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J. Biol. Chem., Vol. 282, Issue 13, 10117-10131, March 30, 2007

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

Kei Ohnuma¹², Masahiko Uchiyama¹, Tadanori Yamochi, Kunika Nishibashi, Osamu Hosono, Nozomu Takahashi, Shinichiro Kina, Hirotoshi Tanaka, Xin Lin, Nam H. Dang^¶, and Chikao Morimoto³

From the Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, the Department of Molecular and Cellular Oncology, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030, and the ^¶Department of Hematologic Malignancies, Nevada Cancer Institute, Las Vegas, Nevada 89135

Received for publication, September 27, 2006 Accepted for publication January 29, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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-B 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-B activation. Ligation of CD26 by caveolin-1 recruits a complex consisting of CD26, CARMA1, Bcl10, and IB kinase to lipid rafts. Taken together, our findings provide novel insights into the regulation of T-cell costimulation via the CD26 molecule.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CD26 is a 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV,⁴ EC 3.4.14.5 [EC] ) activity in its extracellular domain (1-3) and is capable of cleaving N-terminal dipeptides with either L-proline or L-alanine at the penultimate position (2). CD26 activity is dependent on cell type and the microenvironment, factors that can influence its multiple biological roles (reviewed in Refs. 4-8). Although CD26 expression is enhanced following activation of resting T-cells, CD4 + CD26^high T-cells respond maximally to recall antigens such as tetanus toxoid (9, 10). 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 (2, 7, 10). In addition, anti-CD26 antibody treatment of T-cells enhances tyrosine phosphorylation of signaling molecules such as CD3 and p56^lck (11, 12). Moreover, DPPIV activity is required for CD26-mediated T-cell costimulation (13). 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 (14), 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 (14). 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 (15). 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-B activation and functions downstream of protein kinase C (PKC) (16-18). 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 (19, 20). After TCR/CD3-CD28 costimulation or PMA-CD28 stimulation, CARMA1 is phosphorylated by PKC, followed by association with Bcl10 and MALT1, and recruitment of these complexes into lipid rafts (21-23). The recruitment of the CARMA1-Bcl10-MALT1 complex activates IB kinase (IKK) through a ubiquitin-dependent pathway, leading to activation of NF-B (24-27). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (28, 29). The 3' portion of this cassette vector corresponding to human Ig C1 (Fc1) 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-Fc1-EcoRI and SalI-huECDSP-HindIII into SalI/EcoRI-cleaved pCAG-EB6-MCS (pCAG-EB6-huECDSP-Fc1). The N-terminal domain of human caveolin-1 (CavNT) was made by PCR and constructed into pCAG-EB6-huECDSP-Fc1 (pCAG-EB6-huECDSP-CavNT-Fc1). With the same methods, the N-terminal domain with deletion of the scaffolding domain (CavNTSCD) was made by PCR (pCAG-EB6-huECDSP-CavNTSCD-Fc1). 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-Fc1).

For expression of Fc fusion proteins, FreeStyle^TM 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 (2, 13, 14). CARMA1-deficient Jurkat T-cell line, JPM50.6, was developed as described elsewhere (18). 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 (30). Informed consent was obtained from healthy adult volunteers. Biotinylation of recombinant proteins or antibody was generated using EZ-Link^TM 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^TM Analysis of Affinity of Caveolin-1-CD26 Interaction—Experiments were carried out on a Biacore^TM 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). Fc1, NT-Fc, or NTSCD-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 Fc1, NT-Fc, or NTSCD-Fc chips after each experiment. Recombinant soluble CD26 (rsCD26), comprising the extracellular region of human CD26, was prepared as described previously (30, 31). rsCD26 at various concentrations (50, 25, 12.5, 6.3, 3.2, and 1.6 nM) was then injected for 120 s over immobilized Fc1, NT-Fc, or NTSCD-Fc chips. Equilibrium binding analysis was performed as described elsewhere (32), 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 (14), 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^TM), were then selected using standard limiting dilution method.

Flow Cytometric Analysis—For assessment of J.CD26wt that binds biotinylated NT-Fc or NTSCD-Fc, 1 x 10⁶ cells were washed in ice-cold phosphate-buffered saline and incubated with Fc1 and mouse Ig isotypes (1 µg/ml) to block nonspecific binding, followed by reaction with biotinylated NT-Fc or NTSCD-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 NTSCD-Fc. Flow cytometric analysis of 10,000 viable cells was conducted on FACSCalibur^TM. 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^TM; kindly provided by Ihsihara Sangyo Kaisha Ltd.) as described previously (14). After 48 h of transfection, cell were prepared for examination.

T-cell Proliferation and IL-2 Production Assay—For T-cell proliferation assay, 1 x 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 x 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 x 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 Fc1 (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 (15).

