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

TCR- and CD28-Mediated Recruitment of Phosphodiesterase 4to 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 Tasken2*

Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alonedoes not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMPwas generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation,�-arrestinin 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 cytokineproduction. Conversely, overexpression of either PDE4 or�-arrestin augmented TCR/CD28-stimulated cytokine production. Weshow here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipidrafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogatethe negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus.The Journal of Immunology, 2004, 173:4847–4858.

C yclic AMP, generated by G-protein-mediated (Gs�) acti-vation of adenylyl cyclase (AC),3 is a common and ver-satile second messenger controlling numerous cellular

processes including inhibition of mitogenic responses in fibro-blasts and T cells. Although cAMP is known to activate proteinkinase A (PKA) (1), Epac (exchange protein directly activated bycAMP) (2) and cyclic nucleotide-gated ion channels (3), PKA islikely to be the major target of cAMP in T cells because neitherEpac (our unpublished data and Ref. 4) nor cyclic nucleotide-gatedion channels are expressed in lymphocytes (3). Compartmental-ization of receptors, cyclases, and PKA by A-kinase-anchoringproteins (5, 6) as well as generation of local pools of cAMP withinthe cell by the action of anchored cAMP phosphodiesterase (PDE)isoforms (7) underpin the high degree of specificity of action inPKA-mediated signaling.

It has previously been demonstrated that stimulation of the Tcell Ag receptor (TCR) elevates cAMP (8). However, increasedcAMP levels in T cells inhibits T cell function and proliferation (9)

and, as a consequence, TCR-mediated cAMP production must beregulated for T cell activation to occur. Since the only known wayof reducing intracellular cAMP levels is through activation of thelarge family of cAMP PDEs (10–12), they are poised to play a keyregulatory role in regulation of T cell function. The majority of thecAMP-hydrolyzing activity in T cells is known to be mediated bythe PDE3 and PDE4 cAMP PDE families (13–15). Indeed, there iscurrently considerable interest in the PDE4 family as selectivePDE4 inhibitors that exert a profound anti-inflammatory action arebeing developed to treat asthma and chronic obstructive pulmo-nary disease (16–18), where T cells are thought to provide one ofthe key cell targets for their action. Furthermore, there is nowconsiderable evidence demonstrating that individual PDE4 iso-forms display distinct patterns of intracellular targeting, indicatingthat they are likely to play a key role in compartmentalized cAMPsignaling (10, 11). Indeed, PDE4 enzymes have very recently beenshown to interact with the signaling scaffold protein �-arrestin (19)that together serve to regulate signaling through �2-adrenergic re-ceptors. However, the functional role of PDE4 in the proximalTCR signaling in primary T cells is not well known, although itwas recently demonstrated that PDE4B2 stably transfected intoJurkat cells localizes to the immunological synapse upon activa-tion (20), suggesting a role for PDE4 in proximal T cell signaling.

It is generally accepted that proximal TCR-mediated signaling isinitiated in specialized sphingolipid- and cholesterol-enriched mi-crodomains in the cell membrane called lipid rafts (21). Lipid raftsfunction as signaling platforms that are comprised of or recruitprotein complexes involved in the proximal signal transduction inT cells (22). The importance of such membrane microdomains hasbeen demonstrated in T cells, as the integrity of lipid rafts is nec-essary for propagation of TCR-induced signaling to occur (23).One of the most proximal events taking place in T cells after en-gagement of the TCR is activation of the Src family protein ty-rosine kinases, in particular Lck, and phosphorylation of theITAMs present in the CD3 subunits (for review, see Ref. 24). Thisprocess is inhibited by C-terminal Src kinase (Csk) (25, 26). Themolecular mechanism for the inhibitory effect of cAMP on prox-imal T cell signaling involves PKA-mediated phosphorylation of

*Biotechnology Centre and †School of Pharmacy, University of Oslo, Oslo, Norway;‡Institute of Biomedical and Life Sciences, University of Glasgow, United Kingdom;§Infectious and Inflammatory Disease Center, and Program of Signal Transduction,Cancer Research Center, Burnham Institute, La Jolla, CA 92037; and ¶DulbeccoTelethon Institute and Venetian Institute of Molecular Medicine, Padova, Italy

Received for publication March 5, 2004. Accepted for publication August 9, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants to K.T. from the Programme for AdvancedStudies in Medicine, the Research Council of Norway, the Norwegian Cancer Society,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); bygrants to M.Z. from Telethon Italy (TCP00089), the Italian Cystic Fibrosis ResearchFoundation, 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 bygrants to T.M. from the National Institutes of Health (AI48032 and AI53585).2 Address correspondence and reprint requests to Dr. Kjetil Tasken, BiotechnologyCentre, University of Oslo, P.O. Box 1125, N-0317 Oslo, Norway. E-mail address:[email protected] Abbreviations used in this paper: AC, adenylyl cyclase; PKA, protein kinase A;PDE, phosphodiesterase; Csk, C-terminal Src kinase; IBMX, 3-isobutyl-1-methylx-anthine; SH, Src homology.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

serine 364 in Csk, resulting in Csk activation and subsequent in-hibition of Lck (27). Furthermore, PGE2 and other inputs leadingto elevated levels of cAMP before stimulation of the TCR inhibitsignaling through the TCR due to increased association betweenCsk and Csk-binding protein/protein associated with glycosphin-golipid-enriched microdomains residing in lipid rafts (28). How-ever, it is not known to what extent cAMP and PKA may inhibitT cell activation during TCR triggering in the absence of concom-itant ligand-operated stimuli through G protein-coupled receptorssuch as, for example, PGE2 that drive the generation of cAMP.

