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Caveolin-1 Triggers T-cell Activation via CD26 in Association with CARMA1

2007; Elsevier BV; Volume: 282; Issue: 13 Linguagem: Inglês

10.1074/jbc.m609157200

ISSN

1083-351X

Autores

Kei Ohnuma, Masahiko Uchiyama, Tadanori Yamochi, Kunika Nishibashi, Osamu Hosono, Nozomu Takahashi, Shinichiro Kina, Hirotoshi Tanaka, Xin Lin, Nam H. Dang, Chikao Morimoto,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-κB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-κB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IκB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule. CD26 is a widely distributed 110-kDa cell surface glycoprotein with an important role in T-cell costimulation. We demonstrated previously that CD26 binds to caveolin-1 in antigen-presenting cells, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1 in antigen-presenting cells. IRAK-1 is then subsequently phosphorylated to up-regulate CD86 expression, resulting in subsequent T-cell proliferation. However, it is unclear whether caveolin-1 is a costimulatory ligand for CD26 in T-cells. Using soluble caveolin-1-Fc fusion protein, we now show that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-κB activation in a T-cell receptor/CD3-dependent manner. We also demonstrated that the cytoplasmic tail of CD26 interacts with CARMA1 in T-cells, resulting in signaling events that lead to NF-κB activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IκB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule. CD26 is a 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV, 4The abbreviations used are: DPPIV, dipeptidyl peptidase IV; APC, antigen-presenting cells; TCR, T-cell receptor; MAGUK, membrane-associated guanylate kinase; IKK, IκB kinase; aa, amino acid; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; TdR, thymidine; PKC, protein kinase C; GUK, guanylate kinase; pAb, polyclonal antibody; IP, immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; CHO, Chinese hamster ovary; SH, Src homology; IL, interleukin; huECDSP, signal peptide from human E-cadherin; rs, recombinant soluble; RU, response units; 2ME, 2-mercaptoethanol; GFP, green fluorescent protein; SCD, scaffolding domain; cyto, cytoplasmic region of CD10. EC 3.4.14.5) activity in its extracellular domain (1Nanus D.M. Engelstein D. Gastl G.A. Gluck L. Vidal M.J. Morrison M. Finstad C.L. Bander N.H. Albino A.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7069-7073Crossref PubMed Scopus (110) Google Scholar, 2Tanaka T. Camerini D. Seed B. Torimoto Y. Dang N.H. Kameoka J. Dahlberg H.N. Schlossman S.F. Morimoto C. J. Immunol. 1992; 149: 481-486PubMed Google Scholar, 3Fox D.A. Hussey R.E. Fitzgerald K.A. Acuto O. Poole C. Palley L. Daley J.F. Schlossman S.F. Reinherz E.L. J. Immunol. 1984; 133: 1250-1256PubMed Google Scholar) and is capable of cleaving N-terminal dipeptides with either l-proline or l-alanine at the penultimate position (2Tanaka T. Camerini D. Seed B. Torimoto Y. Dang N.H. Kameoka J. Dahlberg H.N. Schlossman S.F. Morimoto C. J. Immunol. 1992; 149: 481-486PubMed Google Scholar). CD26 activity is dependent on cell type and the microenvironment, factors that can influence its multiple biological roles (reviewed in Refs. 4De Meester I. Korom S. Van Damme J. Scharpe S. Immunol. Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 5Fleischer B. Immunol. Today. 1994; 15: 180-184Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 6Kahne T. Lendeckel U. Wrenger S. Neubert K. Ansorge S. Reinhold D. Int. J. Mol. Med. 1999; 4: 3-15PubMed Google Scholar, 7Morimoto C. Schlossman S.F. Immunol. Rev. 1998; 161: 55-70Crossref PubMed Scopus (366) Google Scholar, 8von Bonin A. Steeg C. Mittrucker H.W. Fleischer B. Immunol. Lett. 1997; 55: 179-182Crossref PubMed Scopus (10) Google Scholar). Although CD26 expression is enhanced following activation of resting T-cells, CD4 + CD26high T-cells respond maximally to recall antigens such as tetanus toxoid (9Eguchi K. Ueki Y. Shimomura C. Otsubo T. Nakao H. Migita K. Kawakami A. Matsunaga M. Tezuka H. Ishikawa N. J. Immunol. 