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 x 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 (33). For immunoprecipitation of the pooled lipid raft fraction, fractionated lipid rafts were lysed at 4 °C for 30 min with 1% N-octyl--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 (14, 15, 33).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Expression and purification of caveolin-1 human IgG1 fusion proteins. A, schematic diagrams of human caveolin-1 and its Fc fusion proteins. In human caveolin-1, aa 1-82 includes the N-terminal domain (NT); aa 82-101 includes the scaffolding domain (SCD); aa 101-134 includes membrane-spanning domain (MS); and aa 134-178 includes the C-terminal domain (CT). SP (huECD) depicts the signal peptide (SP) of human E-cadherin. At the 3' portion, the hinge (H) and CH2 and CH3 domains of human IgG1 Fc are also indicated (Fc1). B, expressed fusion proteins were purified as described under "Experimental Procedures." Aliquots (5 µg) of control human IgG (lanes 1 and 5), Fc1 (lanes 2 and 6), Fc fusion proteins of the N-terminal region of human caveolin-1 (NT-Fc) (lanes 3 and 7), and Fc fusion proteins of the N-terminal region with the scaffolding domain of human caveolin-1 being deleted (NTSCD-Fc) (lanes 4 and 8) were subjected to SDS-PAGE (5-20% acrylamide gradient gel) under reducing (+2ME, lanes 2-4) or nonreducing (-2ME, lanes 5-8) conditions. Molecular weight markers are depicted in Mr. Proteins were visualized by staining with Coomassie Brilliant Blue. C, aliquots (50 ng) of Fc1 (lanes 1 and 4), NT-Fc (lanes 2 and 5), and NTSCD-Fc (lanes 3 and 6) were subjected to SDS-PAGE (5-20% acrylamide gradient gel) under reducing (+2ME, lanes 1-3) or nonreducing (-2ME, lanes 4-6) conditions, followed by Western blot analysis, using horseradish peroxidase-conjugated anti-human IgG.

Statistics—Student's t test was used to determine whether the difference between control and sample was significant (p < 0.05 being significant).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 NTSCD-Fc, respectively). The schematic diagrams of the full-length human caveolin-1 protein, NT-Fc, NTSCD-Fc and Fc1 are shown in Fig. 1A. As shown in Fig. 1B, where a band of the recombinant Fc portion of human IgG₁ (Fc1) was observed at 35 kDa under reducing conditions (lane 2), the NT-Fc and NTSCD-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 (34). In nonreducing conditions, Fc1, NT-Fc, or NTSCD-Fc were observed at 60, 100, or 90 kDa, respectively (lanes 6-8 in Fig. 1B), indicating that they were expressed as a homodimer. Fc1, NT-Fc, and NTSCD-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 Procedures." As shown in Fig. 2A, by using lysates of J.CD26wt, CD26 was coimmunoprecipitated with NT-Fc (lane 2) but not with Fc1 (lane 1) nor with NTSCD-Fc (lane 3). We next evaluated binding of NT-Fc to cell surface CD26 using flow cytometry. As shown in Fig. 2B, cell surface CD26 of J.CD26wt was stained with anti-CD26-FITC mAb (panel b, peak 2) (whereas unlabeled CD26 mAb, but not control IgG, blocked staining with anti-CD26-FITC mAb (peak 3 and peak 4 of panel b). J.CD26wt was also stained with biotinylated NT-Fc followed by staining with streptavidin-conjugated FITC (peak 2 of panel c in Fig. 2B). Staining with NT-Fc was blocked by unlabeled anti-CD26 mAb (peak 3 of panel c in Fig. 2B) but not control IgG (peak 4 of panel c in Fig. 2B). On the other hand, J.CD26wt was not stained with NTSCD-Fc (panel d in Fig. 2B). Moreover, native Jurkat T-cells were not stained with anti-CD26 mAb nor with NT-Fc (data not shown). These data suggested that the soluble N-terminal domain of caveolin-1 binds to cell surface CD26 and that the SCD of caveolin-1 is necessary for binding to CD26, as shown in our previous studies (14, 15).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Fc fusion proteins of the N-terminal region of human caveolin-1 (NT-Fc) binds to CD26 and induces IL-2 production. A, lysates of J.CD26wt cells (500 µg) were precleared with Fc1 and protein A-Sepharose beads, and IP assays were conducted with Fc1 (lane 1), NT-Fc (lane 2), or NTSCD (lane 3) (each at 2 µg). IP complexes were then separated using 5-20% SDS-PAGE, followed by immunoblotting with anti-CD26 mAb (upper panel). An aliquot (50 µg) of the input lysate was also analyzed (lane 4). The membrane was stripped and reprobed with horseradish peroxidase-conjugated anti-human IgG (lower panel). Similar results were obtained in three independent experiments. B, J.CD26wt cells were used for binding activity of Fc fusion proteins. Panel a, forward and side scattergrams of the analyzed cells. Solid circle indicates the gated region for analysis. Panel b, cells were stained with FITC-conjugated control mouse IgG (peak 1) or FITC-conjugated anti-CD26 mAb (peak 2). For blocking assay, cells were first reacted with unlabeled anti-CD26 mAb (peak 3) or unlabeled control mouse IgG (peak 4), followed by staining as described in (peak 2). Panel c, cells were stained with biotinylated Fc1 as control (peak 1) or biotinylated NT-Fc (peak 2), followed by reaction with FITC-conjugated streptavidin. For blocking assay, cells were first reacted with unlabeled anti-CD26 mAb (peak 3) or unlabeled control IgG (peak 4), followed by staining as described in peak 2. Panel d, cells were stained with biotinylated Fc1 as control (peak 1) or biotinylated NTSCD-Fc (peak 2), followed by reaction with FITC-conjugated streptavidin. For blocking assay, cells were first reacted with unlabeled anti-CD26 mAb (peak 3) or unlabeled control IgG (peak 4), followed by staining as described in peak 2. All four histograms in panel d were stacked in the same position. C, measuring the affinity of Fc fusion proteins to rsCD26 by equilibrium binding. Injections of rsCD26 at 25 °C started at 50 nM and were followed by five 2-fold dilutions (50, 25, 12.5, 6.3, 3.2, and 1.6 nM), flowing over Fc1 (panel a), NT-Fc (panel b), or NTSCD-Fc (panel c) immobilized at a concentration of 6032, 4996, or 4852 RU, respectively. The curves represent total specific binding after subtraction of the background responses observed in a control flow cell. D, native Jurkat (JKTwt) or J.CD26wt was stimulated with immobilized antibodies and/or Fc fusion proteins (anti-CD3, 1.0 µg/ml; anti-CD28, anti-CD26, Fc1, NT-Fc, NTSCD-Fc, each at 10 µg/ml). After culturing for 48 h, culture supernatants were pooled from the triplicate wells and assayed for IL-2 content. Values shown are means ± S.E. of determinations from triplicate cultures of three independent experiments. * and *** show points of significant increase (p < 0.05), whereas ** and # indicate points of no significant change compared with controls. E, following blocking with soluble anti-CD26, anti-CD28, or control mouse IgG, J.CD26wt cells were stimulated and IL-2 was measured as described in D. Values shown are means ± S.E. of determinations from triplicate cultures of three independent experiments. * and ** show results of significant inhibition obtained following blocking by anti-CD26 mAb (p < 0.05), and # and ## show results of no significant inhibition obtained following blocking by anti-CD28 mAb.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. NT-Fc is costimulatory with anti-CD3 for proliferation of peripheral blood T-cells. A, purified T-cells were stimulated with immobilized antibodies and/or Fc fusion proteins (anti-CD3, 0.05 µg/ml; anti-CD28, anti-CD26, Fc1, NT-Fc, NTSCD-Fc, each at 5 µg/ml). Proliferation was measured by uptake of [3H]TdR as described under "Experimental Procedures." Values shown are means ± S.E. of determinations from triplicate cultures of five independent donors. * shows points of significant increase (p < 0.05), whereas ** indicates points of no significant change compared with controls. B, purified T-cells were stimulated with immobilized Fc fusion proteins at indicated concentrations in the presence of immobilized anti-CD3 (0.05 µg/ml). Proliferation was measured as described in A. Values shown are means ± S.E. of determinations from triplicate cultures of five independent donors. * shows points of significant increase (p < 0.05) compared with control. C, purified T-cells were cultured in the presence of anti-CD3 (0.05 µg/ml) in solution, with varying amounts of CHO transfectants that were fixed with 0.05% glutaraldehyde. Cav-wt+ CHO, Cav-SCD+ CHO, or mock+ CHO represent CHO cells stably transfected with GFP-full-length caveolin-1, GFP-caveolin-1 with the scaffolding domain deleted, or GFP expressing vector, respectively. Proliferation was measured as described in A. Values shown are means ± S.E. of determinations from triplicate cultures of five independent donors. * shows points of significant increase (p < 0.05) compared with control. D, following blocking with soluble anti-CD26, anti-CD28, or control mouse IgG, T-cells were stimulated, and proliferation was measured as described in A. Values shown are means ± S.E. of determinations from triplicate cultures of five independent donors. * and *** show results of significant inhibition obtained following blocking by anti-CD26 mAb (p < 0.05), and ** shows results of significant inhibition obtained following blocking by anti-CD28 mAb (p < 0.05). E, following incubation and blocking with increasing doses (0, 0.5, 5.0, 10.0, 20.0, and 50 µg/ml) of soluble anti-CD26 mAbs, anti-CD28 mAbs, or control mouse IgG, T-cells were stimulated by plate-bound anti-CD3 (0.05 µg/ml) plus NT-Fc (5 µg/ml), and proliferation was measured as described in A. Values shown are means ± S.E. of determinations from triplicate cultures of five independent donors. * shows points of significant decrease (p < 0.05) compared with controls.

