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• W2079804706 abstract "The immunological synapse initiates the clustering and stabilization of the T cell receptor by the formation of a large lipid microdomain that accumulates (e.g. CD4/CD8) and segregates (e.g. CD45 and LFA-1) some proteins of the T cell plasma membrane. This work shows that a fraction of transmembrane glycoproteins CD26 and CD45 (the R0 isoform in particular) is present in the rafts of fresh and activated human T lymphocytes. CD26 is proposed as the costimulator of TCR-dependent activation, and CD45 is essential to the T cell activation process because it dephosphorylates at least the inhibitory site of Src kinases. These findings support a more complex model of compartmentation, depending on the stage of T cell maturation and post-transcriptional and post-translational regulation. In addition, interleukin 12 (IL-12; inducer of TH1 responses) drives CD26 and CD45R0 to particular microdomains, thereby involving interleukins in the rules governing raft inclusion or exclusion. The physical association of CD26 and CD45R0 has long been reported. The results presented in this work fit a model in which IL-12 up-regulates a certain type of CD26 expression that interacts on the cell surface with CD45R0, near but outside of the raft core. The use of antisense oligonucleotides for the CD26 mRNAs demonstrated that both events (enhanced by IL-12), CD26-CD45R0 association and membrane compartment redistribution, are related. Thus, CD26 could be part of a shuttling mechanism for CD45 that regulates membrane tyrosine-phosphatase activities, e.g. to control IL-12 receptor-dependent signal transduction. The immunological synapse initiates the clustering and stabilization of the T cell receptor by the formation of a large lipid microdomain that accumulates (e.g. CD4/CD8) and segregates (e.g. CD45 and LFA-1) some proteins of the T cell plasma membrane. This work shows that a fraction of transmembrane glycoproteins CD26 and CD45 (the R0 isoform in particular) is present in the rafts of fresh and activated human T lymphocytes. CD26 is proposed as the costimulator of TCR-dependent activation, and CD45 is essential to the T cell activation process because it dephosphorylates at least the inhibitory site of Src kinases. These findings support a more complex model of compartmentation, depending on the stage of T cell maturation and post-transcriptional and post-translational regulation. In addition, interleukin 12 (IL-12; inducer of TH1 responses) drives CD26 and CD45R0 to particular microdomains, thereby involving interleukins in the rules governing raft inclusion or exclusion. The physical association of CD26 and CD45R0 has long been reported. The results presented in this work fit a model in which IL-12 up-regulates a certain type of CD26 expression that interacts on the cell surface with CD45R0, near but outside of the raft core. The use of antisense oligonucleotides for the CD26 mRNAs demonstrated that both events (enhanced by IL-12), CD26-CD45R0 association and membrane compartment redistribution, are related. Thus, CD26 could be part of a shuttling mechanism for CD45 that regulates membrane tyrosine-phosphatase activities, e.g. to control IL-12 receptor-dependent signal transduction. In the early events of T cell activation, antigen (Ag) 1The abbreviations used are: Ag, antigen; Ab, antibody; mAb, monoclonal antibody; PTP, protein-tyrosine phosphatase; TCR, T cell receptor; PM, plasma membrane; GEM, ganglioside-enriched membrane; GPI, glycosylphosphatidylinositol; AP, alkaline phosphatase; PBMC, peripheral blood mononuclear cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; HDF, heavy density fraction; LDF, light density fraction; MβCD, methyl-β-cyclodextrin; IL, interleukin; TH1 cell, T helper-1 cell; GAM, goat anti-mouse; HRP, horseradish peroxidase; PHA, phytohemagglutinin; EPEI, ethoxylated polyethileneimine; oligos, oligonucleotides; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid. presentation results in the clustering of protein-tyrosine kinases, which associate with the CD3 and TCR subunits and the co-receptors CD4 or CD8 (1van Leeuwen J.E. Samelson L.E. Curr. Opin. Immunol. 