1989; 142: 4233-4240PubMed Google Scholar, 10Morimoto C. Torimoto Y. Levinson G. Rudd C.E. Schrieber M. Dang N.H. Letvin N.L. Schlossman S.F. J. Immunol. 1989; 143: 3430-3439PubMed Google Scholar). Cross-linking of CD26 and CD3 with solid-phase immobilized monoclonal antibodies (mAbs) can induce T-cell costimulation and IL-2 production by CD26 + T-cells (2Tanaka T. Camerini D. Seed B. Torimoto Y. Dang N.H. Kameoka J. Dahlberg H.N. Schlossman S.F. Morimoto C. J. Immunol. 1992; 149: 481-486PubMed Google Scholar, 7Morimoto C. Schlossman S.F. Immunol. Rev. 1998; 161: 55-70Crossref PubMed Scopus (366) Google Scholar, 10Morimoto C. Torimoto Y. Levinson G. Rudd C.E. Schrieber M. Dang N.H. Letvin N.L. Schlossman S.F. J. Immunol. 1989; 143: 3430-3439PubMed Google Scholar). In addition, anti-CD26 antibody treatment of T-cells enhances tyrosine phosphorylation of signaling molecules such as CD3ζ and p56lck (11Dang N.H. Torimoto Y. Deusch K. Schlossman S.F. Morimoto C. J. Immunol. 1990; 144: 4092-4100PubMed Google Scholar, 12Hegen M. Kameoka J. Dong R.P. Schlossman S.F. Morimoto C. Immunology. 1997; 90: 257-264Crossref PubMed Scopus (75) Google Scholar). Moreover, DPPIV activity is required for CD26-mediated T-cell costimulation (13Tanaka T. Kameoka J. Yaron A. Schlossman S.F. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4586-4590Crossref PubMed Scopus (191) Google Scholar). CD26 may therefore have an important role in T-cell biology and overall immune function. However, the costimulatory ligand of CD26 has not yet been identified, and the proximal signaling events following CD26 engagement in T-cell remain to be determined. In our previous study (14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar), we identified caveolin-1 in antigen-presenting cells (APC) as a binding protein for CD26, and we demonstrated that CD26 on activated memory T-cells directly faces caveolin-1 on tetanus toxoid-loaded monocytes in the contact area, which was revealed as the immunological synapse for T-cell-APC interaction. Moreover, we showed that residues 201-211 of CD26 along with the serine catalytic site at residue 630, which constitute a pocket structure of CD26/DP-PIV, contribute to binding to caveolin-1 scaffolding domain (14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar). More recently, we demonstrated that caveolin-1 binds to Tollip (Toll-interacting protein) and IRAK-1 (interleukin-1 receptor associated serine/threonine kinase 1) in the membrane of tetanus toxoid-loaded monocytes, and following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1, with IRAK-1 being subsequently phosphorylated to up-regulate CD86 expression (15Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Iwata S. Hosono O. Kawasaki H. Tanaka H. Dang N.H. Morimoto C. Mol. Cell. Biol. 2005; 25: 7743-7757Crossref PubMed Scopus (71) Google Scholar). It is conceivable that the interaction of CD26 with caveolin-1 on antigen-loaded monocytes results in CD86 up-regulation, therefore enhancing the subsequent interaction of CD86 and CD28 on T-cells to induce antigen-specific T-cell proliferation and activation. However, it is unclear whether caveolin-1 itself is the costimulatory ligand for T-cell CD26. Recent studies have demonstrated that a newly identified membrane-associated guanylate kinase-like (MAGUK) molecule, CARMA1, is required for TCR/CD3-CD28 costimulation-induced NF-κB activation and functions downstream of protein kinase Cθ (PKCθ) (16Egawa T. Albrecht B. Favier B. Sunshine M.J. Mirchandani K. O'Brien W. Thome M. Littman D.R. Curr. Biol. 2003; 13: 1252-1258Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Hara H. Wada T. Bakal C. Kozieradzki I. Suzuki S. Suzuki N. Nghiem M. Griffiths E.K. Krawczyk C. Bauer B. D'Acquisto F. Ghosh S. Yeh W.C. Baier G. Rottapel R. Penninger J.M. Immunity. 