC that ing events in T cells, we next wanted to assess the effects of PKA occurs upon T cell activation. These data imply that a localized and PDE4 on downstream T cell function. Upon full T cell acti- increase in cAMP is generated in T cells upon activation and that vation, in vivo T cells proliferate and produce different growth- this appears to be concentrated in lipid rafts. ␥ ␤ promoting cytokines like IL-2 and IFN- . To assess the effect of Gs-coupled receptors, such as the 2-adrenergic receptor, invari- PDE4 and PKA activity on T cell function, we treated T cells with ably generate transient increases in cAMP (61). A key regulator of either rolipram or H-89 and measured IL-2 secretion. Pretreatment receptor-stimulated AC activity is the recruitment of ␤-arrestin of cells with rolipram reduced TCR-induced IL-2 production to from the cytosol to the plasma membrane-associated receptor (61). ϳ 50% of control (Fig. 5A), whereas H-89-mediated inhibition of This has the effect of causing the uncoupling of Gs from receptor PKA had the opposite effect (2-fold increase compared with con- stimulation, leading to desensitization of AC. However, regulation trol; Fig. 5A). Similarly, IFN-␥ production was also reduced by of intracellular cAMP levels is not solely the prerogative of AC rolipram and increased by H-89 (data not shown). This is in ac- action, but is also crucially regulated by the action of PDEs, which Downloaded from cordance with previous reports demonstrating that increased levels provide the sole route of cAMP degradation in cells (10, 12). In- of cAMP reduce IFN-␥ production in T cells (37). deed, although ␤-arrestin may serve to terminate AC activation, it Because we found that ␤-arrestin and PDE4 are recruited to is only through the action of PDEs that cAMP levels can return to lipid rafts as a complex, we next investigated the effect of over- their basal state. The importance of PDE action in T cells is clearly expression of ␤-arrestin and various PDE4 isoforms on IL-2 se- evident from both previous studies by other investigators (13Ð15, cretion and IFN-␥ production. Overexpression of PDE4B2 and 20) and also from our experiments (Fig. 1, A and B) showing that http://www.jimmunol.org/ ␤-arrestin augmented anti-CD3/anti-CD28-mediated IL-2 produc- PDE inhibition augments the increase in cAMP caused by T cell tion (Fig. 5B). Overexpression of Csk-SH3-SH2, an interfering activation. Not only are PDE4 enzymes the main contributors of mutant of Csk which is known to potentiate TCR-induced signal- cAMP-PDE activity in T cells (13) but PDE4 selective inhibitors ing (30), is shown as a control (Fig. 5B). Maximal T cell activation exert a major component of their anti-inflammatory action through can be accomplished through full PKC activation, full Ca2ϩ re- attenuation of T cell functioning (17, 18, 54). Thus, one might sponse, and MAPK activation and can be induced upon concom- expect that functioning PDE4 activity would serve to promote T itant treatment with phorbol ester and Ca2ϩ ionophore (PMA and cell signaling, perhaps by reducing cAMP levels in an appropriate ionomycin). Such treatment bypasses the inhibitory effect of compartment. It is interesting then to find that TCR stimulation by guest on October 2, 2021 cAMP (Fig. 5C) and was therefore used as a measure of maximal actually leads to the activation of AC and the generation of the activation of cells that had received various treatments (e.g., dif- second messenger cAMP that plays an inhibitory role in regulating ferent transfections). proximal TCR-induced T cell signaling (9, 28). Presumably it is The effects in Fig. 5B are comparably small because only up to important that T cells have a mechanism to ensure that cAMP 50% of the primary T cells were transfected. However, assessing levels do not become chronically elevated, which would thereby IFN-␥ production in transfected GFP-positive primary T cells by inhibit T cell functioning. In this study, we have shown that the flow cytometry, we found a clear potentiating effect of ␤-arrestin, elevation of cAMP is ablated subsequent to costimulatory activa- PDE4A4, and PDE4B2 (Fig. 5D). We also saw a potentiating ef- tion of T cells. We suggest that an important component in defin- fect of PDE4A4 and PDE4D1 (Fig. 5E). Again, overexpression of ing cAMP levels is recruitment of PDE4 enzymes to lipid raft dominant negative Csk-SH3-SH2 or wild-type Csk are used as fractions, a source for compartmentalized cAMP generation in T control, potentiating and inhibiting TCR-induced signaling, re- cells. Interestingly, such recruitment of PDE4 to rafts is apparently spectively (30) (Fig. 5E). Similar results were also obtained with a accompanied by that of ␤-arrestin, which in various other cell proximal IL-2 promoter reporter construct containing the NFAT types has been shown to play a pivotal role in not only uncoupling and AP-1 elements of the IL-2 promoter (NFAT-AP-1-luciferase; the receptor-mediated stimulation of Gs (71) but also in allowing Fig. 5F). Taken together, these data suggest that the activities of the receptor-mediated recruited of PDE4 isoforms (19, 48). both PKA and PDE4 are important for regulation of TCR-induced Although we clearly identified the recruitment of PDE4 iso- signaling and T cell function. We suggest that recruitment of ␤-ar- forms to rafts upon T cell stimulation, it is also possible that phos- restin and PDE4 to T cell lipid rafts upon concomitant TCR and phorylation could further contribute to the increased