0014-2980/03/0101-204$17.50+.50/0 © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Mechanisms of H4/ICOS costimulation: effects onproximal TCR signals and MAP kinase pathways

Maria Jose Feito1, Rosanna Vaschetto2, Gabriel Criado1, Alejandra Sanchez1,Annalisa Chiocchetti2, Arturo Jimenez-Perianez3, Umberto Dianzani2, Pilar Portoles3

and Jose M. Rojo1

1 Centro de Investigaciones Biologicas, CSIC, Madrid, Spain2 Department of Medical Sciences, “A. Avogadro” University of Eastern Piedmont at Novara,

Novara, Italy3 Centro Nacional de Biologıa Fundamental, Instituto de Salud Carlos III, Madrid, Spain

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals havebeen analyzed in mouse CD4+ Th2 cells, expressing H4/ICOS at higher levels than Th1clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphoryla-tion of ZAP-70, ´ , or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phos-phoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAPkinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, ispartially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulationis partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymeri-zation, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporinA nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAPkinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10secretion. ERK or JNK inhibitors partially inhibited IL-4 and IL-10 secretion, while PKC orp38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our datashow clear similarities of costimulation mechanisms between H4/ICOS and CD28 during theearly steps of TCR activation.

Key words: ZAP-70 / Extracellular signal-regulated kinase / p38 / Jun N-terminal kinase / Phos-phoinositide 3-kinase

Received 29/3/01Revised 22/10/02Accepted 25/11/02

[I 21910]

Abbreviations: MAP kinase: Mitogen-activated proteinkinase ERK: Extracellular signal-regulated kinase JNK:Jun N-terminal kinase p38: p38/HOG, stress-activated pro-tein kinase 2 PI-3K: Phosphoinositide 3-kinase

1 Introduction

Optimal activation of T lymphocyte effector functionsand antigen-specific clonal expansion requires signalsfrom cell surface costimulatory molecules in addition tosignals delivered through the TCR/CD3 complex [1–3].Recently, a new costimulatory molecule termed ICOS(for inducible costimulator), homologous to CD28 andCTLA-4 (CD152) has been identified and cloned in thehuman, mouse, and rat [4–6]. ICOS is the same moleculeas H4, a mouse and human costimulatory protein, whichwe previously described using a hamster monoclonalantibody ([7–9], reviewed in [10]).

H4/ICOS is a disulfide-linked homodimeric glycoproteinof 50–65 kDa, whose expression is characteristicallyinduced upon activation of peripheral resting T lympho-cytes [4, 7, 8]. Like CD28, H4/ICOS enhances TCR/CD3-induced proliferation of peripheral resting T lymphocytes[4, 5, 7]. However, H4/ICOS costimulation produces noincrease of IL-2 secretion, yet enhances the secretion ofcytokines including IL-4, IL-10 and IFN- + [4, 5]. H4/ICOSligand (B7h, B7RP-1) is homologous but distinct to theCD28/CTLA-4 ligands CD80 (B7.1) and CD86 (B7.2) [5,11, 12]. B7h is constitutively expressed by B lympho-cytes and macrophages [5, 11], and its expression canbe induced in cells from different tissues by TNF- § orLPS [11].

Manipulation of ICOS-B7h interactions “in vivo” and “invitro” shows that H4/ICOS is a major costimulator ofactivated T cells [13], important to reactions primarilymediated by Th1 as well as Th2 T cells, including sec-ondary DTH immune responses, experimental allergic

204 M. J. Feito et al. Eur. J. Immunol. 2003. 33: 204–214

Fig. 1. Differences in H4/ICOS expression between Th1 andTh2 mouse T cell clones, and activation-dependent associa-tion of PI-3K to H4/ICOS. (A) H4/ICOS and CD28 were ana-lyzed by flow cytometry in resting cells from the Th1 mouseT cell clones AE103 and D10.Tcr25 or the Th2 cell clonesD10.G4.1, D10.Th2.3, and AK-8. Results show the meanfluorescence above background for each molecule. (B) Anal-ysis by immunoblot of PI-3K co-precipitation in H4/ICOSimmunoprecipitates (i.p.) from the mouse Th2 cell line D10activated (+) or not (–) with pervanadate. H4/ICOS in immu-noprecipitates was detected by blotting with streptavidin-peroxidase using biotin-labeled cells. The presence of PI-3Kin lysates from 2×105 cells was also determined, as indi-cated. Molecular mass is given in kDa.

encephalomyelitis, allograft rejection, and allergic lunginflammation [5, 14–16], or Ig responses [13]. Blockadeor loss of H4/ICOS-B7h interactions induces diminishedIL-4, IL-10 or IL-13 production and Th2-mediated lunginflammation [13, 17–19], as well as enhanced IFN- + pro-duction and incidence of EAE [18, 19] or lower IFN- + pro-duction [13, 17]. ICOS-deficient mice have reduced anti-body responses as well as CD40-mediated antibodyclass switching to foreign antigens [19–21].