We next evaluated whether NT-Fc stimulation had a similar effect as anti-CD26 mAb on CD26-mediated T-cell costimulation in J.CD26wt (13). As shown in Fig. 2D, IL-2 production of J.CD26wt induced by plate-bound anti-CD3 plus NT-Fc was observed to be at a similar level as that induced by anti-CD3 plus anti-CD28 or by anti-CD3 plus anti-CD26 (^*, ^**, and ^*** in the bar graph), whereas IL-2 production was not observed in JKTwt (which is CD26-negative) following stimulation by anti-CD3 plus anti-CD26 nor by anti-CD3 plus NT-Fc (^* and ^*** in Fig. 2D). IL-2 production by J.CD26wt or JKTwt was not observed with the use of control recombinant Fc1 nor NTSCD-Fc (# in Fig. 2D). To further investigate whether the T-cell costimulatory activity of NT-Fc was exerted via CD26, blocking experiments were conducted using CD26-specific mAb, which blocked binding of NT-Fc to J.CD26wt. As shown in Fig. 2E, IL-2 production induced by plate-bound anti-CD3 plus anti-CD26 was blocked by soluble anti-CD26 mAb but not by soluble anti-CD28 mAb (^** and ##). In this experimental condition, IL-2 production induced by plate-bound anti-CD3 plus NT-Fc was blocked by soluble anti-CD26 mAb but not by soluble anti-CD28 mAb (^* and # in Fig. 2E). Control Fc1 or NTSCD-Fc did not have any T-cell costimulatory activity (Fig. 2E). Taken together, our results clearly indicated that caveolin-1 binds directly to CD26 and induces T-cell costimulation via CD26.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. The cytoplasmic tail of dimeric CD26 is necessary for anti-CD3 plus caveolin-1 costimulation. A, Jurkat T-cells stably transfected with V5-tagged full-length CD26 (CD26wt), CD26-CD10 chimeric receptor (CD26 + CD10 cyto), or monomeric CD26 (CD26 H750E) were generated as described under "Experimental Procedures." Cell lysates were resolved in SDS-PAGE under reducing (+2ME) (lanes 1-4) or nonreducing (-2ME) conditions (lanes 5-8), and immunoblotted with anti-CD26 mAb. In nonreducing conditions, the arrowhead shows bands of dimeric CD26 (lanes 6 and 7), and the arrow indicates bands of monomeric CD26 (lane 8). B, cells were lysed and immunoprecipitated with anti-V5 mAb. IPs were resolved in 5-20% gradient SDS-PAGE under reducing conditions and immunoblotted with anti-CD26 mAb. IgH indicates immunoglobulin heavy chain. C, dot plots for expression of cell surface CD3 and CD26. % positive of CD3 is shown in mock vector-transfected Jurkat (panel a), and % positive of CD3 and CD26 is shown in other transfectants (panels b-d). D, Jurkat transfectants, which were stably transfected with full-length CD26 (CD26wt), CD26-CD10 chimeric receptor (CD26 + CD10 cyto), or CD26 containing mutation of histidine residue at amino acid 750 for glutamic acid (CD26 H750E), were stimulated with plate-bound anti-CD3 (1.0 µg/ml) in the presence or absence of plate-bound NT-Fc (10 µg/ml) or PMA (10 ng/ml). Panel a, following 48 h of culture, IL-2 concentration of the culture supernatant was measured by ELISA. Values shown are means ± S.E. of determinations from triplicate cultures. * shows points of significant increase (p < 0.05) compared with control. Panel b, Jurkat transfectants were stimulated as described in panel a, harvested for extraction of nuclear proteins, ad subjected to ELISA-based DNA-binding protein assay. Binding activity to p65 NF-B component was revealed by absorbance value at 450 nm. Data represent mean ± S.E. from triplicate experiments. * shows a point of significant increase (p < 0.05).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. The cytoplasmic tail of dimeric CD26 is associated with CARMA1. A, schematic diagram of Fc fusion proteins of the cytoplasmic tail of CD26 (CD26 aa1-10). MKTPWKVLLG depicts amino acid residues of human CD26 at 1-10 positions. At the 3' portion, the hinge (H) and CH2 and CH3 domains of human IgG1 Fc are also indicated (Fc1). B, expressed fusion proteins were purified as described under "Experimental Procedures." Aliquots (5 µg) of control human IgG (lanes 1 and 4), Fc1 (lanes 2 and 5), and CD26 aa1-10-Fc (lanes 3 and 6) were subjected to SDS-PAGE under reducing (+2ME, lanes 1-3) or nonreducing (-2ME, lanes 4-6) conditions. Molecular weight markers are depicted in Mr. Proteins were visualized by staining with Coomassie Brilliant Blue. C, an aliquot (50 µg) of Jurkat lysates was separated by two-dimensional PAGE using pH 3.0-10 nonlinear (NL) IPG (isoelectric focusing of proteins using immobilized pH gradient) stripped in the first dimension and 4-12% SDS-PAGE, and the gels were stained with Coomassie Brilliant Blue (panel a). Aliquots (1 mg) of lysates were precleared by human IgG (2 µg) and proteins A-Sepharose, followed by immunoprecipitation with Fc1 (1 µg) (panel b) or CD26 aa1-10-Fc (1 µg) (panel c). IPs were analyzed by two-dimensional PAGE, and six spots were clearly detected in IP complex of CD26 aa1-10-Fc (1-6 in panel c). * and ** were spots of Fc1 and CD26-Fc (aa1-10), respectively. Similar results were obtained in five independent experiments, and the panels shown are the representative results. D, J.CD26wt were lysed, and IP assays were conducted with anti-CARMA1 pAb (goat), anti-CD26 mAb (mouse (ms)), or control Ig (cIgG). IP complexes as well as 10% of input lysates were then separated using SDS-PAGE, immunoblotted with indicated antibodies. Similar results were obtained in three independent experiments. E, 293FT cells were transiently transfected with V5-tagged full-length CD26 (CD26wt), CD26-CD10 chimeric receptor (CD26 + CD10 cyto), or CD26 containing mutation of histidine residue at amino acid 750 for glutamic acid (CD26 H750E), together with Xpress-tagged full-length CARMA1 (CARMA1wt). Cells were lysed with TBSD buffer and immunoprecipitated with anti-V5 mAb. IPs were separated using 5-20% SDS-PAGE and immunoblotted with anti-Xpress mAb (upper panel), followed by stripping and reprobing with anti-V5 mAb (lower panel). Similar results were obtained in three independent experiments. F, schematic diagrams of Xpress-tagged CARMA1 and its deletion mutants: CARAM1wt, Xpress-tagged full-length CARMA1; CARMA1-(1-742), Xpress-tagged CARMA1 minus the SH3 + GUK domains; CARMA1-(1-660), Xpress-tagged CARMA1 minus the PDZ + SH3 + GUK domains. G, 293FT cells were transiently transfected with Xpress-tagged CARMA1wt, CARMA1 with the SH3 + GUK domains deleted (residues 1-742), or CARMA1 with the PDZ + SH3 + GUK domains deleted (residues 1-660), together with V5-tagged CD26wt. Cells were lysed with TBSD buffer and immunoprecipitated with anti-Xpress mAb. IPs were separated using SDS-PAGE and immunoblotted with anti-V5 mAb (upper panel), followed by stripping and reprobing with anti-Xpress mAb (lower panel). Similar results were obtained in three independent experiments.