1999; 11: 242-248Google Scholar). The transmembrane tyrosine-phosphatase CD45 is essential in this process because it dephosphorylates at least the inhibitory site of Src family kinases, responsible for the phosphorylation of ITAMs (immunoreceptor tyrosine-based activation motifs) (2Baker M. Gamble J. Tooze R. Higgins D. Yang F.T. O'Brien P.C. Coleman N. Pingel S. Turner M. Alexander D.R. EMBO J. 2000; 19: 4644-4654Google Scholar). This extremely active phosphatase does not require ligand binding for optimum catalytic activity; later in the process, CD45-dependent dephosphorylation of key substrates, Src, or other protein-tyrosine kinases (e.g. ZAP-70/Syk) and ITAMs must be avoided. An advance in the understanding of CD45 function was the discovery of specialized membrane domains, called rafts, ganglioside-enriched membranes (GEMs), or detergent-resistant membranes (DRMs). These membranes contain a high density of sphingolipids and cholesterol (2Baker M. Gamble J. Tooze R. Higgins D. Yang F.T. O'Brien P.C. Coleman N. Pingel S. Turner M. Alexander D.R. EMBO J. 2000; 19: 4644-4654Google Scholar, 3Xavier R. Seed B. Curr. Opin. Immunol. 1999; 11: 265-269Google Scholar, 4Horejsi V. Drbal K. Cebecauer M. Cerny J. Brdicka T. Angelisova P. Stockinger H. Immunol. Today. 1999; 20: 356-361Google Scholar) and serve as attachment sites for a variety of lipid-modified proteins (including GPI) and also integral membrane and cytoplasmic proteins (e.g. Src family kinases Lck and Fyn, CD4, CD8, and LAT (linker for activation of T cells)). A compartmentation model has been proposed in which the immunological synapse initiates the clustering and stabilization of the TCR by the formation of a large lipid microdomain that accumulates (e.g. CD4 and CD8) and segregates (e.g. CD45 and LFA-1) several membrane proteins (–6). As the phosphorylation decreases within minutes after the initial response, other phosphatases (not CD45) should be recruited to these rafts later (5Thomas M.L. Curr. Opin. Immunol. 1999; 11: 270-276Google Scholar). However, the role of the extracellular domain of CD45 remains elusive despite its high Mr and structure, which strongly suggest ligand-receptor interactions (6Penninger J.M. Irie-Sasaki J. Sasaki T. Oliveira-dos-Santos A.J. Nat. Immunol. 2001; 2: 389-396Google Scholar). The diversity of the structures and sizes of different CD45 isoforms is cell type-dependent and developmentally regulated. Upon T cell activation, naive T cells switch from isoforms containing A, B, or C epitopes, with post-translational (O- and N-linked glycosylation) information, to the lowest Mr isoform, CD45R0, which lacks sequences coded by 4/A, 5/B, or 6/C exons (6Penninger J.M. Irie-Sasaki J. Sasaki T. Oliveira-dos-Santos A.J. Nat. Immunol. 2001; 2: 389-396Google Scholar, 7Johnson P. Maiti A. Ng D.H.W. Herzenberg L.A. Blackwell C. Weir's Handbook of Experimental Immunology: Cell Surface and Messenger Molecules of the Immune System. 2. Blackwell Science, Oxford, UK1996: 62.1-62.16Google Scholar, 8Dustin M.L. Bromley S.K. Davis M.M. Zhu C. Annu. Rev. Cell Dev. Biol. 2001; 17: 133-157Google Scholar). Several experiments reported distinct CD45 interactions on naive and memory/effector (CD45R0+) cells. CD45– T cell lines transfected with cDNAs of different CD45 isoforms or cells from transgenic and knock-out mice had differential responses to Ag. CD45 has also been reported to associate with several surface molecules such as Thy-1, TCR, CD2, CD3, CD4, CD7, CD8, CD26, CD28, LFA-1, B cell receptor, lymphocyte phosphatase-associated protein, endoplasmic reticulum protein glucosidase II, and CD45 itself (6Penninger J.M. Irie-Sasaki J. Sasaki T. Oliveira-dos-Santos A.J. Nat. Immunol. 2001; 2: 389-396Google Scholar, 7Johnson P. Maiti A. Ng D.H.W. Herzenberg L.A. Blackwell C. Weir's Handbook of Experimental Immunology: Cell Surface and Messenger Molecules of the Immune System. 