The precise mechanisms of H4/ICOS costimulation arenot known. However, its functional and sequence homol-ogy with CD28 [7, 8, 13], and particularly the conserva-tion of a Tyr-Met-X-Met sequence motif in the cytoplas-mic domain suggests that H4/ICOS and CD28 might useoverlapping signaling pathways.

Several mechanisms of TCR/CD3/CD28 signal integra-tion have been proposed, including the activation ofZAP-70, Vav, and MAP kinases [22–26]. To determine ifsome of these mechanisms of signal integration extendto H4/ICOS, we have used mouse T cells constitutivelyexpressing H4/ICOS to analyze its effect on early TCR/CD3 signaling and MAP kinase pathways. Our resultsshow that H4/ICOS strongly synergizes with CD3 fortyrosine phosphorylation of TCR/CD3 ´ chains, ZAP-70and Vav, as well as for the activation of the MAP kinasesERK, JNK and p38. Inhibition of actin polymerizationinhibit these effects, while inhibition of phosphoinositide3-kinase (PI-3K) inhibits activation of the ERK and JNKMAP kinases but not ZAP-70 phosphorylation, showingthat the integration of TCR/CD3 and H4/ICOS signalscan occur at different levels in the early steps of T cellactivation by PI-3K- and actin polymerization-dependentmechanisms.

2 Results

2.1 H4/ICOS expression in Th1 and Th2 mouse Tcell lines: association of PI-3K

To determine adequate experimental models to analyzeH4/ICOS costimulation, we checked several CD4+ T celllines for H4/ICOS expression. Although, as describedpreviously [7], all mouse T cell lines examined expressedH4/ICOS, lines of Th2 phenotype expressed higher lev-els of H4/ICOS than Th1 cell lines (Fig. 1A). These dataare in agreement with recent reports [13, 18]. In view ofthese results, the SR.D10 subclone of the D10.G4.1 Th2cell line was chosen for our study.

It has been suggested that H4/ICOS constitutively asso-ciates with PI-3K regardless of cross-linking [13]. Fig. 1Bshows that immunoprecipitation of H4/ICOS from the

cell surface of unstimulated D10 cells recruited a smallamount of the 85-kDa regulatory subunit of PI-3K. How-ever, the amount of H4/ICOS-associated PI-3K wasenhanced more than 30-fold by pervanadate activationof the cells (Fig. 1B). These results suggest a differencebetween our system, involving cell lines naturallyexpressing H4/ICOS, and those used by Coyle et al. [13],using ICOS-transfected Jurkat.

2.2 H4/ICOS synergizes with TCR/CD3 for earlyT cell activation

H4/ICOS-mediated costimulation was analyzed usingantibody-coated latex microspheres essentially asdescribed in Viola et al. ([27], see Methods). Several MAPkinase pathways involved in TCR-mediated activationand CD28 costimulation were analyzed by determiningthe activation (i.e. dual phosphorylation) of downstreamtarget kinases including ERK1/2, JNK, and p38. The

Eur. J. Immunol. 2003. 33: 204–214 Mechanisms of H4/ICOS costimulation 205

Fig. 2. H4/ICOS-mediated costimulation of early TCR acti-vation. (A) Immunoblot of cell lysates of SR.D10 cells incu-bated for 5 min with polystyrene beads coated with anti-CD3 (1 ? g/ml), anti-CD4 (5 ? g/ml) or anti-H4/ICOS antibody(5 ? g/ml). Parallel samples of cell lysates were analyzed byimmunoblot for the presence of active forms of the MAPkinases ERK, JNK, and p38 using antibodies specific fordually phosphorylated kinases. The same blots were thenstripped and re-probed with kinase-specific antibodies tocheck for protein loading. (B) Lysates from the cells shown in(A) (5×106 cells/determination) were immunoprecipitatedwith anti-ZAP-70 antibodies coupled to Sepharose. Theimmunoprecipitates were separated by electrophoresis,transferred to PVDF membranes and then analyzed byimmunoblotting with anti-pTyr antibody, stripped and re-probed with anti-ZAP-70 antibody. (C) Lysates form cellsactivated as described in (A) were immunoprecipitated withanti- ´ or anti-Vav antibodies, as indicated. After electropho-resis, the immunoprecipitates were analyzed by immunoblotwith anti-phosphotyrosine, stripped, and re-probed withanti- ´ or anti-Vav antibodies.

results shown in Fig. 2 indicate that joint ligation of CD3and H4/ICOS strongly synergized for the activation of allthree kinases. Ligation of CD3 or H4/ICOS alone induced

a low level of ERK and p38 activation (which usuallycould only be detected upon long exposures, data notshown); however, under the activation conditions used,JNK activation was only observed upon ligation of bothCD3 and H4/ICOS. Thus, while all MAP kinase pathwayswere targets for H4/ICOS costimulation, the JNK/SAPKpathway might be a specific target for TCR-H4/ICOS sig-nal integration, as previously shown for CD28 [23, 26].