In this study, we investigate TCR-mediated cAMP productionand subsequent PKA activation in primary T cells and the role ofPDE4 in the propagation of T cell signaling in the absence andpresence of CD28 coreceptor stimuli. We show that the TCR-me-diated increase in cAMP levels in rafts is generated by recruitmentand dissociation of G proteins and that PKA activation serves todown-modulate signal transduction through the TCR. We showhere for the first time that concurrent TCR and CD28 stimulationacts to increase raft-localized PDE4 enzymes, enhancing the deg-radation of cAMP and, thereby, potentiating T cell activation.Thus, recruitment of PDE4 isoforms to lipid rafts is required andnecessary for a maximal T cell immune response to occur.

Materials and MethodsCells and transfections

Human peripheral blood T cells were purified by negative selection (95–98% pure) as described previously (29) and transfected in accordance withthe manufacturer’s instructions (Amaxa, Cologne, Germany) nucleofectorkit for peripheral T cells, catalogue no. VPA-1002). The human leukemicT cell line Jurkat TAg was cultured and transfected by electroporation aspreviously described (30) (10–40 �g DNA for each transfection). Unlessotherwise indicated, all experiments were conducted in normal primaryperipheral blood T cells.

Reagents and Abs

Rolipram (R-6520), Brij 98 (P-5641), PMA (P-8139), ionomycin (I-0634),mFLAG M2 Ab (F-3165), and pFLAG Ab (F-7425) were purchased fromSigma-Aldrich (St. Louis, MO), n-octyl-�-D-glucoside (19775) was ob-tained from USB (Cleveland, OH). 8-Bromoadenosine-3�,5�-cyclic mono-phosphorothioate (Rp-8-Br-cAMPs) was obtained from BIOLOG Life Sci-ence Institute (Bremen, Germany). Anti-Fc F(ab�)2 for cross-ligation ofbound Ab and peroxidase-conjugated secondary reagents were obtainedfrom Jackson ImmunoResearch Laboratories (West Grove, PA). H-89(371963) was obtained from Calbiochem (San Diego, CA). Protein A/G-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 CruzBiotechnology (Santa Cruz, CA). Anti-CD3� (��3) Ab was affinity-purified from a hybridoma cell line from American Type Culture Collec-tion (CRL-8001; Manassas, VA). Magnetic beads coated with anti-CD14(Dynabeads M-450 CD14, 111.12), anti-CD19 (Dynabeads M-450 CD19,111.04), and anti-CD3/anti-CD28 Abs (Dynabeads CD3/CD28 T cell ex-pander, 111.31) were obtained from Dynal Biotech (Oslo, Norway). mAbtoward CD28 (clone CD28.2) was purchased from Immunotech (Marseille,France). Anti-linker for activation of T cells (LAT; 06H807), anti-p44/42MAPK (06-182), and anti-Tyr(P) mAb (4G10) were purchased from Up-state Biotechnology (Lake Placid, NY). Abs recognizing phospho-416 Srcfamily (phospho-394 Lck/phospho-417 FynT) (2101), p42/p44 MAPK(9106), and PKA substrate phospho-specific Ab (abbreviated anti-RXXPS/PT, 9621) were purchased from Cell Signaling Technology (Berverly,MA). Abs toward PDE4A, PDE4B, PDE4D, and �-arrestin were as pre-viously described (19, 31–33).

Purification and stimulation of lipid rafts

Isolation of Triton X-100 (0.7%)- and Brij 98 (1%)-insoluble rafts wereperformed as described in detail elsewhere (34, 35) except that cells werelysed in standard lysis buffer (30). Brij 98 lysis was performed at 37°C for7 min. After lysis both Triton X-100 lysates and Brij 98 lysates werecompletely homogenized in a Dounce homogenizer, mixed with 80% su-crose, loaded at the bottom of a 40–5% sucrose gradient (in 25 mM MES(pH 6.5), 5 mM EDTA, and 150 mM NaCl), centrifuged at 200,000 � g for20 h at 4°C, and fractionated into 12 fractions from the top. Western blot

with LAT Abs revealed that fractions 2–5 normally contained the majorityof LAT and were used as a measurement for successful separation. Peakraft fractions were made by mixing fractions 2–5. For all cAMP measure-ments, fractions were prepared with Brij 98 as detergent. All other sepa-rations were done in the presence of Triton X-100. After Brij 98 cell sep-aration, all fractions were stimulated with anti-CD3 (5 �g/ml) alone or withanti-CD3 in combination with anti-CD28 (1 �g/ml) in the presence of ATP(1 mM) and MgCl2 (15 mM). After 1 min of Ab incubation F(ab�)2 (20�g/ml) were added for Ab cross-ligation. F(ab�)2 (20 �g/ml) were alsoadded to controls. Reactions were stopped in reaction termination buffersupplied with the cAMP radioimmunoassay. Activity was normalized forprotein content.

Stimulation of T cells for time course studies

PBL were incubated at 37°C for 10 min and then stimulated with anti-CD3alone (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 for1–10 min as described in relevant figures. Reactions were ended by addingice-cold RPMI 1640 followed by cell pelleting and lysis in standard lysisbuffer (30) containing n-octyl-�-D-glucoside (50 mM). Equal cell numberswere used at each time point for lipid raft purification after stimulation.