2. Blackwell Science, Oxford, UK1996: 62.1-62.16Google Scholar, 8Dustin M.L. Bromley S.K. Davis M.M. Zhu C. Annu. Rev. Cell Dev. Biol. 2001; 17: 133-157Google Scholar, 9Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Google Scholar). As the different CD45 isoforms have similar PTP activities, these data suggest that they may interact differentially with other surface molecules that alter PTP accessibility to substrates, modifying in this way the signals received not only through Ag receptors but also through cytokine receptors and integrin-mediated adhesion, to either augment or inhibit T cell activation (9Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Google Scholar, 10Arroyo A.G. Campanero M.R. Sánchez-Mateos P. Zapata J.M. Ursa M.A. del Pozo M.A. Sánchez-Madrid F. J. Cell Biol. 1994; 126: 1277-1286Google Scholar, 11Baldwin T.A. Gogela-Spehar M. Ostergaard H.L. J. Biol. Chem. 2000; 275: 32071-32076Google Scholar, 12Irie-Sasaki J. Sasaki T. Matsumoto W. Opavsky A. Cheng M. Welstead G. Griffiths E. Krawczyk C. Richardson C.D. Aitken K. Iscove N. Koretzky G. Johnson P. Liu P. Rothstein D.M. Penninger J.M. Nature. 2001; 409: 349-354Google Scholar). The surface CD26 glycoprotein is identical to dipeptidyl-peptidase IV (EC 3.4.14.5). T cells expressing high levels of CD26 constitute a subpopulation of CD45R0+ cells with helper activities in B cell Ig synthesis, proliferative responses to soluble Ags and allogeneic cells, secretion of TH1-type cytokines, and transendothelial migration capacity (13Fleischer B. Immunol. Today. 1994; 15: 180-184Google Scholar, 14De Meester I. Korom S. Van Damme J. Scharpé S. Immunol. Today. 1999; 20: 367-375Google Scholar). This study describes the distribution of CD26 and CD45R0 molecules in plasma membrane microdomains of fresh and activated human T cells; it also shows that IL-12 (an inducing TH1 response cytokine) dramatically changes CD45R0 membrane compartmentation through a CD26-CD45R0 association. The significance of this finding is discussed. Cytokines, Antibodies, and Reagents—Recombinant human IL-12 was purchased from PeproTech (London, UK). Lectin from Phaseolus vulgaris (PHA-P) was obtained from Sigma. Three different anti-human CD26s were used. Anti-CD26-FITC (or -PE) Ta1 mAb (murine IgG1) was from Coulter (Hialeah, FL), and 1F7 mAb (murine IgG1) was kindly donated by Prof. S. F. Schlossman (15Morimoto C. Torimoto Y. Levinson G. Rudd C.E. Schrieber M. Dang N.H. Letvin N.L. Schlossman S.F. J. Immunol. 1989; 143: 3430-3439Google Scholar). Anti-CD26 TP1/16 hybridoma, donated by Prof. F. Sánchez-Madrid, was obtained from a fusion with splenocytes from mice immunized with activated human T lymphocytes. Its precise specificity was studied by Western blot and immunoprecipitation as described in this report, comparing the results with those of 1F7 mAb, and by flow cytometry of cDNA-transfected (or not) Jurkat cell lines (clone 11) (16Tanaka T. Camerini D. Seed B. Torimoto Y. Dang N.H. Kameoka J. Dahlberg H.N. Schlossman S.F. Morimoto C. J. Immunol. 1992; 149: 48-486Google Scholar, 17Morimoto C. Lord C.I. Zhang C. Duke-Cohan J.S. Letvin N.L. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9960-9964Google Scholar, 18Herrera C. Morimoto C. Blanco J. Mallol J. Arenzana F. Lluis C. Franco R. J. Biol. Chem. 2001; 276: 19532-19539Google Scholar), comparing the results with Ta1 and 1F7 staining. TP1/16 mAb was used as a hybridoma supernatant or purified from ascitic fluid using affinity chromatography in protein A-Sepharose columns (Amersham Biosciences), isotyped as IgG1 (Sigma; ImmunoType mouse monoclonal antibody isotyping kit), and labeled with FITC (Sigma, Fluorotag FITC conjugation kit). F(ab′)2 goat anti-mouse (GAM), labeled with FITC or PE, and ascitic fluid containing IgG2a and IgG1 isotype control mAbs (UPC10 and MOPC21) were purchased from Sigma. Mouse anti-human CD3 (IgG1, clone UCTH-1), CD71 (transferrin R; IgG2a, clone M-A712), CD4 (IgG1, clone RPA-T4), CD8 (IgG1, clone RPA-T8), HLA-DR (IgG2b, clone TU36), common CD45 (anti-HLe-1, IgG1, clone 2D1), which recognizes a sialic acid-independent epitope, and CD45R0 (IgG2a, clone UHTL-1) mAbs were purchased from Pharmingen or BD Biosciences. Goat anti-mouse H+L was from Caltag, and mouse anti-human CD3 (clone OKT3) mAb was kindly provided by Prof. J. R. Regueiro and Dr. A. Pacheco. Mouse anti-common CD45 (clone D3/9), CD45RA (clone RP1/11), CD45RB (MC5/2), and CD45RC (RP2/19) mAbs, used to study isoform switching, have been described previously (10Arroyo A.G. Campanero M.R. Sánchez-Mateos P. Zapata J.M. Ursa M.A. del Pozo M.A. Sánchez-Madrid F. J. Cell Biol. 1994; 126: 1277-1286Google Scholar, 19Pulido R. Sánchez-Madrid F. Eur. J. Immunol. 