Data on CD28 suggest that signal integration of costimu-latory molecules can occur upstream in the TCR activa-tion cascade, pointing to ZAP-70, Vav, and even TCR ´chains as key molecular targets in this process [24, 25,28]. Consequently, we checked for upstream activationtargets for H4/ICOS (Fig. 2B). CD3 ligation alone inducedlow level tyrosine phosphorylation of the tyrosine kinaseZAP-70 in the conditions of the assay. However, H4/ICOS clearly synergized with CD3 for ZAP-70 phosphor-ylation. Furthermore, H4/ICOS and CD3 coligationinduced enhanced ´ chain and Vav tyrosine phosphory-lation (Fig. 2C). A low level of tyrosine phosphorylation ofVav could be observed by H4/ICOS cross-linking alone(Fig. 2C), a phenomenon previously described for CD28[29, 30]. Similar results were obtained in other Th2 clonesanalyzed (data not shown).

Since CD4 co-localizes with H4/ICOS upon cross-linking[7, 8], it is involved in early TCR/CD3 signals in D10 cells[31–33], and mutual modification of CD4- and CD28-mediated signals have been described [34–36], the roleof CD4 in H4/ICOS costimulation was also analyzed.Fig. 2 shows that CD3-CD4 coligation enhanced CD3signals, inducing strong tyrosine phosphorylation ofZAP-70 (Fig. 2B) as well as ERK, p38 and JNK activationin these cells (Fig. 2A). Using D10 cells expressing or notCD4, we observed that H4/ICOS costimulation of ERKactivation is largely independent of CD4 expression,whereas JNK activation is clearly stronger in cellsexpressing CD4 (Fig. 3A). Intriguingly, costimulation ofp38 activation is stronger in cells lacking CD4 (Fig. 3A).

The role of CD28 in H4/ICOS costimulation was alsoanalyzed in CD4+ T cells from CD28-deficient mice.Since resting T lymphocytes express negligible levels ofH4/ICOS, CD4+ T cell blasts were used. H4/ICOSexpression in cells from CD28–/– mice was about onefourth of that of cells from congenic normal mice(Fig. 3B), confirming previous data showing CD28dependence of optimal H4/ICOS expression [18, 37, 38].This difference was not observed in other cell surfacemarkers, including CD3 (Fig. 3B) or CD4 (data notshown). Despite the low-level H4/ICOS expression, ZAP-70 phosphorylation was enhanced by CD3 and H4/ICOSco-ligation in these CD28-deficient cells (Fig. 3C),although H4/ICOS-costimulation of ZAP-70 phosphory-

206 M. J. Feito et al. Eur. J. Immunol. 2003. 33: 204–214

Fig. 3. Role of CD4 or CD28 expression onH4/ICOS costimulation. (A) Cells expressing (CD4.wt) or not (CD4–) were activated asdescribed in Fig. 2. Cell lysates were analyzed by immunoblot to detect active forms of ERK, JNK, or p38, then stripped and re-probed for protein loading with kinase-specific antibodies. (B) Surface expression of CD3, CD28, and H4/ICOS in CD4+ T cellblasts from CD28-deficient (CD28–/–) or normal (CD28+/+) C57BL.6 mice, as determined by flow cytometry. (C) Effect of H4/ICOScostimulation on ZAP-70 tyrosine phosphorylation and MAP kinase activation determined by immunoblot of ZAP-70 immunopre-cipitates or cell lysates from CD28+/+ or CD28–/– CD4+ T cell blasts, as described in Fig. 2.

lation was stronger in cells from CD28+/+ mice (Fig. 3C).These differences in costimulation extended to MAPkinase activation, and were particularly clear in the caseof ERK (Fig. 3C). Whether these differences are due tothe differences in ICOS expression between CD28+/+ andCD28–/– cells, or there is some degree of functionaldependence of ICOS costimulation on CD28 expression,needs to be further ascertained.

2.3 PI-3K dependence of H4/ICOS-mediatedcostimulus

The conservation of a PI-3K association motif and theassociation of this kinase to H4/ICOS suggest a primerole of this kinase in the biological effects of H4/ICOS.Pre-treatment of SR.D10 cells with the PI-3K inhibitorsLY294002 (LY, 20 ? M) or Wortmannin (0.1 ? M) clearlyinhibited ERK-1/2 and JNK activation, but not p38 acti-vation induced by combined anti-CD3 plus anti-H4/ICOS antibodies (Fig. 4A). However, some differenceswere noted, as Wortmannin blocked both ERK and JNKactivation (i.e. n 90% inhibition, Fig. 4), whereas LY-induced inhibition was partial under the same conditions(Fig. 4A, top).