The proximal signaling molecules of CD26-mediated T-cell costimulation by caveolin-1 were next determined. We first examined whether the cytoplasmic tail of CD26 is responsible for T-cell costimulation by NT-Fc in the presence of anti-CD3 mAb. For this purpose, costimulation experiments were performed on Jurkat T-cells transfected with CD26-CD10 chimeric receptor. Moreover, whereas CD26 is reported to form homodimers on cell surface (36, 37), it remains to be determined whether dimeric CD26 is responsible for CD26-mediated T-cell costimulation. Therefore, costimulation experiments were conducted using Jurkat T-cells transfected with monomeric CD26 (CD26 H750E). We first verified the Jurkat-stable transfectants, as shown in Fig. 4A, and CD26wt, CD26 + CD10 cyto, and CD26 H750E were detected at 100 kDa in reducing conditions (lanes 2-4), bands of CD26wt and CD26 + CD10 cyto migrated at 200 kDa (lanes 6 and 7), and a band of CD26 H750E migrated at 100 kDa (lane 8) in nonreducing conditions, indicating that CD26wt and CD26 + CD10 cyto exist as dimers, and CD26 H750E exists as monomers in the Jurkat transfectants. Moreover, as shown in Fig. 4B, by immunoprecipitation using anti-V5 mAb, V5-tagged CD26 was detected by anti-CD26 mAb in each transfectant (CD26wt, CD26 + CD10 cyto, or CD26 H750E) in reducing SDS-PAGE (lanes 2-4). Furthermore, Fig. 4C showed the cell surface expression of CD3 and CD26 in Jurkat transfectants. CD3 was expressed at similar intensity among transfectants (horizontal axis of panels a-d in Fig. 4C), whereas the intensity of cell surface CD26 expression was different between CD26wt/CD26 + CD10 cyto and CD26 H750E (vertical axis of panels b-d in Fig. 4C), suggesting a difference between dimeric expression and monomeric expression. CD26 was not observed in mock vector-transfected Jurkat (JKT/mock; panel a in Fig. 4C). Using these Jurkat transfectants, costimulation experiments by NT-Fc were conducted. As shown in Fig. 4D, IL-2 production was observed in CD26wt-transfected Jurkat T-cells by stimulation with anti-CD3 plus NT-Fc (^* in panel a), but not in CD26-CD10 chimera nor in CD26 H750E-transfected Jurkat. Moreover, p65, one of NF-B components, was activated in CD26wt-transfected Jurkat T-cells following stimulation with anti-CD3 plus NT-Fc (^* in panel b) but not in CD26-CD10 chimera nor in CD26 H750E-transfected Jurkat. Furthermore, IL-2 production or p65 induction by stimulation with anti-CD3 plus PMA was equally observed in either of CD26wt, CD26-CD10 chimera, or CD26 H750E transfected Jurkat (panels a and b of Fig. 4D). These data strongly suggested that the cytoplasmic tail of dimeric CD26, but not of monomeric CD26, is responsible for T-cell costimulation by NT-Fc in the presence of anti-CD3 mAb.