Immunoprecipitations were as before (30).

Densitometric scanning analysis

Scanned x-ray films were subjected to densitometric analysis with the pro-gram Scion image (www. scioncorp.com).

PDE activity assay

After ultracentrifugation and lipid raft separation, the collected fractionswere analyzed for PDE activity (36). Assays were conducted in duplicatewith the presence and absence of the PDE4 selective inhibitor rolipram (10�M) to determine the rolipram-dependent PDE4 activity. PDE3 activitywas similarly assessed but in this instance through chemical ablation in thepresence of the PDE3 selective inhibitor cilostimide (1 �M, C 79781;Sigma-Aldrich). Protein concentrations were quantified according to themethod of Bradford using BSA as a standard. Activity was normalized forprotein content.

IL-2 assay and cAMP assay

For IL-2 production, T cells were stimulated with beads coated with anti-CD3 and anti-CD28 Abs for 18 h (two beads per cell). For inhibition ofPKA or PDE4, cells were treated with 10 �M H-89 or 10 �M rolipram,respectively, for 20 min at 37°C before stimulation. Standard IL-2 assay(S2050; R&D Systems, Minneapolis, MN) was used to measure secretedIL-2. cAMP radioimmunoassays (cAMP kit from NEN, Boston, MA, cat-alogue no. SP004) were performed in accordance with the manufacturer’sinstructions.

NFAT-AP-1 luciferase assay

Jurkat Tag cells were transfected with NFAT-AP-1 luciferase reporter con-struct (10 �g) and Renilla-TK luciferase (1 �g) in combination with vec-tors encoding �-arrestin 2, PDE4B2, PDE4A4, PDE4D1, or empty vector(20 �g) as indicated in Fig. 5D. Sixteen hours after transfection cells wereeither left unstimulated, stimulated with anti-CD3 (2.5 �g/ml) alone, stim-ulated with a combination of anti-CD3/anti-CD28 (2.5 �g/ml/0.5 �g/ml),or stimulated with PMA (40 nM) and ionomycin (10 �M). Six hours afterstimulation, cells were harvested and lysed. Thereafter, luciferase activitywas measured by a dual luciferase reporter assay system from Promega(E1960; Madison, WI). NFAT-AP-1 luciferase activity in any sample wasnormalized against Renilla luciferase activity in the same sample. Mea-surements were done in triplicates. NFAT-AP-1 activity in each series wascalculated as percentage of control (PMA/ionomycin) that was found to beunaffected by increased cAMP levels (see Fig. 5C).

Intracellular staining and FACS analysis

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 withF(ab�)2 (10 �g/ml), or a mix of PMA (25 nM) and ionomycin (10 �M).Stimulation proceeded for 6 h in a 96-well plate. Pelleted cells were fixed,stained and analyzed as previously described (37).

Constructs

FLAG �-arrestin 2 (19), PDE4A4 (38), PDE4B2 (32), PDE4D1 (39),Csk-wt and Csk-SH3-SH2 (30) were as previously described.

4848 PDE4 RECRUITMENT TO RAFTS INCREASES T CELL ACTIVATION

ResultsTCR-mediated cAMP production is increased upon3-isobutyl-1-methylxanthine (IBMX) treatment andmay occur in lipid rafts

The TCR-induced elevation of cAMP levels has been observedpreviously in T cells (8). We demonstrate here that TCR stimula-tion in the absence of PDE inhibitors resulted in a modest butconsistent cAMP production (Fig. 1A). However, in the presenceof the nonselective PDE inhibitor IBMX, both basal and TCR-induced cAMP levels were further increased (Fig. 1A). In contrast,in T cells activated by TCR and CD28 cross-ligation, cAMP levelsactually decreased (Fig. 1B). An increase in cAMP levels was onlyobserved in the presence of IBMX (Fig. 1B). Since the cAMPlevels in total cell lysates were low, we next explored the possi-bility that the TCR-stimulated accumulation of cAMP might belocalized to lipid rafts since these membrane domains have beenshown to provide a key focus for initiation of TCR-mediated sig-naling in T cells (23). To evaluate this, we purified lipid rafts fromprimary T cells using Brij 98 as a detergent. As previously shown,lysis of cells with Brij 98 at 37°C before sucrose gradient ultra-centrifugation prevents the TCR-CD3 complex from being dis-solved from rafts (35). Upon lipid raft purification with Brij 98, weidentified CD3�, Gs� (Fig. 1C), and low levels of the coreceptorCD28 (data not shown) in the fractions corresponding to lipid rafts.In comparison, lipid raft purification with Triton X-100 dissolvesthe TCR-CD3� from the rafts (data not shown). The fact that CD3and Gs� copurifies with these purified cell-free fractions indicatesthat at least part of the cAMP production machinery may alreadybe present in lipid rafts, without the need for recruitment of othercomponents. Indeed, when lipid raft fractions isolated by sucrosegradient centrifugation were reconstituted with Mg2�/ATP andsubsequently stimulated with anti-CD3/anti-CD28, high levels ofcAMP production were detected in lipid raft fractions (up to 3-foldabove control in 1 min) (Fig. 1D). This reflects increased AC ac-tivity occurring in isolated lipid rafts in response to TCR stimu-lation in purified raft fractions. Comparable results were obtainedby in vitro TCR stimulation by anti-CD3 treatment alone (data notshown). Furthermore, PKA-mediated phosphorylation of putativeraft-localized PKA substrates was detected immediately after TCRstimulation of primary T cells (Fig. 1E). Densitometric analysisrevealed a 4- to 5-fold increase in PKA-mediated phosphorylationof a protein of �80 kDa. Phosphorylation of the same protein asshown in Fig. 1E was reduced upon H-89 pretreatment (data notshown). This provides further evidence of the activation of thecAMP signaling pathway upon T cell activation.