LY294002 or Wortmannin did not inhibit tyrosine phos-phorylation of ZAP-70 induced by anti-CD3 plus anti-H4/ICOS, indicating that optimal ZAP-70 recruitment andactivation is independent on PI-3K activity under theseconditions. Both LY294002 and Wortmannin stronglyinhibited IL-4 or IL-10 secretion in SR.D10 cells activatedby anti-CD3 plus anti-H4/ICOS (Fig. 4C). This suggeststhat p38 activation is not relevant to IL-4 or IL-10 secre-tion by these cells. On the other hand, the data show thatLY294002 is as effective as Wortmannin in inhibiting lym-phokine secretion (Fig. 4C), whereas Wortmannin inhibitsERK or JNK activation more efficiently (Fig. 4A). On theother hand, the ERK inhibitor U0126 (U0, 5 ? M) or theJNK inhibitor L-JNKI1 (JNKI, 20 ? M) partially inhibited IL-4 and IL-10 secretion, whereas SB203580, a p38 inhibi-tor (SB, 2 ? M) did not produce significant effects (Fig. 5).Taken together, these data suggests that PI-3K isinvolved in ERK and JNK activation as well as in addi-tional pathways relevant to lymphokine secretion.

2.4 Role of actin cytoskeleton, PKC andcalcineurin in H4/ICOS costimulation

Further experiments were performed to analyze theimportance of actin cytoskeleton, calcineurin, and pro-

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Fig. 4. Effect of PI-3K inhibitors on H4/ICOS costimulation.(A) Lysates of SR.D10 cells activated as in Fig. 2 with orwithout the PI-3K inhibitors LY 294002 (LY, 20 ? M) or Wort-mannin (Wort, 0.1 ? M) were probed by immunoblot usingantibodies specific for active forms of the ERK, JNK and p38kinases. These blots were stripped and re-probed with anti-bodies specific for each kinase, as indicated. (B) Blots ofZAP-70 precipitates of the same cell lysates were probedwith anti-phosphotyrosine antibodies (pTyr), stripped, andblotted with anti-ZAP-70 antibodies. (C) IL-4 and IL-10secretion by cells activated for 5 h in the presence of differ-ent drugs, as determined by capture ELISA in culture super-natants. Data represent the mean ±SE of triplicate cultures.Significant differences (p X 0.05) between samples with orwithout drug are indicated by asterisks.

Fig. 5. Effect of the ERK inhibitor U0126 (U0, 1 ? M), the JNKinhibitor L-JNKI1 (JNKI, 20 ? M), or the p38 inhibitorSB203580 (SB, 2 ? M) on IL-4 and IL-10 secretion by SR.D10cells. Activation as described in Fig. 4C.

tein kinase C on H4/ICOS-mediated costimulation ofTCR/CD3 signals in D10 cells (Fig. 6). Activation of ERKand ZAP-70 tyrosine phosphorylation were stronglyinhibited by cytochalasin D, an inhibitor of actin polymer-ization. Cytochalasin D also blocked the appearance ofthe activated 46-kDa form of JNK, possibly the lowmolecular mass product of JNK1, [39, 40]. Intriguingly, astrong phosphorylated form of 48 kDa appeared. Thismight correspond to the low molecular mass form ofJNK2, whose dual phosphorylation was occasionallydetectable as a faint band upon activation of untreatedcells (Fig. 6A). The functional meaning of the phosphory-lation of this JNK isoform is obscure, as it did not induceactivation of lymphokine secretion, as shown below.Cytochalasin D also induced a strong inhibition of ZAP-70 phosphorylation (Fig. 6B), and blocked IL-4 and IL-10secretion (Fig. 6C). Taken together, these results showthat actin polymerization is required for H4/ICOS co-stimulation, acting upstream of ZAP-70 phosphorylation.

Neither the calcineurin phosphatase inhibitor cyclospo-rin A, nor the PKC inhibitors RO-31-8220 (RO) and GF109203X (GF) produced detectable changes in ZAP-70

phosphorylation or in ERK and JNK activation (Fig. 6A,B). On the other hand, cyclosporin A blocked IL-4 secre-tion, and it did not produce significant changes of IL-10secretion. As shown in Fig. 6C, the PKC inhibitors ROand GF produced no significant effect on IL-4 or IL-10secretion in this experimental system.

3 Discussion

H4/ICOS is an activation-induced molecule homologousto the T cell regulatory proteins CD28 and CTLA-4 [4–6].Its ligand (Bh7) is also homologous to the CD28/CTLA-4ligands B7.1 and B7.2 [5, 11, 12]. Although its function isnot fully understood, experiments performed using H4/ICOS-specific antibodies show a costimulatory effect ofH4/ICOS ligation, enhancing T cell proliferation and alter-ing the pattern of lymphokine secretion ([4, 5, 7],reviewed in [10]). Particularly, H4/ICOS might be impor-tant for IL-4- and IL-10-mediated responses includingthe homeostasis of inflammatory reactions, the balanceof Th1/Th2 differentiation, or B lymphocyte differentia-tion to plasma cells [13, 17–21].