1990; 20: 2667-2671Google Scholar). Cell Isolation and Culture—Buffy coats were kindly provided by the Centro de Transfusión de Galicia (Santiago, Spain). Blood was donated by healthy volunteers and human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque PLUS (Amersham Biosciences) density gradient centrifugation as described elsewhere (20Cordero O.J. Salgado F.J. Viñuela J.E. Nogueira M. Immunol. Lett. 1998; 61: 7-13Google Scholar). Cells were cultured (106 PBMCs/ml) in RPMI 1640 (Sigma) supplemented with 10% inactivated fetal calf serum (Invitrogen), 100 μg/ml streptomycin, and 100 IU/ml penicillin (Sigma) in a humidified atmosphere of 5% CO2 at 37 °C. PBMCs were activated with 1–1.5 μg/ml P. vulgaris lectin in the presence or absence of cytokines for the indicated time. The cell lines used were cultured under the same conditions except that Geneticin (100 μg/ml, Sigma) was added to the medium of CD26-transfected cell lines. Inhibition of CD26 Expression with Antisense Morpholino Oligos— Antisense morpholino oligos bound to partially complementary DNA and ethoxylated polyethileneimine (EPEI) were provided by Gene Tools as a Special Delivery Protocol kit. Morpholino subunits are assembled by phosphorodiamidate linkages to obtain an oligo with a modified non-ionic nuclease-resistant backbone. The CD26 antisense sequence, 5′-GAACCTTCCACGGTGTCTTCATCGT-3′, was designed to bind to the target sequence in the region containing AUG of the post-spliced mRNA for the highest effectivity of translation blocking. The fluoresceinated 5′-CCTCTTACCTCAGTTACAATTTATA-3′ morpholino oligo was used as a standard control for nonspecific effects. EPEI electrostatically binds the anionic morpholino-DNA duplex, generating a cationic complex that approaches the anionic cell surface, leading to endocytosis. Subsequent acidification within the endosome increases EPEI ionization driving to membrane permeabilization and oligo release into the cytosol. The ratio of EPEI/morpholino oligos/PBMCs was tested for optimal internalization. Briefly, Morpholino-DNA duplex and EPEI (1.6 nm and 0.64 nm, final concentrations) were mixed and preincubated for 20 min in MilliQ (Millipore) water at room temperature, the mixture was added to the cells (4 × 106) in serum-free RPMI medium, and the samples were incubated for 3 h at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were then washed with RPMI and cultured as described above. Immunostaining and Immunofluorescence—Cell surface Ag expression was measured by direct or indirect immunofluorescence as described (21Cordero O.J. Salgado F.J. Fernández-Alonso C.M. Herrera C. Lluis C. Franco R. Nogueira M. J. Leukocyte Biol. 2001; 70: 920-930Google Scholar). CD45RA, CD45RB, CD45RC, and sometimes TP1/16 mAbs were used as hybridoma supernatant and revealed with FITC- or PE-labeled GAM Abs. The other Abs were applied as primary antibodies. Viable lymphocytes were identified according to their forward and right angle scattering. The percentage of cells positive for the Ag was evaluated by setting negative controls as the omission of primary antibody or the inclusion of isotype controls. Direct immunofluorescent protocol was used for two-color experiments (21Cordero O.J. Salgado F.J. Fernández-Alonso C.M. Herrera C. Lluis C. Franco R. Nogueira M. J. Leukocyte Biol. 2001; 70: 920-930Google Scholar). For studies of detergent resistance of proteins associated with rafts, the work of Janes et al. (22Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Google Scholar) was adapted to flow cytometry. Briefly, cells were treated with 1% Triton X-100 for 5 min on ice, with 