Gi, Gs, and AC have been reported to segregate into lipid rafts(40, 41). Therefore, we next examined whether these G proteinsplayed a role in the raft-associated cAMP increase. In resting Tcells, Gs� was detected both in lipid rafts as well as in nonraftfractions (Fig. 1, C and F). However, the level of Gs� in rafts wasrapidly and transiently increased upon anti-CD3/anti-CD28 cross-ligation of whole cells, with a decline to basal levels occurringafter 2 min (Fig. 1F, first panel). Conversely, the G protein Gi�,which acts to inhibit AC, was present at high levels in rafts fromresting T cells but dissociated rapidly from rafts following TCRstimulation (Fig. 1F, second panel). As shown previously by oth-ers (42), the G protein Gq� is recruited to rafts upon engagementof the TCR (Fig. 1F, third panel). However, this G protein is notconsidered to be directly involved in regulating cAMP signaling(43). Since both AC and its activator, Gs�, are present in lipid rafts,we next set out to hamper the anti-CD3/anti-CD28-induced cAMPproduction in vitro by using specific blocking Abs that inhibit Gs�

function by interfering with effector coupling (44). Pretreatment of

the isolated lipid raft fractions with these inhibitory Gs� Abs be-fore stimulation reduced the in vitro anti-CD3/anti-CD28-inducedcAMP production almost back to control levels (Fig. 1G). Con-versely, incubation with Ab blocking Gi� effector coupling in-creased both the basal and stimulated cAMP levels to 1.6 and 2pmol cAMP/106 cells, respectively, demonstrating the specificityof action of these reagents (data not shown). Together these dataindicate that in vitro TCR stimulation induces cAMP production inlipid rafts through a Gs-mediated process. Additionally, in intactcells, generation of cAMP seems to be increased by concurrentrecruitment of additional Gs� to rafts and dissociation of inhibitoryGi� from rafts. Furthermore, by blocking PDE activity using thePDE inhibitor IBMX, TCR-induced cAMP production is increasedidentifying a key role of PDEs in regulation of cAMP levels uponT cell activation.

T cell stimulation increases PDE4 activity in lipid rafts

Localization of PDE isoforms to specific cellular compartments(10) has been shown to provide a pivotal contribution to the gen-eration of local intracellular cAMP gradients in various cell types(7, 45). A number of PDE isoforms are expressed in T cells(PDE3B, 4A, 4B, 4D, 7A1, 7A3, 8A), of which PDE3 and PDE4provide the majority of cAMP PDE activity (13, 15, 46). The factthat the nonselective PDE inhibitor IBMX augmented cAMP pro-duction upon T cell stimulation indicates a role for PDEs in reg-ulating cAMP levels in T cells (Fig. 1, A and B). Given that PDE4selective inhibitors are considered to mediate part of their thera-peutic potential by acting on T cells and that a key feature of PDE4isoforms is their intracellular targeting (10, 11), we set out to de-termine whether PDE4 activity was localized to lipid rafts where Tcell signaling is initiated (47) and where the majority of cAMPseems to be produced. Doing this we see that although anti-CD3stimulation induces PDE4 activity in fractions corresponding tolipid rafts (Fig. 2, A and A�), CD28 costimulation leads to a pro-found raft-associated increase in PDE4 activity (Fig. 2, B and B�).In marked contrast to this, lipid raft fractions even from T cellssubjected to anti-CD3/anti-CD28 stimulation contained little or noPDE3 activity (Fig. 2, C and C�).

Enhanced recruitment of PDE4 and �-arrestin to lipid raftsafter TCR and CD28 costimulation

The specific increase in PDE4 activity in lipid raft fractions uponTCR/CD28 engagement might suggest that temporal changes inPDE4 activity can play a key role in tuning intracellular activation-induced gradients of cAMP in T cell lipid rafts and thereby in-crease signal propagation upon costimulation. Since a key featureof T cell activation is to induce the redistribution of componentsboth to and from lipid rafts, we next explored whether there wasany specific recruitment of PDE4 enzymes to lipid raft fractionsupon concomitant TCR and CD28 engagement. The PDE4 familyconsists of four subfamilies (A, B, C, and D), each of which gen-erates a series of isoforms by the use of distinct promoters andalternative mRNA splicing (10, 11). However, within any one sub-family, the C-terminal regions of all active isoforms are identical.This has been effectively exploited to generate antisera that are notonly specific for each subfamily but thereby identify all isoformswithin a particular subfamily (39). Using antisera specific for thefour PDE4 subfamilies, we found that the long PDE4A4 isoform,the short PDE4B2 isoform, and the short PDE4D1/2 isoforms wereall rapidly and concomitantly recruited to lipid rafts upon TCR andCD28 costimulation (Fig. 3A). This suggests that the increasedPDE4 activity seen in lipid rafts upon TCR/CD28 stimulation atleast in part is a result of increased levels of PDE4 in membranemicrodomains.