CD28 and H4/ICOS share a Y-M-x-M sequence motif intheir cytoplasmic domain, which, in CD28, can efficientlybind PI-3K upon phosphorylation of the tyrosine residue.Unlike one recent report showing that PI-3K associationto ICOS is largely independent on activation [13], wehave observed that it is strongly enhanced by cell activa-tion (Fig. 1).

208 M. J. Feito et al. Eur. J. Immunol. 2003. 33: 204–214

Fig. 6. Effect of the actin polymerization inhibitor cytochala-sin D (CytD, 10 ? M), the calcineurin inhibitor cyclosporin A(CyA, 1 ? M), or the PKC inhibitors RO-31–8220 (RO, 0.5 ? M)and GF109203X (GF, 0.5 ? M) on (A) the activation of the ERKand JNK MAP kinases, as determined by immunoblot oflysates from cells activated as described in Fig. 2, usingantibodies specific for active forms of the kinases, or (B) byimmunoblot of immunoprecipitated ZAP-70 withphosphotyrosine-specific antibodies. (C) IL-4 and IL-10secretion by cells activated for 5 h in the presence of differ-ent stimuli and drugs, as indicated. Conditions, as describedin Fig. 4.

Our results show that H4/ICOS produces a strongenhancement of early TCR/CD3-mediated signals,including enhanced tyrosine phosphorylation of TCRzeta, ZAP-70, and Vav (Fig. 2–4, 6). The mechanismsinvolved in enhanced tyrosine phosphorylation of sub-strates by H4/ICOS are not clear, although similar find-ings have been observed in CD28-mediated costimula-tion [24, 25].

It is intriguing that inhibitors of events thought to occurlater in the activation process, like the inhibitor of actinpolymerization cytochalasin D, can also inhibit events asearly as ZAP-70 phosphorylation. One possible explana-tion is a positive feedback mechanism whereby H4/ICOSfavors TCR-mediated activation of Vav and Rho, produc-ing enhanced TCR/CD3 recruitment and proximity by anactin polymerization-dependent mechanism (reviewed in[41]), perhaps by the reorganization of membrane micro-domains as recently observed in CD28 [27]. CD28 isneeded for efficient induction of H4/ICOS surfaceexpression (Fig. 3B, [18, 37, 38]), and hence H4/ICOSsignaling is at least indirectly dependent on CD28expression (Fig. 3C). The process of reorganization ofmembrane molecules would also help in localizing inclose range key activating enzymes like the tyrosinekinase Lck, as well as ZAP-70 substrates including Vavitself and the Vav-associated scaffolds LAT and SLP-76.The role of these molecules in H4/ICOS-mediated costi-mulation needs to be further ascertained.

In addition, H4/ICOS ligation synergized with TCR/CD3signals for activation of the ERK and p38 MAP kinases,although a low-level activation was detected by separateligation of TCR/CD3 or H4/ICOS in some experiments(Fig. 2, 3, and data not shown). JNK activation wasclearly observed by coligation of TCR/CD3 plus H4/ICOS, but was never detected using either stimulusalone (Fig. 2–4, 6). This phenomenon resembles previousdata on the mechanisms of CD28 costimulation, pointingto JNK as a key molecule in the integration of TCR andCD28 signals for secretion of lymphokines like IL-2 [23,26], although recent data challenges JNK as the soleintegration point of TCR and CD28 [39, 40, 42]. Further-more, integration of TCR and CD28 signals can alsooccur at Vav and ZAP-70, upstream in the activation cas-cade [22, 24, 25, 28]. These data suggest several inte-gration points between TCR and CD28 signals, and pos-sibly between TCR and H4/ICOS, depending on the celland the phenomenon considered.

It has been reported that, in some models, CD28 activa-tion can be only achieved in T cells with abundant freeLck, i.e. lacking CD4 [36]. In addition, the results shownin Fig. 2 suggest that CD4 and H4/ICOS might haveoverlapping costimulatory mechanisms in SR.D10 cells.The analysis of H4/ICOS costimulation in D10 cellsexpressing or not CD4 indicates that CD4 expression isnot an absolute need for H4/ICOS costimulation,although MAP kinase activation can be higher (p38) orlower (JNK) in the CD4– cells (Fig. 3).

In this study, IL-4 and IL-10 where chosen as two lym-phokines showing positive, but distinct, responses toH4/ICOS costimulation [4, 5], and being under different

Eur. J. Immunol. 2003. 33: 204–214 Mechanisms of H4/ICOS costimulation 209

mechanisms of transcription control [43–46]. Our resultssupport the contention that efficient lymphokine secre-tion and H4/ICOS costimulation is partially dependent onERK and JNK activation (Fig. 4, 5). So, ERK and JNKinhibitors partially inhibited IL-4 and IL-10 secretion(Fig. 5). Although p38 activation was enhanced by H4/ICOS costimulation, this was not relevant to IL-4 or IL-10secretion by these Th2 cells (Fig. 2–5). Nevertheless,enhanced p38 activity might be important to the block-ing effect of anti-ICOS reactions in Th1-mediatedimmune reactions [15, 16].