2003; 18: 763-775Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 18Wang D. You Y. Case S.M. McAllister-Lucas L.M. Wang L. DiStefano P.S. Nunez G. Bertin J. Lin X. Nat. Immun. 2002; 3: 830-835Crossref Scopus (253) Google Scholar). CARMA1, which is predominantly expressed in thymus, spleen, and peripheral blood leukocytes, contains an N-terminal caspase-recruitment domain followed by a coiled-coil domain, a PDZ domain, an SH3 domain, and a guanylate kinase (GUK)-like domain (19Bertin J. Wang L. Guo Y. Jacobson M.D. Poyet J.L. Srinivasula S.M. Merriam S. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 276: 11877-11882Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 20Gaide O. Martinon F. Micheau O. Bonnet D. Thome M. Tschopp J. FEBS Lett. 2001; 496: 121-127Crossref PubMed Scopus (169) Google Scholar). After TCR/CD3-CD28 costimulation or PMA-CD28 stimulation, CARMA1 is phosphorylated by PKCθ, followed by association with Bcl10 and MALT1, and recruitment of these complexes into lipid rafts (21Che T. You Y. Wang D. Tanner M.J. Dixit V.M. Lin X. J. Biol. Chem. 2004; 279: 15870-15876Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 22Gaide O. Favier B. Legler D.F. Bonnet D. Brissoni B. Valitutti S. Bron C. Tschopp J. Thome M. Nat. Immun. 2002; 3: 836-843Crossref Scopus (292) Google Scholar, 23Hara H. Bakal C. Wada T. Bouchard D. Rottapel R. Saito T. Penninger J.M. J. Exp. Med. 2004; 200: 1167-1177Crossref PubMed Scopus (77) Google Scholar). The recruitment of the CARMA1-Bcl10-MALT1 complex activates IκB kinase (IKK) through a ubiquitin-dependent pathway, leading to activation of NF-κB (24Sun L. Deng L. Ea C.K. Xia Z.P. Chen Z.J. Mol. Cell. 2004; 14: 289-301Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 25Wang D. Matsumoto R. You Y. Che T. Lin X.Y. Gaffen S.L. Lin X. Mol. Cell. Biol. 2004; 24: 164-171Crossref PubMed Scopus (179) Google Scholar, 26Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2004; 427: 167-171Crossref PubMed Scopus (451) Google Scholar, 27Wegener E. Oeckinghaus A. Papadopoulou N. Lavitas L. Schmidt-Supprian M. Ferch U. Mak T.W. Ruland J. Heissmeyer V. Krappmann D. Mol. Cell. 2006; 23: 13-23Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, it remains to be determined whether CARMA1 is associated with lipid rafts directly or is recruited to lipid rafts via undetermined lipid raft-interacting proteins in the immunological synapse of T-cells. In this study, using recombinant immunoglobulin-caveolin-1 fusion proteins, we identify caveolin-1 as the costimulatory ligand for CD26, and we demonstrate that the N-terminal domain of caveolin-1 induces T-cell proliferation and cytokine production via CD26 costimulation. Furthermore, we show that CARMA1 is bound to the cytoplasmic tail of dimeric CD26 on T-cells and that this interaction of CD26 and CARMA1 plays a pivotal role in CD26-mediated T-cell costimulation. Our data hence identify a previously unknown ligand for CD26 as a costimulatory molecule while elucidating the mechanisms involved in CD26-mediated T-cell activation and differentiation. Expression and Purification of Fc Proteins—For initial attempts at expression of soluble forms of Fc fusion proteins, human IgG1 Fc cassette vector was made using pCAG-EB6-MCS vector (28Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4597) Google Scholar, 29Tanaka J. Miwa Y. Miyoshi K. Ueno A. Inoue H. Biochem. Biophys. Res. Commun. 1999; 264: 938-943Crossref PubMed Scopus (48) Google Scholar). The 3′ portion of this cassette vector corresponding to human Ig Cγ1 (Fcγ1) sequencing (comprising hinge + CH2 + CH3 regions) was made by PCR. All the primer information used in this study is described in the Supplemental Material. The 5′ portion of the cassette vector containing the signal peptide from human E-cadherin (huECDSP) was made by PCR. Final constructs were assembled by ligating both fragments of HindIII-Fcγ1-EcoRI and SalI-huECDSP-HindIII into SalI/EcoRI-cleaved pCAG-EB6-MCS (pCAG-EB6-huECDSP-Fcγ1). The N-terminal domain of