10 mm methyl-β-cyclodextrin (MβCD; Sigma), which depletes cellular cholesterol, for 15 min at 37 °C, or with MβCD followed by Triton X-100 extraction, before fixation (3% PFA in PBS for 30 min at room temperature) and staining as described above. Samples were processed on a BD Biosciences FACScalibur cytometer, except where indicated. WinMDI software (a kind gift of J. Trotter, Scripps Institute, La Jolla, CA) was used to analyze data. Protein Concentration Determination—The protein concentration of samples was determined using the Bradford procedure (Sigma). Bovine serum albumin was used as a standard. Membrane Phosphatase and Peptidase in Vitro Assays—Cells were washed twice in RPMI 1640 and resuspended at 3 × 106 cells/ml in hypotonic lysis buffer (HLB) (25 mm Tris-HCl, pH 7.5, 25 mm sucrose, 0.1 mm EDTA, 5 mm MgCl2, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin) before sonication. Individual samples were made up in duplicate in the presence and absence of 1 mm Na3VO4. The membranes were sedimented from post-nuclear supernatants at 100,000 × g for 60 min at 4 °C, the resulting pellet was resuspended in 200 μl HLB by sonication, and the protein concentration was determined. Plasma membrane (PM) protein (20 μg) was incubated as described by Cayota et al. (23Cayota A. Vuillier F. González G. Dighiero G. Clin. Exp. Immunol. 1996; 104: 11-17Google Scholar) in a reaction mixture of 5 mmp-nitrophenyl phosphate (Sigma 104® substrate), 80 mm MES, pH 5.5, 10 mm EDTA, and 10 mm dithiothreitol at 37 °C. After a 20-min incubation, reaction was stopped by the addition of 1 ml of 0.2 n NaOH. PTP activity was expressed as absorbance units at 405 nm after removal of values of the samples duplicated in the presence of vanadate. Dipeptidyl-peptidase IV activity was measured as described previously (24Cordero O.J. Salgado F.J. Viñuela J.E. Nogueira M. Immunobiology. 1997; 197: 522-533Google Scholar) and expressed as absorbance units at 405 nm. Briefly, PM protein (20–40 μg) was incubated for 1 h at 37 °C in the presence of 0.5 mg/ml Gly-Pro p-nitroanilide tosylate (Sigma). Isolation of GPI-enriched Membranes by Equilibrium Density Gradient Centrifugation—The following steps were carried out at 4 °C unless indicated otherwise, basically following the works of Ilangumaran et al. (25Ilangumaran S. Briol A. Hoessli D.C. Biochim. Biophys. Acta. 1997; 1328: 227-236Google Scholar, 26Ilangumaran S. Arni S. van Echten-Deckert G. Borisch B. Hoessli D.C. Mol. Biol. Cell. 1999; 10: 891-905Google Scholar). Cells (50 × 106) were washed twice in PBS and once in TKM buffer (50 mm Tris-HCl, pH 7.5, 25 mm KCl, 5 mm MgCl2, and 1 mm EDTA). Detergent lysates were prepared in TKM containing 0.5% Triton X-100 and the protease inhibitors Pefabloc SC (2 mm), leupeptin (10 μg/ml), and aprotinin (5 μg/ml) for 20 min on ice. For equilibrium gradient centrifugation, cell extracts were adjusted to 40% sucrose, loaded into SW55Ti tubes (Beckman L8-M), overlaid with 2.7 ml of 36% sucrose, and finally completed with 1.575 ml of 5% sucrose in TKM buffer. After centrifugation at 200,000 × g for 18 h, 450-μl fractions were collected and stored at –20 °C. Fraction proteins were evaluated by Western blotting or immunoprecipitation or were detected by dot immunoassay. Serial dilutions in PBS (200 μl) of detergent-lysates or sucrose density fractions were applied to the wells and dotted onto nitrocellulose filter sheets using a Bio-Rad dot-blot apparatus (25Ilangumaran S. Briol A. Hoessli D.C. Biochim. Biophys. Acta. 1997; 1328: 227-236Google Scholar). Western Blotting Analysis and Immunoprecipitation—Differentially activated cells were washed in cold PBS and harvested in lysis buffer at 4 × 106 cells/100 μl (20 mm Tris, pH 7.4, 0.15 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm EDTA, 10 μg/ml leupeptin, 5 μg/ml aprotinin, and 1% Triton X-100; or alternatively, 20 mm triethanolamine, pH 7.8, 0.15 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 0.12% Triton X-100, and 1% digitonin as described by Torimoto et al. (27Torimoto Y. Dang N.H. Vivier E. Tanaka T. Schlossman S.F. Morimoto C. J. Immunol. 