The calcineurin inhibitor cyclosporin A had no effect onZAP-70 or MAP kinase activation (Fig. 6). However, asexpected from the importance of NFAT in IL-4, but not inIL-10 transcription, cyclosporin A blocked IL-4 secretionwhile leaving IL-10 secretion unaffected (Fig. 6), inagreement with previous data [47]. Also in agreementwith previously published data using Th2 cells [47], inhib-itors of protein kinase C barely modified IL-4 or IL-10secretion induced by TCR plus H4/ICOS (Fig. 6). The ele-ments involved in the H4/ICOS-mediated costimulationof IL-10 secretion are obscure, since IL-10 transcriptionis primarily under the control of the ubiquitous Sp1 andSp2 transcription factors [44, 46]. Furthermore, previousdata indicate that basal IL-10 mRNA in unstimulated D10cells is very low [47], making mRNA changes [48] anunlikely IL-10 regulatory mechanism in this system. JNKactivation might exert a positive role on IL-10 secretionthrough Sp1 and Sp2 regulation [49]. In fact, IL-10 secre-tion is inhibited by JNK-specific inhibitors (Fig. 5) or byPI-3K inhibitors that block JNK activation (Fig. 4).

It is know that the IL-4 promoter possesses several AP-1and NF-AT sites [50]. It is not surprising, then, that ERKand JNK inhibitors induce significant inhibition of IL-4secretion (Fig. 5). Interestingly, PI-3K inhibitors arestrong inhibitors of IL-4 and IL-10 secretion. This mightbe due to inhibition of ERK and JNK activation (Fig. 4A).In addition, PI-3K might activate other pathways relevantto lymphokine secretion. For instance, one important tar-get of PI-3K is Akt-1, whose activation is stronglyenhanced by H4/ICOS costimulation [51]. However,recent data indicates that Akt is not involved in the regu-lation of Th2 cytokines [52]. Another possible target isthe activation of PLC- + mediated by the Tec familykinase Itk, as recently observed in CD28 costimulation[53].

There are clear differences between H4/ICOS and CD28.One major difference is their constitutive (CD28) orinducible (H4/ICOS) expression in resting peripheral Tcells, the differences in expression between Th1 and Th2CD4+ T cell clones (Fig. 1, [13, 18]), and the expressionpatterns of their ligands. These differences might influ-

ence the expansion of T cells in different activationstages, the differentiation of T and B cells and/or favorthe secretion of particular sets of lymphokines. In addi-tion, differences in the cytoplasmic Y-M-x-M motif(YMNM in CD28, YMFM in ICOS) or the C-terminalregion of the cytoplasmic domain might affect the asso-ciation of molecules, like Grb-2, involved in certain CD28downstream signaling pathways [13, 30, 54]. Althoughthe molecular mechanisms underlying these differencesneed to be further ascertained, our results using Th2 celllines suggest that they are not relevant to at least somemechanisms of signal integration between TCR/CD3 andthe costimuli, involving ZAP-70 and MAP kinases.

4 Materials and methods

4.1 Cells and plasmids

The SR.D10 subclone of the mouse Th2 clone D10.G4.1 wasused and was maintained in Click’s EHAA medium supple-mented with 10% heat-inactivated FCS (culture medium)containing 5 U/ml mouse IL-2, 10 U/ml mouse IL-4, and 25pg/ml mouse IL-1 § [55]. To obtain the clones CD4–.Neo.2H2,and CD4FL.4F3, the CD4–.F1 mutant of SR.D10 [32] wastransfected by electroporation with pSR § Neo, or with thesame vector containing mouse wild-type CD4 (FL.4F3).These cells were maintained as described for SR.D10 cells.The Th1 clones AE103 [56] and D10.Tcr25 [57] and the Th2clones D10.Th2.3 [57] and AK-8 [58] were grown as previ-ously described [59].

4.2 Antibodies and other reagents

The following monoclonal antibodies were used: YCD3–1(rat IgG2b anti-mouse CD3 [60]); M1/70 (rat IgG2b anti-mouse CD11b [61]); GK1.5 (rat IgG2b anti-mouse CD4 [62]);C398.4A (hamster IgG anti-mouse and human H4/ICOS [7,8]; and 37.51 (hamster IgG anti-mouse CD28 [63]). Theywere used as protein A- or protein G-purified preparations ofhybridoma supernatants. Horseradish peroxidase coupledanti-phosphotyrosine monoclonal antibody PY-20 wasobtained from Amersham Pharmacia Biotech (Little Chal-font, GB). A mouse IgG1 monoclonal antibody specific forthe PI-3K 85 kDa subunit was from Upstate Biotechnology(Upstate, NY, cat. no. 05-212).