human caveolin-1 (CavNT) was made by PCR and constructed into pCAG-EB6-huECDSP-Fcγ1 (pCAG-EB6-huECDSP-CavNT-Fcγ1). With the same methods, the N-terminal domain with deletion of the scaffolding domain (CavNTΔSCD) was made by PCR (pCAG-EB6-huECDSP-CavNTΔSCD-Fcγ1). The Fc fusion protein containing amino acids 1-10 of human CD26 cytoplasmic tail (CD26 aa1-10) was constructed in identical fashion, using the primers described in the Supplemental Material (pCAG-EB6-huECDSP-CD26 aa1-10-Fcγ1). For expression of Fc fusion proteins, FreeStyle™ 293 expression system was used according to the manufacturer's instruction (Invitrogen). The Fc fusion proteins expressed in the culture supernatant were then purified by affinity chromatography on protein A-Sepharose (Bio-Rad) followed by size-exclusion purification on Microcon® centrifugal filter devices (Millipore), and sterilized using inner diameter 0.22-μm filter microcentrifugation tube Spin-X (Corning Glass). Cells and Reagents—HEK293FT human embryonal kidney, Jurkat T-cell line (JKTwt), and Jurkat T-cells stably transfected with human CD26 (J.CD26wt) were grown as described previously (2Tanaka T. Camerini D. Seed B. Torimoto Y. Dang N.H. Kameoka J. Dahlberg H.N. Schlossman S.F. Morimoto C. J. Immunol. 1992; 149: 481-486PubMed Google Scholar, 13Tanaka T. Kameoka J. Yaron A. Schlossman S.F. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4586-4590Crossref PubMed Scopus (191) Google Scholar, 14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar). CARMA1-deficient Jurkat T-cell line, JPM50.6, was developed as described elsewhere (18Wang D. You Y. Case S.M. McAllister-Lucas L.M. Wang L. DiStefano P.S. Nunez G. Bertin J. Lin X. Nat. Immun. 2002; 3: 830-835Crossref Scopus (253) Google Scholar). Human peripheral blood T-cells were purified from peripheral blood mononuclear cells using MACS Pan T-cell isolation kit II (Miltenyi), collected from healthy adult volunteers and incubated according to the methods described previously (30Ohnuma K. Munakata Y. Ishii T. Iwata S. Kobayashi S. Hosono O. Kawasaki H. Dang N.H. Morimoto C. J. Immunol. 2001; 167: 6745-6755Crossref PubMed Scopus (63) Google Scholar). Informed consent was obtained from healthy adult volunteers. Biotinylation of recombinant proteins or antibody was generated using EZ-Link™ Sulfo-NHS-LC-Biotin reagents according to the manufacturer's instruction (Pierce). Protease inhibitor mixture, phosphatase inhibitor mixture, and poly-l-lysine were from Sigma. Water-soluble digitonin was purchased from Wako Pure Chemicals Industries, Ltd. Biacore™ Analysis of Affinity of Caveolin-1-CD26 Interaction—Experiments were carried out on a Biacore™ J (Biacore, Japan) using HBS buffer (25 mm HEPES (pH 7.4), 150 mm NaCl, 3.4 mm EDTA, 0.005% surfactant P20) supplied by the manufacturer (Biacore AB). Fcγ1, NT-Fc, or NTΔSCD-Fc was coupled in 10 mm sodium acetate (pH 5.0) to a research grade CM5 sensor chip (Biacore AB) using the amine coupling kit (Biacore AB), with an activating time of 5 min, resulting in immobilization of ∼5,000-6,000 response units (RU). The surface of the chip was washed with 5 mm NaOH after coupling. NaOH (5 mm) was used also to regenerate immobilized Fcγ1, NT-Fc, or NTΔSCD-Fc chips after each experiment. Recombinant soluble CD26 (rsCD26), comprising the extracellular region of human CD26, was prepared as described previously (30Ohnuma K. Munakata Y. Ishii T. Iwata S. Kobayashi S. Hosono O. Kawasaki H. Dang N.H. Morimoto C. J. Immunol. 2001; 167: 6745-6755Crossref PubMed Scopus (63) Google Scholar, 31Tanaka T. Duke-Cohan J.S. Kameoka J. Yaron A. Lee I. Schlossman S.F. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3082-3086Crossref PubMed Scopus (129) Google Scholar). rsCD26 at various concentrations (50, 25, 12.5, 6.3, 3.2, and 1.6 nm) was then injected for 120 s over immobilized Fcγ1, NT-Fc, or NTΔSCD-Fc chips. Equilibrium binding analysis was performed as described elsewhere (32Nath D. van der Merwe P.A. Kelm S. Bradfield P. Crocker P.R. J. Biol. Chem. 1995; 270: 26184-26191Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), using the BIAevaluation software version 2.1 (Biacore AB). Generating Stable Transfectants—The construct of V5-tagged full-length human CD26 (pEF6/V5-CD26wt) was made by PCR, using the primers described in the Supplemental Material. The amplified products were cloned into the pEF6/V5-His B vector (Invitrogen) at the BamHI/EcoRI site. The CD26-CD10 chimeric receptor was composed of the N-terminal cytoplasmic region of human CD10 (1-23-amino acid position) ligated to the transmembrane and extracellular regions of human CD26 (7-766-amino acid position), which were made by PCR. The construct of V5-tagged monomeric human CD26 (CD26H750E), which has histidine replacing glutamic acid as a point mutation at amino acid position 750, was made by site-directed mutagenesis method using pEF6/V5-CD26w as a template with the primers described in the Supplemental Material. After constructs were confirmed by DNA sequencing, plasmids were transfected to Jurkat T-cells using Nucleofector II device according to the manufacturer's instruction (Amaxa Biosystems). Two days after transfection of indicated plasmids, the cells were selected for blasticidin (1 μg/ml) resistance for 4 weeks. Single clone cells expressing CD26wt (V5-CD26wt), CD26-CD10 (V5-CD26 + CD10 cyto), and CD26H750E were then selected using standard limiting dilution method. For rescue experiments, the CARMA1-deficient Jurkat cell line JPM50.6 was transfected with expression vectors of CD26 and/or CARMA1. The constructs of Xpress-tagged CARMA1 and its deletion mutant (CARMA1wt, CARMA1-(1-742), or CARMA1-(1-660), respectively) were made by PCR, using primers described in the Supplemental Material. The PCR products were ligated into pcDNA4/HisMax-TOPO (Invitrogen). After constructs were confirmed by DNA sequencing, plasmids were transfected to JPM50.6 cells using the Nucleo-fector II device according to the manufacturer's instruction. Two days after transfection of the indicated plasmids, the cells were selected for blasticidin (1 μg/ml, for cells transfected with pEF6/V5 vectors) or Zeocin (10 μg/ml, for cells transfected with pcDNA4/HisMax vectors) resistance for 4 weeks. Single clone cells expressing CD26wt (JPM50.6/CD26wt), CARMA1wt (JPM50.6/CARMA1wt), CD26wt and CARMA1wt (JPM50.6/CD26wt + CARMA1wt), or CD26wt and CARMA1-(1-660) (JPM50.6/CD26wt + CARMA1-(1-660)) were then selected using standard limiting dilution method. For stimulation experiments using the expression system, CHO-K1 cells were transfected with GFP-fused full-length caveolin-1 or SCD-deleted caveolin-1 expression plasmids, with the constructs being described previously (14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar), using Lipo-fectamine2000 reagent (Invitrogen). Two days after transfection of the indicated plasmids, the cells were selected for G418 (500 μg/ml) resistance for 4 weeks. Single clone cells expressing GFP and caveolin-1, detected by anti-caveolin-1 pAb (N20) recognizing the N-terminal region of caveolin-1 using flow cytometry (FACSCalibur™), were then selected using standard limiting dilution method. Flow Cytometric Analysis—For assessment of J.CD26wt that binds biotinylated NT-Fc or NTΔSCD-Fc, 1 × 106 cells were washed in ice-cold phosphate-buffered saline and incubated with Fcγ1 and mouse Ig isotypes (1 μg/ml) to block nonspecific binding, followed by reaction with biotinylated NT-Fc or NTΔSCD-Fc (1 μg/ml), and subsequently stained with FITC-conjugated streptavidin (1:500). For blocking experiments, unlabeled mouse IgG (20 μg/ml) or unlabeled anti-CD26 mAb (20 μg/ml) was incubated with cells prior to reaction with bio-tinylated NT-Fc or NTΔSCD-Fc. Flow cytometric analysis of 10,000 viable cells was conducted on FACSCalibur™. Each experiment was repeated at least three times, and the results were provided in the form of a histogram or dot plots of a representative experiment. Small