4849The Journal of Immunology

FIGURE 1. cAMP levels increase upon T cell activation. A, Increase in total cellular cAMP levels upon TCR stimulation. Purified primary T cells werestimulated 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 measuredby radioimmunoassay. The experiment is representative of three similar experiments. B, TCR-induced cAMP levels in T cells are reduced upon cross-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 98as detergent. Lysates from the 11 first fractions were analyzed with the indicated Abs. D, Anti-CD3/anti-CD28-mediated cAMP increase occurs in lipidrafts. Insoluble membranes from T cells were prepared as in C (Brij 98), whereupon the TCR is not dissolved from rafts. Each fraction harvested from thesucrose 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 threerepresentative experiments. E, T cell activation increases PKA activity in rafts. Primary T cells were stimulated with anti-CD3 and F(ab�)2 and subsequentlysubjected to sucrose gradient separation with Triton X-100. Peak raft fractions were analyzed for proteins phosphorylated by PKA (Anti-RXXPS/PT). Theblot shown is representative of four experiments. Densitometric scanning revealed about a 4-fold induction in PKA activity (average, 4.2 � 1.2, n 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 separatedas 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 towardGs� reduces anti-CD3/anti-CD28-induced cAMP production. T cell lysates were subjected to sucrose gradient fractionation with Brij 98 as in A. Raftfractions were reconstituted with Mg/ATP, not pretreated or pretreated with Abs inhibiting Gs�, stimulated as indicated, and cAMP production comparedwith control was calculated.


It has recently been shown that upon �2-adrenergic stimulationof HEK293 cells a cytoplasmic complex consisting of �-arrestinand PDE4 is recruited to membranes where the phosphorylated�2-adrenergic receptor is localized (19, 48). Intriguingly, we foundhere that anti-CD3/anti-CD28 stimulation caused a clear recruit-ment of �-arrestin to T cell lipid raft fractions concurrently withPDE4 (Fig. 3A). �-Arrestin serves as a cytosolic scaffold proteinthat can bind a variety of signaling molecules and recruit them tothe plasma membrane upon appropriate receptor stimulation (49).One such species is the MAPK/ERK (50–52) and we demonstratehere (Fig. 3A) that anti-CD3/anti-CD28 stimulation also serves toconcomitantly recruit ERK along with �-arrestin to lipid rafts in Tcells. Furthermore, the fraction of ERK present in lipid rafts wasevidently active, as determined by phospho-specific Abs towardThr(P)202 and Tyr(P)204 in ERK (Fig. 3A).

To examine whether PDE4 and �-arrestin exist in a complexboth before and after T cell stimulation, we transfected T cells witha vector encoding a FLAG-tagged �-arrestin, and immunoprecipi-tated �-arrestin from both unstimulated cells and cells stimulatedwith anti-CD3/anti-CD28. Interestingly, �-arrestin and PDE4D co-immunoprecipitated from both unstimulated and stimulated cells(Fig. 3B), indicating that they exist in a preassembled complex inT cells as has been shown to occur in other cell types (19). OtherPDE4 isoforms were not tested here, as they have been describedto coimmunoprecipitate with �-arrestin before.

Stimulation using anti-CD3 alone was not as effective as CD28costimulation in increasing PDE4 activity in lipid raft fractions(Fig. 2). Indeed, compared with anti-CD3 stimulated cells, 2.5-fold

more PDE4/�-arrestin was recruited upon costimulation withCD28 (Fig. 3C). This led us to evaluate the impact of costimula-tion on recruitment of the PDE4/�-arrestin complex to lipid rafts.Indeed, cross-linking of CD3 alone was far less effective in re-cruiting both �-arrestin and ERK to lipid rafts compared with co-stimulation with anti-CD3/anti-CD28 (Fig. 3C). In the same ex-periment, PDE4 activity was lower upon CD3 stimulationcompared with CD3/CD28 stimulation (data not shown) as de-scribed in Fig. 2.

The diterpene forskolin interacts directly with the catalytic unitof AC to cause its activation. Treating T cells with this agentclearly fails to show any recruitment of PDE4 to rafts (Fig. 3D),although forskolin always gave a robust cellular increase in cAMPcompared with CD3 stimulation (Fig. 3E). This demonstrates thatPDE4 recruitment is not elicited through any increase in intracel-lular cAMP levels and argues toward a specific role for the TCR-and CD28-mediated signaling in recruitment of PDE4 to rafts. An-ti-CD3 stimulation was used as a control in these experiments asonly low levels of PDE4 are recruited upon anti-CD3 stimulation,allowing detection of potentially low PDE4 recruitment upon for-skolin treatment.

Only a few TCRs are concomitantly ligated under normal cir-cumstances in vivo, generating incomplete activation events thateventually lead to anergy or cell death (53). However, CD28 co-stimulation amplifies the weak TCR-induced signals leading to fullT cell activation and clonal expansion in vivo. When T cells werestimulated with anti-CD28 Abs alone here, we found that this wassufficient to induce recruitment of �-arrestin to lipid rafts (Fig. 3F).

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 raftfractions. 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 andF(ab�)2 (B) for 2 min. Lysates were subjected to sucrose gradient centrifugation and each of the harvested fractions was subsequently analyzed for PDE4activity and protein content (A� and B�). Time-dependent increase in PDE4 activity in T cell rafts after stimulation. Cells were stimulated for 0, 2, 3, and5 min with either anti-CD3 alone (A�) or anti-CD3/anti-CD28 in combination (B�), separated, and analyzed as in A. Data are presented as induction of PDE4activity at different time points (average � SEM) compared with control (set to 1). Induction of PDE4 activity at 2 min upon anti-CD3 stimulation was2.0 � 0.8-fold (average � SEM, n 3). Induction of PDE4 activity at 2 min upon anti-CD3/anti-CD28 stimulation was 4.5 � 0.8-fold (average � SEM,n 4; C and C�). No significant increase in PDE3 activity in rafts upon anti-CD3/anti-CD28 stimulation. Cells were stimulated with anti-CD3/anti-CD28for 3 min, separated as in A, and analyzed for PDE3 activity. PDE3 activity following anti-CD3/anti-CD28 treatment was 1.1 � 0.1-fold (average � SEM,n 3) compared with untreated cells.