Rabbit anti-ZAP-70 antibodies were specific for a fusionprotein of GST and residues 266 and 344 of mouse ZAP-70.Rabbit anti- ´ antibodies were specific for residues 132–144of mouse ´ chains conjugated to Ovalbumin. Anti-ZAP-70and anti- ´ antibodies were purified by affinity chromatogra-phy over the immunizing protein or peptide coupled toSepharose. For immunoprecipitation, purified anti-ZAP-70or anti- ´ antibodies were coupled to CNBr-Sepharose. Rab-bit anti-Active ERK antibodies were from New England Bio-

210 M. J. Feito et al. Eur. J. Immunol. 2003. 33: 204–214

labs, (Beverly, MA). Rabbit anti-active JNK, and anti-activep38 antibodies were from Promega (Madison, WI). Rabbitanti-ERK, anti-JNK1, and anti-p38 were from Santa CruzBiotechnology (Santa Cruz, CA). Rabbit anti-Vav serum(301-S) was a gift of Dr. Xose Bustelo (Salamanca, Spain).FITC-conjugated goat anti-hamster IgG was from SouthernBiotechnology (Birmingham, AL). Horseradish peroxidase-conjugated anti-mouse Ig or anti-rabbit Ig antibodies werefrom Sigma. Normal Armenian hamster IgG was obtainedfrom Armenian hamster sera by protein A-Sepharose affinitychromatography.

Cyclosporin A and LY 294002 were from Sigma ChemicalCo. (St. Louis, MO); cytochalasin D, Wortmannin, SB203580, U0126, GF 109203X (GÖ 6850, bisindolylmalei-mide) and RO-31–8220 were from Calbiochem (San Diego,CA). The JNK-specific inhibitor L-JNKI1 was obtained fromAlexis Biochemicals (San Diego, CA).

4.3 Cell culture and activation

For activation, the antibodies were adsorbed to polystyrenebeads (5- ? m diameter, Polysciences) by overnight incuba-tion at 4°C in PBS (2×107–4×107 beads/ml plus 1 ? g/ml anti-CD3 plus 5 ? g anti-H4/ICOS or the filling antibodies M1/70and normal Armenian Ig, respectively). Cells (108 cells/ml)were resuspended in culture medium and mixed 1:1 withantibody-coated polystyrene beads. After 5 min at 37°C,1 ml of ice-cold PBS, 500 ? M EDTA, 200 ? M sodium ortho-vanadate was added, and the cells were spun for 2 min at1,000×g before lysis. Where indicated, cells (107/ml in cul-ture medium) were incubated with metabolic inhibitors for30 min at 37°C, washed, and then activated as described.For lymphokine determinations, 3–5 ? l of cells plus beadswere taken after the 5 min activation and incubated for fur-ther 5 h at 37°C in 0.2 ml of complete culture medium sup-plemented with metabolic inhibitors were appropriate.

For the experiments described in Fig. 1B, SR.D10 cells(2×107/point) were incubated for 30 min with anti-H4/ICOSor control antibody (10 ? g/ml, 2 ml) in ice-cold PBS plus 5%FCS. To detect surface H4/ICOS, each sample included5×106 cells that had previously been biotinylated by incuba-tion (15 min in the cold) with freshly prepared sulfo-NHS bio-tin (Pierce) in phosphate-buffered saline, pH 8.0 (50 ? g/mlbiotin, 107 cells/ml) and washed. The cells were washed andresuspended in culture medium and kept in the cold. Onesample was activated for 3 min at 37°C with freshly pre-pared 0.5 mM pervanadate. Then, all the samples receivedrabbit anti-mouse Ig, cross-reactive with hamster Ig (50 ? g/ml). After further 5 min, the activation was stopped by add-ing ice-cold PBS, 500 ? M EDTA, 200 ? M sodium orthovana-date, and lysed as described above.

4.4 T cell blasts

CD4+ T cells were isolated from the spleens of CD28–/–

(Jackson, Bar Harbor, ME) or congenic C57BL/6 mice byimmunoglobulin-anti-immunoglobulin columns as described[59, 64]. These cells were activated 106 cells/ml in culturemedium with 2.5 ? g/ml concanavalin A, plus 10 U/ml IL-2and T cell-depleted, mitomycin C-treated spleen cells asfeeders (5×105/ml). After 48 h, the cells were washed andseparated in a discontinuous Percoll gradient. Blasts fromthe 0–50% Percoll interface were taken, washed thoroughly,and expanded for further 48 h at 0.4×106 cells/ml in culturemedium plus 20 U/ml IL-2. The resulting cells were n 98%CD3+ CD4+ cell blasts, as determined by flow cytometry, andused in activation experiments with antibody-coated poly-styrene beads as described above.