Because the cytoplasmic tail of CD26 appears to play a key role for CD26-mediated T-cell costimulation as shown in Fig. 4, we next explored signaling molecules associated with the cytoplasmic tail of dimeric CD26. For this purpose, we prepared Fc fusion protein containing the first 10 aa of the N-terminal residues of CD26 (CD26 aa1-10-Fc) (Fig. 5A). As shown in Fig. 5B, purified protein of CD26 aa1-10-Fc was observed at 37 kDa in reducing conditions (lane 3) and at 70 kDa in nonreducing conditions (lane 6), suggesting that CD26 aa1-10-Fc formed homodimers. Following pulldown by CD26 aa1-10-Fc of Jurkat T-cell lysates, molecules that interacted with CD26 aa1-10-Fc were analyzed by two-dimensional SDS-PAGE. The gel of two-dimensional PAGE using input lysates is shown in Fig. 5C (panel a). Compared with two-dimensional gel analyzing IP complex by control Fc1 (panel b in Fig. 5C), six spots were detected by pulldown assays with CD26 aa1-10-Fc (panel c in Fig. 5C). Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the proteins were determined to be as follows: spot 1, epidermal cytokeratin 2 (66 kDa); spot 2, glutamyl-tRNA synthetase (70 kDa); spot 3, tubulin (50 kDa); spot 4, unnamed protein (84 kDa); spot 5, CARMA1 (120 kDa); or spot 6, HSP70 (55 kDa), respectively (panel c in Fig. 5C). Following five independent repeats of these experiments with similar results, the unnamed protein (spot 4) was not identified more precisely by this procedure, and the other spots other than CARMA1 were ubiquitously expressed as housekeeping proteins. Therefore, CARMA1 was identified as an interacting protein with the cytoplasmic tail of CD26 and subjected to further examination.

For further confirmation, we next performed IP studies using lysates of J.CD26wt. As shown in Fig. 5D, CD26 was detected in a complex of lysates coprecipitated with anti-CARMA1 pAb (lane 2 of upper panel), and not coprecipitated with control goat IgG (lane 1 of upper panel). Moreover, CARMA1 was detected in a complex of lysates coprecipitated with anti-CD26 mAb (lane 4 of lower panel in Fig. 5D), and not coprecipitated with control mouse IgG (lane 3 of lower panel in Fig. 5D). These data suggested that CARMA1 binds to CD26 in cells. To determine the binding domain between CD26 and CARMA1, coimmunoprecipitation assay was next performed using 293FT-cells cotransfected with Xpress-tagged human full-length CARMA1 (CARMA1wt) and with CD26wt, CD26 + CD10 cyto, or CD26 H750E. As shown in Fig. 5E, CARMA1 was coprecipitated with CD26wt (lane 2) but not with CD26 + CD10 cyto nor CD26 H750E (lanes 3 and 4). These data strongly suggested that the cytoplasmic tail and dimerization of CD26 are necessary to interact with CARMA1. We next explored the binding domain of CARMA1 to CD26. For this purpose, we prepared the C-terminal truncated deletion mutants of CARMA1 (Fig. 5F). As shown in Fig. 5G, CD26 was coprecipitated with CARMA1wt or with CARMA1-(1-742) (lanes 2 and 3) but not with CARMA1-(1-600) (lane 4), suggesting that the PDZ domain in CARMA1 was necessary for binding to CD26.