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 lipidrafts 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 andsubjected to sucrose gradient ultracentrifugation with Triton X-100. Peak raft fractions (fractions 2–5) from cells activated for various periods of time wereanalyzed with the indicated Abs. B, Jurkat TAg cells were transfected with empty vector or with a construct coding for FLAG-�-arrestin, stimulated asindicated, and lysed in standard lysis buffer. Immune complexes were precipitated with Abs toward FLAG and analyzed for the presence of PDE4D andFLAG �-arrestin. C, Enhanced recruitment of PDE4 and �-arrestin to T cell lipid rafts after anti-CD28 costimulation. T cells were stimulated either withanti-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 indicatedtimes of activation. Recruitment of �-arrestin to rafts with anti-CD3/anti-CD28 compared with anti-CD3 alone was 2.5 � 0.2-fold (average � SEM, n 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-CD3and F(ab�)2 (3 min) that give low levels of PDE4 in rafts and thereafter subjected to sucrose gradient ultracentrifugation. Peak raft fractions (fractions 2–5)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 stimulatedwith indicated combinations of anti-CD3, anti-CD28, and anti-CD4 and analyzed with Abs detecting �-arrestin and LAT.


In contrast, upon cross-ligation of the TCR or CD28 with CD4, aT cell surface marker to which the Src kinase Lck binds, no ad-ditional effect on recruitment of �-arrestin was found (Fig. 3F).Altogether, these results suggest that signals generated upon CD28stimulation are critical for enhancing the recruitment of the PDE4and �-arrestin to lipid rafts in a cAMP-independent fashion.

PKA is activated upon TCR stimulation and inhibits proximal Tcell signaling

The fact that PDE4 selective inhibitors inhibit T cell functioningtherapeutically (17, 54) might suggest that our novel demonstra-

tion of the recruitment of PDE4 isoforms to lipid raft fractionsupon T cell activation and regulation of raft-associated cAMP sig-naling has particular functional significance. We thus set out togain insight into the possible physiological consequences of TCR-induced cAMP production by analyzing the effect of PKA-mediated signaling on proximal TCR activation in T cells. As astart, we began to analyze whether TCR stimulation increasesPKA-mediated signaling. It has previously been demonstrated thatthe phosphatase HePTP is directly phosphorylated at serine 23 byPKA in T cells (55), making serine 23 phosphorylation of HePTPa good readout for PKA activity. Stimulation of Jurkat cells with

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


FIGURE 5. PKA and PDE4 are activated following T cell activation and play opposite regulatory roles in titrating T cell immune functions. A, IL-2secretion is impaired upon rolipram and H-89 pretreatment. Primary T cells pretreated with rolipram or H-89 were left unstimulated, stimulated with beadscoated 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 measuredwith ELISA (average � SEM, n 3). B, Expression of PDE4B2 and �-arrestin increases IL-2 production in T cells. Primary T cells were transfected withindicated constructs. At 3 h after transfection, cells were stimulated with anti-CD3/anti-CD28-coated beads and 18 h after stimulation cells were pelletedand IL-2 secreted was analyzed by ELISA (average � half range, n 2). C, Increased cAMP does not affect PMA/ionomycin-induced proximal IL-2promoter (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 werestimulated 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, andpermeabilized, then stained with Abs detecting intracellular IFN-�. FACS analysis was subsequently performed to determine (Figure legend continues)


anti-CD3-IgM (C305) resulted in rapid phosphorylation of serine23 in this protein (Fig. 4A), clearly demonstrating TCR-inducedPKA activation. Next, we studied phosphorylation of CREB (56),another direct target of PKA, through a pathway that was largelyinhibited by pretreatment with H-89 (Fig. 4B, upper panel). Like-wise, pretreatment of primary T cells with the PKA antagonistRp-8-Br-cAMPS reduced the activation-induced CREB phos-phorylation (Fig. 4B, lower panel). Altogether, this demonstratesTCR-induced PKA activation in primary T cells.

Next, we explored what functional role TCR-induced PKA ac-tivity plays in the proximal TCR signaling. One of the most prox-imal events taking place upon TCR stimulation is activation of theSrc family kinases Lck and FynT and phosphorylation of the CD3�-chains (57). The phosphorylated �-chains function as dockingsites for the Src homology (SH) 2 domain of the Syk family mem-

ber ZAP-70 (58), which upon Lck phosphorylation mediates thephosphorylation of the important adapter molecule linker for ac-tivated T cells (LAT) (for review, see Ref. 24). Phosphotyrosineson LAT in turn attract complexes of adapters and signaling mol-ecules like SH2 domain-containing leukocyte protein of 76 kDa(SLP-76) necessary to mediate a functional T cell response (59).Indeed, upon pretreatment of T cells with the PKA inhibitor H-89,early T cell activation markers such as �-chain phosphorylationand phosphorylation in the catalytic domain of Lck/FynT wereincreased (Fig. 4C). Densitometric analysis revealed that in thepresence of H-89, TCR-induced �-chain phosphorylation was in-creased 2- to 4-fold compared with control cells (Fig. 4D). Fur-thermore, phosphorylation of other molecules important for T cellactivation such as LAT and SLP-76 (24) were also increased underconditions of low PKA activity (data not shown). Lastly, we