4.5 Flow cytometry

Cells were stained by incubation with antibody at 107 cells/ml in staining buffer (PBS, 0.1% NaN3, 2% heat-inactivatedfetal bovine serum). After 30 min at 4°C, the cells werewashed and incubated for further 30 min in the cold withFITC-coupled anti-hamster IgG antibodies in staining buffer.The cells were then washed four times with staining bufferand analyzed on an EPICS-XL flow cytometer (Coulter Elec-tronics).

4.6 Cell lysis and immunoprecipitation

Cells were lysed on ice for 30 min at 2×107 cells/ml with50 mM Tris/HCl, pH 7.6, 10 mM 3-[(3-cholamido propyl)-dimethyl ammonium]-1-propane sulfonate (CHAPS),150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 10 ? g/ml aproti-nin, 10 ? g/ml leupeptin, 1 mM PMSF, and 1 mM NaVO4.Post nuclear lysates were used for immunoblot, or immuno-precipitated as described [59, 64]. For immunoprecipitationof cell surface H4/ICOS, cell lysis and immunoprecipitationwas performed as described above, except that cells werelysed at 107 cells/ml, and that immunoprecipitation was car-ried out using Sepharose-conjugated protein A.

4.7 Immunoblot

For immunoblot of cell lysates, 10 ? l (equivalent to 2×105

cells) were mixed V:V with 2× reducing SDS Laemmli samplebuffer. Immunoprecipitates were extracted with 1× reducingsample buffer. The samples were boiled for 2 min and sepa-rated by SDS-PAGE in 10% acrylamide gels. Proteins weretransferred to PVDF membranes (Immobilon-P, Millipore),and blocked for 2 h with 1% BSA in TBST containing200 ? M sodium orthovanadate. The membranes werewashed with TBST, incubated overnight in the cold with anti-active MAP kinase antibodies, horseradish peroxidase(HRP)-coupled anti-phosphotyrosine antibodies, or HRP-

Eur. J. Immunol. 2003. 33: 204–214 Mechanisms of H4/ICOS costimulation 211

streptavidin in 1% BSA in TBST, and washed with TBST.Then, the blot was visualized using the Supersignal WestPico chemiluminiscence substrate (Pierce) for phosphotyro-sine or biotin determination. For detection of anti-activeMAP kinase antibodies, the membranes were incubated forfurther 2 h at room temperature with HRP-conjugated anti-rabbit IgG in 1% non-fat dry milk in TBST, washed, anddeveloped with chemoluminiscence substrate as above.The membranes were washed, stripped and re-probed withantibodies specific for MAP kinases, ´ , ZAP-70, or Vav. Anti-PI-3K detection in immunoprecipitates or cell lysates wasperformed as described above, except that membraneblocking and incubation with antibodies were performed in1% non-fat dry milk in TBST, and HRP-conjugated anti-mouse Ig was used as second antibody.

4.8 Cytokine detection by ELISA

IL-4 and IL-10 were detected in culture supernatants byspecific capture ELISA. 11B11 was used for capture andbiotinylated BVD6–24G2 (PharMingen) was used for detec-tion of IL-4. JES5–2A5 and biotinylated JES5–16E3 (Phar-Mingen) were used as capture and detection antibodies,respectively, in IL-10 assays. The assays were performedaccording to the instructions of the supplier and developedusing streptavidin-HRP (Amersham) and Sigma-fast o-phenylenediamine substrate (Sigma). Cytokine content wascalculated from reference standard curves set up withrecombinant cytokines.

Acknowledgements: The authors thank Charles A. Jane-way, Jr., for generously providing many cell lines and anti-bodies used in this study, Dr. Xose Bustelo for anti-Vav anti-serum, and Maria Luisa del Pozo for her skillful technicalassistance. This work was supported by Fellowships from“Fondo de Investigacion Sanitaria” (A.S., and G.C.), I.S. Car-los III (A. J.-P), Ministerio de Educacion y Cultura (G.C.), andComunidad Autonoma de Madrid, (A.S. and M.J.F.), andgrants FIS 98/0037-CO2–01 and FIS 98/0037-CO2–02 from“Fondo de Investigacion Sanitaria” and ISCIII-01/30 (Minis-terio de Sanidad y Consumo, Spain), grant BMC2001–2177from Ministerio de Ciencia y Tecnologıa, Spain, and grantsfrom Associazzione Italiana Ricerca Cancro (AIRC, Milan,Italy).

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Correspondence: Jose M. Rojo, Departamento de Inmuno-logıa, Centro de Investigaciones Biologicas, CSIC, Velaz-quez 144, E-28006 Madrid, SpainFax: +34-91-562-7518e-mail: jmrojo — cib.csic.es

G. Criado’s present address: J. P. Robarts Research Insti-tute, London, Ontario, Canada

214 M. J. Feito et al. Eur. J. Immunol. 2003. 33: 204–214