PDE4 activity CD28 stimulation down-regulates the inhibitory effect of cAMP in lipid raft fractions. Thus, the long PDE4A4 isoform has been induced upon TCR stimulation. Through this mechanism ␤-arres- shown to be activated by ϳ25% through phosphorylation by PKA tin and PDE4 potentiate T cell signaling upon CD28 costimulation (62, 63) and the short PDE4B2 and PDE4D1 isoforms can be (Fig. 6). similarly activated through the action of ERK (33), which becomes activated upon TCR stimulation (64). However, the paucity of Discussion PDE4 protein in rafts of these cells militates against a direct dem- cAMP plays an inhibitory role in regulating proximal TCR-in- onstration of this using either direct phosphorylation or the use of duced T cell signaling (28). On this basis, one might expect the phospho-Abs. There are other PDEs found in T cells (14, 65); intracellular level of cAMP to fall upon T cell activation in the however, no change in the activity of PDE3 was detected in lipid compartment relevant for regulation of proximal T cell signaling. rafts upon TCR costimulation (Fig. 2, C and CЈ). PDE7 has been Interestingly, however, increased intracellular cAMP levels fol- suggested to be important for T cell proliferation with its expres- lowing T cell activation in the presence of the PDE inhibitor sion up-regulated during the first 8 h of T cell activation. However, The Journal of Immunology 4857 it is absent from resting T cells (65) and T cell functioning has 12. Manganiello, V. C., and E. Degerman. 1999. Cyclic nucleotide phosphodiester- recently been shown to be normal in knockout mice (66), indicat- ases (PDEs): diverse regulators of cyclic nucleotide signals and inviting molecular targets for novel therapeutic agents. Thromb. Haemost. 82:407. ing that PDE7 is unlikely to be involved in the regulation of the 13. Erdogan, S., and M. D. Houslay. 1997. Challenge of human Jurkat T-cells with initial T cell signaling events. the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 Stimulation of the TCR is known to induce a signal that is too and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice weak to fully activate T cells (67). The signal can, however, be variant. Biochem. J. 32:165. amplified by CD28 costimulation and together these two signals 14. Ekholm, D., J. C. Mulloy, G. Gao, E. Degerman, G. Franchini, and V. C. Manganiello. 1999. Cyclic nucleotide phosphodiesterases (PDE) 3 and 4 in can induce full activation and clonal expansion (68). In this regard, normal, malignant, and HTLV-I transformed human lymphocytes. Biochem. we show here that ␤-arrestin recruitment appears to be mainly Pharmacol. 58:935. induced by CD28 stimulation and this may play a key role in 15. Seybold, J., R. Newton, L. Wright, P. A. Finney, N. Suttorp, P. J. Barnes, I. M. Adcock, and M. A. Giembycz. 1998. Induction of phosphodiesterases 3B, constraining the inhibitory consequence of TCR-induced AC ac- 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T- tivation through the recruitment of PDE4. The recruited ␤-arrestin lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists: potential role ␤ may also serve an uncoupling role, although the demonstration of in 2-adrenoreceptor desensitization. J. Biol. Chem. 273:20575. 16. Giembycz, M. A. 2002. Development status of second generation PDE4 inhibi- this and the identification of its partner allowing recruitment will tors for asthma and COPD: the story so far. Monaldi Arch. Chest Dis. 57:48. be a challenge for future studies. Since the combination of anti- 17. Burnouf, C., and M. P. Pruniaux. 2002. Recent advances in PDE4 inhibitors as immunoregulators and anti-inflammatory drugs. Curr. Pharm. Des. 8:1255. CD3 and anti-CD28 stimulation recruits and activates PDE4 to a 18. Spina, D. 2003. Theophylline and PDE4 inhibitors in asthma. Curr. Opin. Pulm. greater extent than anti-CD3 stimulation alone, signal amplifica- Med. 9:57. tion by costimulation may be mediated through activation of un- 19. Perry, S. J., G. S. Baillie, T. A. Kohout, I. McPhee, M. M. Magiera, K. L. Ang, W. E. Miller, A. J. McLean, M. Conti, M. D. Houslay, and R. J. Lefkowitz. 2002. known molecules in addition to identified species such as PI3K, Targeting of cyclic AMP degradation to ␤2-adrenergic receptors by ␤-arrestins. Downloaded from Itk, and Vav-1 (67). We suggest an additional role for CD28 as a Science 298:834. molecular amplifier of TCR-induced signals whereby CD28-me- 20. Arp, J., M. G. Kirchhof, M. L. Baroja, S. H. Nazarian, T. A. Chau, C. A. Strathdee, E. H. Ball, and J. Madrenas. 2003. Regulation of T-cell activa- diated PDE4 recruitment to lipid rafts serves to down-modulate tion by phosphodiesterase 4B2 requires its dynamic redistribution during immu- inhibitory cAMP signals. The inhibitory function of cAMP might, nological synapse formation. Mol. Cell. Biol. 23:8042. for example, be mediated through the action of PKA on the Csk- 21. Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387:569. Lck pathway as previously demonstrated (27, 28). Upon TCR 22. Montixi, C., C. Langlet, A. M. Bernard, J. Thimonier, C. Dubois, M. A. Wurbel, stimulation alone however, PDE4 recruitment may be too low to J. P. Chauvin, M. Pierres, and H. T. He. 1998. Engagement of T cell receptor http://www.jimmunol.org/ triggers its recruitment to low-density detergent-insoluble membrane domains. fully reduce the cAMP levels and therefore maximal T cell acti- EMBO J. 17:5334. vation cannot occur. 23. 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