Interfering RNA (siRNA) against Human CARMA1—We selected two target sequences from nucleotides +305 to +325 (ss1) and +792 to + 802 (ss2) downstream of the start codon of human CARMA1 mRNA (sense1 siRNA (ss1-siRNA), 5′ AAGAGCCCACUCGGAGAUUCUdTdT, and sense2 siRNA (ss2-siRNA), 5′ AACUGGAGCGGGAGAAUGAAAdTdT). Moreover, mis-siRNA at four nucleotides was prepared to examine nonspecific effects of siRNA duplexes (mis-siRNA, 5′ UAGUGGCCACACGGUGATTCdTdT). These selected sequences also were submitted to a BLAST search against the human genome sequence to ensure that only one gene of the human genome was targeted. siRNAs were purchased from Qiagen. Transfection of siRNA into purified T-cells were conducted using HVJ-E vector (GenomeONE™; kindly provided by Ihsihara Sangyo Kaisha Ltd.) as described previously (14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar). After 48 h of transfection, cell were prepared for examination. T-cell Proliferation and IL-2 Production Assay—For T-cell proliferation assay, 1 × 105 purified T-cells were cultured in 96-well flat-bottomed plates (COSTAR) in a volume of 200 μl of AIM-V medium (Invitrogen). For solid-phase stimulation, anti-CD3 (OKT3, 0.05 μg/ml) and/or anti-CD26 mAb (5 μg/ml), anti-CD28 mAb (4B10, 5 μg/ml), or Fc fusion proteins (5 μg/ml) were bound on the plates. For stimulation with caveolin-1-transfected CHO cells, purified T-cells were cultured in the presence of soluble anti-CD3 (OKT3, 0.05 μg/ml) at 1 × 105 cells/well with varying amounts (T-cells: CHO = 800, 400, 200, 100, 50, 25:1 or no CHO cells as background control) of CHO cell transfectants. Before coculturing with T-cells, CHO transfectants were fixed with 0.05% glutaraldehyde for 30 s at room temperature, followed by washing three times with phosphate-buffered saline. T-cell proliferation was measured by [3H]TdR (ICN Radiochemicals) uptake. Cells were incubated for 96 h and were pulsed with 1 μCi/well of [3H]TdR, 16 h prior to harvesting onto a glass fiber filter (Wallac), and the incorporated radioactivity was quantified by a liquid scintillation counter (Wallac). For blocking experiments, cells were treated with soluble anti-CD26 mAb (1F7), anti-CD28 (4B10), or control mouse Ig (each at 20 μg/ml) before being cultured in plates coated with stimulatory antibodies and/or Fc proteins. For IL-2 production assay using Jurkat T-cell lines, JPM50.6, or their transfectants, 5 × 105 cells/well in 200 μl of culture media were incubated at 37 °C in the presence of the indicated plate-bound antibodies and/or NT-Fc proteins. Cells were also stimulated with PMA (10 ng/ml) in anti-CD3-coated wells. After 48 h of incubation, culture supernatants were pooled from the triplicate wells and assayed for IL-2 content using Human IL-2 Biotrack Easy ELISA (Amersham Biosciences) according to the manufacturer's instruction. Two-dimensional PAGE—For two-dimensional PAGE analysis of cytosolic proteins, Jurkat cells were lysed in TBSD buffer (50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 2 mm EDTA, 0.1% digitonin, 102-fold diluted protease inhibitor mixture, 102-fold diluted phosphatase inhibitor mixture), and then an aliquot (50 μg) of lysates was subjected to two-dimensional PAGE. For pulldown by CD26 aa1-10-Fc, aliquots (1 mg) of lysates were precleared by human IgG (2 μg) and protein A-Sepharose, followed by immunoprecipitation with Fcγ1 (1 μg) or CD26 aa1-10-Fc (1 μg). Total lysates or IPs were boiled at 95 °C for 3 min, and supernatants were then resuspended in rehydration lysis buffer (RHB; 8 m urea, 2 m thiourea, 4% CHAPS, 50 mm dithiothreitol, 0.5% ZOOM carrier ampholyte (pH range 3-10) (Invitrogen), 0.002% bromphenol blue). Two-dimensional PAGE and peptide mass mapping were conducted as described previously (15Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Iwata S. Hosono O. Kawasaki H. Tanaka H. Dang N.H. Morimoto C. Mol. Cell. Biol. 2005; 25: 7743-7757Crossref PubMed Scopus (71) Google Scholar). Preparation of Lysates or