FIGURE 6. PKA and PDE4 have opposing func-tions in proximal T cell signaling and titrates theactivation-induced response. We suggest a modelwhereby TCR triggering through G protein couplingto the AC generates cAMP and prevents full T cellactivation response (upper panel). However, CD28costimulation serves to amplify TCR-induced sig-nals by recruiting the PDE4/�-arrestin complexleading to cAMP degradation, down-modulation ofinhibitory signals, and full T cell activation (lowerpanel).

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-� productionwas 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 andNFAT-AP-1-luciferase reporter constructs. Eighteen hours after transfection, cells were stimulated with anti-CD3, anti-CD3/anti-CD28, or PMA/ionomycinfor 6 h and then analyzed for luciferase activity. NFAT-AP-1 luciferase activity was normalized against Renilla luciferase in the same sample. Theexperiment shown has triplicate measurements (average � SD, n 3) and is representative of three independent experiments.


wanted to investigate whether expression of �-arrestin or PDE4A4influenced the proximal signaling events as well. As shown in Fig.4E, �-arrestin clearly potentiated proximal events like �-chainphosphorylation (Fig. 4E). Overexpression of PDE4A4 also po-tentiated �-chain phosphorylation although the induction was notas evident as when �-arrestin was expressed (Fig. 4E).

PKA and PDE4 control T cell function

Since �-arrestin and PDE4 expression potentiate proximal signal-ing events in T cells, we next wanted to assess the effects of PKAand PDE4 on downstream T cell function. Upon full T cell acti-vation, in vivo T cells proliferate and produce different growth-promoting cytokines like IL-2 and IFN-�. To assess the effect ofPDE4 and PKA activity on T cell function, we treated T cells witheither rolipram or H-89 and measured IL-2 secretion. Pretreatmentof cells with rolipram reduced TCR-induced IL-2 production to�50% of control (Fig. 5A), whereas H-89-mediated inhibition ofPKA had the opposite effect (2-fold increase compared with con-trol; Fig. 5A). Similarly, IFN-� production was also reduced byrolipram and increased by H-89 (data not shown). This is in ac-cordance with previous reports demonstrating that increased levelsof cAMP reduce IFN-� production in T cells (37).

Because we found that �-arrestin and PDE4 are recruited tolipid rafts as a complex, we next investigated the effect of over-expression of �-arrestin and various PDE4 isoforms on IL-2 se-cretion and IFN-� production. Overexpression of PDE4B2 and�-arrestin augmented anti-CD3/anti-CD28-mediated IL-2 produc-tion (Fig. 5B). Overexpression of Csk-SH3-SH2, an interferingmutant of Csk which is known to potentiate TCR-induced signal-ing (30), is shown as a control (Fig. 5B). Maximal T cell activationcan be accomplished through full PKC activation, full Ca2� re-sponse, and MAPK activation and can be induced upon concom-itant treatment with phorbol ester and Ca2� ionophore (PMA andionomycin). Such treatment bypasses the inhibitory effect ofcAMP (Fig. 5C) and was therefore used as a measure of maximalactivation of cells that had received various treatments (e.g., dif-ferent transfections).

The effects in Fig. 5B are comparably small because only up to50% of the primary T cells were transfected. However, assessingIFN-� production in transfected GFP-positive primary T cells byflow cytometry, we found a clear potentiating effect of �-arrestin,PDE4A4, and PDE4B2 (Fig. 5D). We also saw a potentiating ef-fect of PDE4A4 and PDE4D1 (Fig. 5E). Again, overexpression ofdominant negative Csk-SH3-SH2 or wild-type Csk are used ascontrol, potentiating and inhibiting TCR-induced signaling, re-spectively (30) (Fig. 5E). Similar results were also obtained with aproximal IL-2 promoter reporter construct containing the NFATand AP-1 elements of the IL-2 promoter (NFAT-AP-1-luciferase;Fig. 5F). Taken together, these data suggest that the activities ofboth PKA and PDE4 are important for regulation of TCR-inducedsignaling and T cell function. We suggest that recruitment of �-ar-restin and PDE4 to T cell lipid rafts upon concomitant TCR andCD28 stimulation down-regulates the inhibitory effect of cAMPinduced upon TCR stimulation. Through this mechanism �-arres-tin and PDE4 potentiate T cell signaling upon CD28 costimulation(Fig. 6).

DiscussioncAMP plays an inhibitory role in regulating proximal TCR-in-duced T cell signaling (28). On this basis, one might expect theintracellular level of cAMP to fall upon T cell activation in thecompartment relevant for regulation of proximal T cell signaling.Interestingly, however, increased intracellular cAMP levels fol-lowing T cell activation in the presence of the PDE inhibitor

IBMX was reported 15 years ago (8, 60). Despite this, the sig-nificance of activation-induced cAMP production has been ill-un-derstood to date, as has the location in the cell where cAMP isgenerated. In this study, we show that T cell activation rapidlyinduces the production of the immunoinhibitory signaling mole-cule cAMP in lipid rafts, resulting in raft-associated PKA activa-tion. Our data indicate that the concomitant recruitment to lipidrafts of the stimulatory G protein, Gs, and dissociation of the in-hibitory G protein, GI, play a key role in the activation of AC thatoccurs upon T cell activation. These data imply that a localizedincrease in cAMP is generated in T cells upon activation and thatthis appears to be concentrated in lipid rafts.