To explore the role of CARMA1 in CD26-mediated T-cell costimulation, we used CARMA1-deficient Jurkat T-cell lines JPM50.6 to conduct rescue experiments (18). As shown in Fig. 6A, CARMA1 and CD26 were not detected in JPM50.6 (lane 3 of upper and lower panels), whereas CARMA1 was expressed in native Jurkat and J.CD26wt (lanes 1 and 2 of lower panel), and CD26 was expressed in J.CD26wt (lane 2 of upper panel) but not in native Jurkat (lane 1 of upper panel). We next generated the stable transfectants using JPM50.6 as described under "Experimental Procedures." Fig. 6B shows that transfected Xpress-tagged CARMA1 was expressed in JPM50.6/CARMA1wt, JPM50.6/CD26wt + CARMA1wt, and JPM50.6/CD26wt + CARMA1-(1-660) and that transfected V5-tagged CD26 was expressed in JPM50.6/CD26wt, JPM50.6/CD26wt + CARMA1wt, and JPM50.6/CD26wt + CARMA1-(1-660). Fig. 6C shows the cell surface expression of CD3 and CD26 in JPM50.6 transfectants. CD3 was expressed at similar intensity among transfectants (horizontal axis of panels a-e in Fig. 6C). Although the intensity of cell surface CD26 expression was similar among JPM50.6/CD26wt, JPM50.6/CD26wt + CARMA1wt, and JPM50.6/CD26wt + CARMA1-(1-660) (panels b, d, and e in Fig. 6C), CD26 was not observed in JPM50.6/V (mock vector) and JPM50.6/CARMA1wt (panels a and c in Fig. 6C). Using these transfectants, IL-2 production and NF-B activation assays were performed with stimulation by anti-CD3 alone or anti-CD3 plus NT-Fc. As shown in Fig. 6D, IL-2 production induced by anti-CD3 plus NT-Fc was clearly observed in JPM50.6/CD26wt + CARMA1 but not in JPM50.6, JPM50.6/CD26wt, JPM50.6/CARMA1, nor JPM50.6/CD26wt + CARMA1-(1-660) (panel a). Similarly, NF-B activation induced by anti-CD3 plus NT-Fc was clearly observed in JPM50.6/CD26wt + CARMA1 but not in JPM50.6, JPM50.6/CD26wt, JPM50.6/CARMA1, nor JPM50.6/CD26wt+ CARMA1-(1-660) (panel b in Fig. 6D). Furthermore, IL-2 production or NF-B activation by stimulation with anti-CD3 plus PMA was equally observed in either of the transfectants (panels a and b of Fig. 6D). Taken together, these results suggested that CARMA1 is necessary to exert CD26-mediated costimulation by NT-Fc.

As shown above, costimulation of CD26 is observed to be exerted via interaction of CD26 with CARMA1 in the cytoplasm in Jurkat cells. To confirm this interaction more profoundly, we performed biochemical assays using human T-cells purified from healthy adult peripheral blood mononuclear cells (APB-T-cells). For this purpose, we first conducted IP studies using lysates of APB-T-cells. As shown in Fig. 7A, CD26 was detected in a complex of lysates coprecipitated with anti-CARMA1 pAb (lane 2 of upper panel), and not coprecipitated with control goat IgG (lane 1 of upper panel). Moreover, CARMA1 was detected in a complex of lysates coprecipitated with anti-CD26 mAb (lane 4 of lower panel in Fig. 7A), and not coprecipitated with control mouse IgG (lane 3 of lower panel in Fig. 7A). These data suggested that CARMA1 binds to CD26 in normal T-cells.