Lipid Raft Fractionation, Immunoprecipitation, and Western Blotting—Stimulated or unstimulated cells were pelleted and lysed with TBSD buffer (50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 2 mm EDTA, 0.1% digitonin, 102-fold diluted protease inhibitor mixture (Sigma), 102-fold diluted phosphatase inhibitor mixture (Sigma)) and subjected to immunoprecipitation, followed by SDS-PAGE and Western blot analysis. To obtain the lipid raft fraction, purified T-cells (1 × 108) that were stimulated for 10 min with anti-CD3 alone or with anti-CD3 plus NT-Fc were lysed with 1 ml of 1% Triton X-100 and protease inhibitor mixture in ice-cold MNE buffer (25 mm MES (pH 6.5) (Sigma), 150 mm NaCl, 5 mm EDTA), and then fractionated by sucrose gradient centrifugation as described previously (33Ishii T. Ohnuma K. Murakami A. Takasawa N. Kobayashi S. Dang N.H. Schlossman S.F. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12138-12143Crossref PubMed Scopus (145) Google Scholar). For immunoprecipitation of the pooled lipid raft fraction, fractionated lipid rafts were lysed at 4 °C for 30 min with 1% N-octyl-β-d-glucoside (Nakalai Tesque) and subjected to immunoprecipitation experiment, followed by SDS-PAGE and Western blot analysis. Immunoprecipitation and Western blot analysis were conducted as described previously (14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar, 15Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Iwata S. Hosono O. Kawasaki H. Tanaka H. Dang N.H. Morimoto C. Mol. Cell. Biol. 2005; 25: 7743-7757Crossref PubMed Scopus (71) Google Scholar, 33Ishii T. Ohnuma K. Murakami A. Takasawa N. Kobayashi S. Dang N.H. Schlossman S.F. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12138-12143Crossref PubMed Scopus (145) Google Scholar). Nuclear Protein Extraction and DNA-binding Protein Assay—Nuclear extracts were prepared from Jurkat cells or transfectants stimulated as indicated, and ELISA-based DNA-binding protein assays for NF-κB p65 were performed using Mercury TransFactor kits (BD Biosciences) as described previously (14Ohnuma K. Yamochi T. Uchiyama M. Nishibashi K. Yoshikawa N. Shimizu N. Iwata S. Tanaka H. Dang N.H. Morimoto C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14186-14191Crossref PubMed Scopus (104) Google Scholar). Statistics—Student's t test was used to determine whether the difference between control and sample was significant (p < 0.05 being significant). We prepared soluble caveolin-1 protein consisting of the putative extracellular N-terminal region or the N-terminal region minus the SCD of human caveolin-1, fused with human IgG1 Fc (NT-Fc or NTΔSCD-Fc, respectively). The schematic diagrams of the full-length human caveolin-1 protein, NT-Fc, NTΔSCD-Fc and Fcγ1 are shown in Fig. 1A. As shown in Fig. 1B, where a band of the recombinant Fc portion of human IgG1 (Fcγ1) was observed at ∼35 kDa under reducing conditions (lane 2), the NT-Fc and NTΔSCD-Fc proteins migrated under reducing conditions predominantly as single bands of 50 and 48 kDa, respectively (lanes 3 and 4). Because immuno-globulins are glycosylated post-translationally, the recombinant Fc fusion proteins produced with mammalian cells had higher a molecular weight in SDS-PAGE than as calculated from their amino acid composition (34Ciccimarra F. Rosen F.S. Schneeberger E. Merler E. J. Clin. Investig. 1976; 57: 1386-1390Crossref PubMed Scopus (31) Google Scholar). In nonreducing conditions, Fcγ1, NT-Fc, or NTΔSCD-Fc were observed at ∼60, 100, or 90 kDa, respectively (lanes 6-8 in Fig. 1B), indicating that they were expressed as a homodimer. Fcγ1, NT-Fc, and NTΔSCD-Fc were also evaluated by Western blot analysis using anti-human IgG antibody (Fig. 1C). We examined whether the generated NT-Fc fusion protein binds to CD26. For this purpose, we used the Jurkat T-cell line that was stably transfected with full-length human CD26 (J.CD26wt) as described under "Experimental Proc

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