Gs-coupled receptors, such as the �2-adrenergic receptor, invari-ably generate transient increases in cAMP (61). A key regulator ofreceptor-stimulated AC activity is the recruitment of �-arrestinfrom the cytosol to the plasma membrane-associated receptor (61).This has the effect of causing the uncoupling of Gs from receptorstimulation, leading to desensitization of AC. However, regulationof intracellular cAMP levels is not solely the prerogative of ACaction, but is also crucially regulated by the action of PDEs, whichprovide the sole route of cAMP degradation in cells (10, 12). In-deed, although �-arrestin may serve to terminate AC activation, itis only through the action of PDEs that cAMP levels can return totheir basal state. The importance of PDE action in T cells is clearlyevident from both previous studies by other investigators (13–15,20) and also from our experiments (Fig. 1, A and B) showing thatPDE inhibition augments the increase in cAMP caused by T cellactivation. Not only are PDE4 enzymes the main contributors ofcAMP-PDE activity in T cells (13) but PDE4 selective inhibitorsexert a major component of their anti-inflammatory action throughattenuation of T cell functioning (17, 18, 54). Thus, one mightexpect that functioning PDE4 activity would serve to promote Tcell signaling, perhaps by reducing cAMP levels in an appropriatecompartment. It is interesting then to find that TCR stimulationactually leads to the activation of AC and the generation of thesecond messenger cAMP that plays an inhibitory role in regulatingproximal TCR-induced T cell signaling (9, 28). Presumably it isimportant that T cells have a mechanism to ensure that cAMPlevels do not become chronically elevated, which would therebyinhibit T cell functioning. In this study, we have shown that theelevation of cAMP is ablated subsequent to costimulatory activa-tion of T cells. We suggest that an important component in defin-ing cAMP levels is recruitment of PDE4 enzymes to lipid raftfractions, a source for compartmentalized cAMP generation in Tcells. Interestingly, such recruitment of PDE4 to rafts is apparentlyaccompanied by that of �-arrestin, which in various other celltypes has been shown to play a pivotal role in not only uncouplingthe receptor-mediated stimulation of Gs (71) but also in allowingthe receptor-mediated recruited of PDE4 isoforms (19, 48).

Although we clearly identified the recruitment of PDE4 iso-forms to rafts upon T cell stimulation, it is also possible that phos-phorylation could further contribute to the increased PDE4 activityin lipid raft fractions. Thus, the long PDE4A4 isoform has beenshown to be activated by �25% through phosphorylation by PKA(62, 63) and the short PDE4B2 and PDE4D1 isoforms can besimilarly activated through the action of ERK (33), which becomesactivated upon TCR stimulation (64). However, the paucity ofPDE4 protein in rafts of these cells militates against a direct dem-onstration of this using either direct phosphorylation or the use ofphospho-Abs. There are other PDEs found in T cells (14, 65);however, no change in the activity of PDE3 was detected in lipidrafts upon TCR costimulation (Fig. 2, C and C�). PDE7 has beensuggested to be important for T cell proliferation with its expres-sion up-regulated during the first 8 h of T cell activation. However,


it is absent from resting T cells (65) and T cell functioning hasrecently been shown to be normal in knockout mice (66), indicat-ing that PDE7 is unlikely to be involved in the regulation of theinitial T cell signaling events.

Stimulation of the TCR is known to induce a signal that is tooweak to fully activate T cells (67). The signal can, however, beamplified by CD28 costimulation and together these two signalscan induce full activation and clonal expansion (68). In this regard,we show here that �-arrestin recruitment appears to be mainlyinduced by CD28 stimulation and this may play a key role inconstraining the inhibitory consequence of TCR-induced AC ac-tivation through the recruitment of PDE4. The recruited �-arrestinmay also serve an uncoupling role, although the demonstration ofthis and the identification of its partner allowing recruitment willbe a challenge for future studies. Since the combination of anti-CD3 and anti-CD28 stimulation recruits and activates PDE4 to agreater extent than anti-CD3 stimulation alone, signal amplifica-tion by costimulation may be mediated through activation of un-known molecules in addition to identified species such as PI3K,Itk, and Vav-1 (67). We suggest an additional role for CD28 as amolecular amplifier of TCR-induced signals whereby CD28-me-diated PDE4 recruitment to lipid rafts serves to down-modulateinhibitory cAMP signals. The inhibitory function of cAMP might,for example, be mediated through the action of PKA on the Csk-Lck pathway as previously demonstrated (27, 28). Upon TCRstimulation alone however, PDE4 recruitment may be too low tofully reduce the cAMP levels and therefore maximal T cell acti-vation cannot occur.

In conclusion, our results suggest opposing functions of PKAand PDE4 isoforms during proximal T cell signaling, thereby ti-trating the activation-induced response. In this study, we then pro-pose a novel facet of the CD28 costimulation effect, namely, inachieving down-modulation of TCR-induced cAMP-mediated in-hibitory signals through recruitment of PDE4 to lipid rafts.

AcknowledgmentsWe are grateful to Guri Opsal and Gladys Tjørholm for technical assis-tance, Sondre Myklebust for scientific contribution, and Jorrit M. Enserinkfor critical reading of this manuscript.

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