We previously showed that nonactivated peripheral blood T-cells treated with the anti-CD26 mAb 1F7 resulted in CD26 recruitment to lipid rafts, concomitant with increased tyrosine phosphorylation of ZAP70, p56^lck, and TCR (33). Other investigators have reported that in the process of activation of NF-B via CD3 costimulation, CARMA1 was recruited to lipid rafts along with Bcl10 and IKK (18, 25, 27, 38, 39). We therefore examined whether CD26 and CARMA1 are recruited to lipid rafts by anti-CD3 plus caveolin-1 costimulation in normal T-cells. For this purpose, using a sucrose gradient separation method, we prepared lipid raft fractions of APB-T-cell lysates in the presence or absence of anti-CD3 plus NT-Fc costimulation. As shown in Fig. 7B, following stimulation with anti-CD3 plus NT-Fc, CD26, CARMA1, Bcl10, and IKK were detected in the lipid raft fractions, whereas CD26, CARMA1, Bcl10, and IKK were not detected in the lipid raft fractions after stimulation with anti-CD3 alone. Moreover, time course analysis revealed that CD26, CARMA1, Bcl10, and IKK were migrated into lipid rafts after stimulation with anti-CD3 plus NT-Fc (Fig. 7C), whereas CD26, CARMA1, Bcl10, and IKK were not detected after anti-CD3 treatment (data not shown). Furthermore, to examine whether CD26 and CARMA1 forms a complex with Bcl10 and IKK in lipid rafts, coprecipitation assay was performed using lipid raft fractions of APB-T-cell lysates from cells costimulated with anti-CD3 plus NT-Fc. As shown in Fig. 7D, CD26, CARMA1, Bcl10, and IKK in lipid rafts were coprecipitated with CD26 (lane 4), although not detected in the lysates of APB-T-cells following stimulation with anti-CD3 alone (lane 2). Taken together, these data indicated that ligation of CD26 by caveolin-1 recruits a complex of CARMA1, Bcl10, and IKK to lipid rafts in normal T-cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. CARMA1 is necessary for CD26-mediated costimulation by caveolin-1. A, cell lysates of native Jurkat, J.CD26wt, or JPM50.6 were resolved in SDS-PAGE under reducing conditions, followed by immunoblotting with anti-CD26 mAb (upper panel) or anti-CARMA1 pAb (lower panel). B, JPM50.6 cells stably transfected with V5-tagged full-length CD26 (CD26wt) and/or Xpress-tagged full-length CARMA1 (CARMA1wt) or CARMA1 with the PDZ + SH3 + GUK domains deleted (CARMA1-(1-660)) were generated as described under "Experimental Procedures." Lysates were resolved in SDS-PAGE and immunoblotted with anti-Xpress mAb (CARMA1) (upper panel) or anti-V5 mAb (CD26) (lower panel). C, dot plots for cell surface expression of CD3 and CD26. % positive of CD3 is shown in mock vector-transfected JPM (panel a) and CARMA1-transfected JPM 50.6 (panel c), and % positive of CD3 and CD26 is shown in other transfectants (panels b, d, and e). D, JPM50.6 transfectants, which were described in B, were stimulated with plate-bound anti-CD3 in the presence or absence of plate-bound NT-Fc as described in Fig. 4D. Panel a, following 48 h of culture, IL-2 concentration of the culture supernatant was measured by ELISA. Values shown are means ± S.E. of determinations from triplicate cultures. * shows points of significant increase (p < 0.05) compared with control. Panel b, JPM50.6 transfectants were stimulated as described in panel a and harvested for nuclear extract. Each 5 µg of nuclear extract was subjected to ELISA-based DNA-binding protein assay. Binding activity to p65 NF-B component was revealed by absorbance value at 450 nm. Data represent mean ± S.E. from triplicate experiments. * shows a point of significant increase (p < 0.05). Xpress vec. or V5 vec. depicts pcDNA4/HisMax or pEF6/V5 empty vector as a mock, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we showed that caveolin-1 is the costimulatory ligand for CD26, and that ligation of CD26 by caveolin-1 induces T-cell proliferation and NF-B activation with costimulation of TCR/CD3. Moreover, we showed that the cytoplasmic tail of CD26 in T-cell interacts with CARMA1, resulting in signal transduction leading to NF-B activation and that ligation of CD26 by caveolin-1 recruits a complex of CD26, CARMA1, Bcl10, and IKK to lipid rafts.

View larger version (42K): [in this window] [in a new window]   FIGURE 7. CARMA1 plays an important role in CD26-mediated costimulation by caveolin-1 in human peripheral blood T-cells. A, purified T-cells were lysed, and IP assays were conducted with anti-CARMA1 pAb (goat), anti-CD26 mAb (mouse (ms)), or control Ig (cIgG). IP complexes as well as 10% of input lysates were then separated using SDS-PAGE and immunoblotted with the indicated antibodies. Similar results were obtained in three independent experiments. B, purified T-cells were stimulated for 10 min with anti-CD3 alone (0.05 µg/ml) or with anti-CD3 plus NT-Fc (5.0 µg/ml), and lysates were prepared by sucrose gradient centrifugation as described under "Experimental Procedures." The distribution of CD26, CARMA1, Bcl10, and IKK was determined by immunoblotting with specific antibodies. Similar results were obtained in three independent experiments. C, purified T-cells were stimulated for 0, 10, and 30 min with anti-CD3 plus NT-Fc, and lysates were prepared by sucrose gradient centrifugation as described under "Experimental Procedures." The distribution of CD26, CARMA1, Bcl10, and IKK was determined by immunoblotting with specific antibodies. Similar results were obtained in three independent experiments. D, purified T-cells were stimulated with anti-CD3 alone (lanes 1 and 2) or with anti-CD3 plus NT-Fc (lanes 3 and 4), and lipid raft fractions were prepared as described in A, and immunoprecipitation of lipid rafts with control IgG (cIgG) (lanes 1 and 3) or anti-CD26 mAb (lanes 2 and 4) was performed as described under "Experimental Procedures." IPs were resolved in SDS-PAGE and immunoblotted with indicated antibodies. Similar results were obtained in three independent experiments. E, purified T-cells were transfected with sense-siRNA (ss1 and ss2) of CARMA1 gene or mismatched siRNA (control) using HVJ-E vector. Cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies, followed by stripping and reprobing with anti--actin antibody (inside box). Purified T-cells treated with siRNA were stimulated and subjected to T-cell proliferation assay as described in Fig. 3A. Values shown are means ± S.E. of determinations from triplicate cultures of five independent donors. * shows points of significant decrease (p < 0.05), and ** indicates points of no significant change compared with controls.

View larger version (30K): [in this window] [in a new window]   FIGURE 8. A model for signaling by TCR and CD26 costimulation. Stimulation of cells through TCR complexes leads to phosphorylation of cytoplasmic immunoreceptor tyrosine-based activation motifs by ligation of peptide-loaded major histocompatibility complex class II (bold arrows) and recruitment and activation of phosphatidylinositol 3-kinase and PKC (gray arrowhead). Meanwhile, caveolin-1, of which the N-terminal extracellular regions are presented on antigen-loaded APC, ligates CD26, which exists as dimers on the cell surface and recruits lipid rafts (gray arrows) while interacting with CARMA1 (gray double-headed arrow). The recruitment of CARMA1 along with CD26 to lipid rafts also recruits the CARMA1-Bcl10-IKKs complex (black double-headed arrows), leading to activation of the IKK complex (black double-headed arrows) and finally activation of NF-B.

We have demonstrated previously that ligation of CD26 by the anti-CD26 mAb 1F7 induces T-cell costimulation and IL-2 production by CD26-transfected Jurkat T-cell lines, while increasing tyrosine phosphorylation of signaling molecules such as ZAP70, p56^lck, and CD3 was observed (2, 7, 12). In addition, we have shown that ligation of the CD26 molecules by the anti-CD26 mAb 1F7 increases the recruitment of CD26 molecules with CD45RO to lipid rafts, resulting in increased tyrosine phosphorylation of signaling molecules (33). However, the precise proximal signaling pathway of CD26 has not yet been identified, particularly in view of the fact that the cytoplasmic tail of CD26 contains only 6 amino acid residues without a common signaling motif structure. Moreover, it has been unclear whether the short cytoplasmic tail is responsible for signal transduction associated with CD26-mediated costimulation. In this study, using recombinant CD26-CD10 chimeric receptor, we showed that the cytoplasmic tail of CD26 is indeed responsible for T-cell costimulation induced by anti-CD3 plus caveolin-1 (Fig. 4D). Furthermore, to explore the proximal signaling molecules interacting with the cytoplasmic tail of dimeric CD26, we used proteomic analyses with Fc fusion proteins containing the cytoplasmic amino acid residues of CD26 (Fig. 5, A and B) to identify that CARMA1 binds to the cytoplasmic tail of dimeric CD26 (Fig. 5C). Moreover, we demonstrated here that a PDZ domain in CARMA1 is necessary for binding to CD26 (Fig. 5G). The importance of CARMA1 in CD26-mediated costimulation is also shown by rescue experiments using the CARMA1-deficient Jurkat T-cell line JPM50.6 (Fig. 6D) and using siRNA against CARMA1 in APB-T-cells (Fig. 7E). CARMA1, containing caspase-recruitment domain and MAGUK domains, plays an essential role in the NF-B activation and IL-2 expression induced by CD3-CD28 or CD28-PMA stimulation (18, 22). After being phosphorylated, CARMA1 functions as a signaling intermediate downstream of PKC and upstream of IKK in the TCR signaling transduction pathway leading to NF-B activation (39, 51). Because MAGUK domain-containing proteins are generally involved in the organization of multiprotein complexes at the interface of the cytoplasmic membrane (52), it is possible that CARMA1 associates with as yet undefined membrane proteins in the immunological synapse of T-cells. In this regard, our present data suggest a novel mechanism for CAMRA1 function as it complexes with Bcl10 and IKK to transduce CD26-costimulatory signals. Moreover, as shown Fig. 5C, cytoskeletal proteins were also observed in the complex in the pulldown assays with CD26 aa1-10-Fc. Because MAGUK domain-containing proteins are generally involved in the organization of multiprotein complexes in the cytoskeleton (52), the downstream signaling of CD26 may also be associated with cytoskeletal assembly via CARMA1. The association of CD26, CARMA1, and the cytoskeleton will be elucidated in future studies.

CD26/DDPIV is reported to exist as homodimers, a structural organization that allows access of substrates to DPPIV catalytic activity (36, 37). Although DPPIV activity is crucial for CD26-mediated T-cell costimulation (13, 30), the exact role played by DPPIV in this process is unclear. Our previous study showed that the enzymatic pocket structure of the DPPIV catalytic site is necessary for binding of CD26 to caveolin-1, leading to the up-regulation of CD86 expression on APC (14, 15). In this study, we found that monomeric CD26 H750E, which has a 300-fold decrease in catalytic activity (36), does not bind to CARMA1 (Fig. 5E), resulting in loss of CD26-mediated T-cell costimulation by anti-CD3 plus caveolin-1 (Fig. 4D). Therefore, dimerization of CD26 is not only necessary for binding to caveolin-1 but also serves as a scaffolding structure for the cytoplasmic signaling molecule CARMA1. The precise binding position of CARMA1 in the cytoplasmic domain of CD26 remains to be elucidated in future work, because PDZ domains bind primarily to specific C-terminal motifs (X(S/T)X(V/L), where X depicts any amino acid) or internal target motifs as well as other PDZ domains (52).

Based upon this study, we propose the following model to explain the sequence of events leading from CD26-CD3 costimulation to NF-B activation (Fig. 8). In CD3-CD26 costimulation, TCR engagement by peptide-loaded major histocompatibility complex class II presented on APC activates phosphatidylinositol 3-kinase via phosphorylation of immunoreceptor tyrosine-based activation motifs in TCR, leading the recruitment of PKC and IKK complex in lipid rafts (16, 18, 25, 38). Concomitantly, CD26 ligation by caveolin-1 on APC recruits CD26-interacting CARMA1 to lipid rafts, resulting in the formation of a CARMA1-Bcl10-MALT1-IKK complex, and this membrane-associated Bcl10 complex then activates IKK through ubiquitination of the NF-B essential modulator. This study involving Jurkat T-cell lines and human peripheral T-cells represents a different cellular system than those with murine T-cells, where other investigators previously described a role for CD26 in thymic development of murine T-cells (53, 54). Our objective with this study was to define a costimulatory ligand for CD26 and proximal signaling molecule of CD26 in human T-cells, with a future aim of analyzing the in vivo role of CD26-mediated T-cell immunity.

In conclusion, we have now demonstrated that CD26 on the T-cell<