1991; 147: 2514-2517Google Scholar)). After 30 min on ice, nuclei and debris were removed by centrifugation at 13,000 × g for 15 min, and cleared lysates were assayed for protein content. Samples, normalized for total protein or for number of cells, were run on 7.5% SDS-PAGE and electrotransferred to nitrocellulose (Schleicher & Schuell) or polyvinylidene difluoride membranes (Amersham Biosciences) for analysis with the appropriate primary Ab (TP1/16, D3/9, or UCHL-1) and HRP-labeled secondary Ab. For Western blotting with D3/9 or TP1/16 Abs, samples were treated under nonreducing conditions with SDS-PAGE buffer at 37 °C for 15 min. Detection was carried out by the chemiluminescent system (ECL, Amersham Biosciences). For immunoprecipitation studies, cell surface proteins were sometimes biotinylated following the manufacturer's instructions (Pierce). Briefly, 25 × 106 lymphocytes/ml were resuspended in PBS, pH 8.0, containing 0.5 mg/ml sulfo-NHS-biotin for 30 min at room temperature. When required, the depletion of microdomain-linked proteins was carried out according to Cheng et al. (28Cheng P.C. Dykstra M.L. Mitchell R.N. Pierce S.K. J. Exp. Med. 1999; 190: 1549-1560Google Scholar). Briefly, washed cells (50 × 106) were resuspended in 1 ml of ice-cold Hanks' balanced salt solution with 1 μg/ml HRP-conjugated cholera toxin B subunit (which binds membrane GM1; Sigma) for 30 min at 37 °C. Then cells were washed twice and resuspended in 1 ml of 0.5 mg/ml 3–3′-diaminobenzidine in Hanks' balanced salt solution with or without H2O2 for 45 min at 4 °C. After lysis, polymerized raft proteins were removed as described above. Immunoprecipitation was performed with anti-CD26 TP1/16 (in contrast to 1F7, anti-CD26 TP1/16 was more effective with Triton than digitonin lysates), anti-CD45 D3/9, or anti-CD45R0 mAbs, previously coupled for 1 h at 4 °C to anti-mouse-agarose beads (Sigma; 50 μl of 1:1 lysis buffer). Occasionally, precleared lysates were incubated first with antibodies. Precipitated protein was recovered by centrifugation, washed three times in lysis buffer, eluted by boiling in SDS-PAGE sample buffer, and analyzed as described above, except for the filters with biotinylated proteins, which were incubated sequentially with streptavidin, biotinylated alkaline phosphatase, and CDP-Star substrate (New England Biolabs). Developed filters were exposed on X-Omat S film (Eastman Kodak Co.) several times depending on the intensity of the signal. When needed, the blots were stripped as described elsewhere (29Mirabet M. Herrera C. Cordero O.J. Mallol J. Lluis C. Franco R. J. Cell Sci. 1999; 112: 491-502Google Scholar). Dot blots were treated as described above, except when measuring alkaline phosphatase (AP) levels. AP activity was developed directly with bromochloroindolyl phosphate/nitro blue tetrazolium substrate (BCIP/NBT, BioRad). The spots were quantitated by scanning the filters and densitometry (ImageMaster 1D, Amersham Biosciences). Cell Proliferation and Calcium Assays—Three-day PHA-blasts were cultured in 96-well culture plates at 1 × 105 cells/well in a total volume of 100 μl of complete medium with/without 20% conditioned medium (medium in which PBMCs were activated previously for 3 days). In the preincubation assays, cells were cultured with the anti-CD45R0 and CD45RA Abs for 30 min before adding the cytokines, whereas in the postincubation assays, the Abs were added 30 min after the cytokines. Controls were cells incubated in the absence of cytokines. After 2 days of culture, 20 μl/well CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) was added to the plate 4 h before the absorbance was recorded at 492 nm in a Labsystems Multiskan MS plate reader. All cultures were performed in triplicate. For calcium measurements, 5 × 106/ml washed cells were resuspended in calcium-containing assay buffer (145 mm NaCl, 5 mm KCl, 0.5 mm MgSO4,5mm glucose, 1 mm CaCl2,10mm HEPES, 1 mm Na2HPO4) with 4 μg/ml fluo-3-penta-acetoxymethylester (Fluo-3 AM; Molecular Probes, Eugene, OR) in the presence of 0.025% Pluronic F-127 (Molecular Probes) and 2 mm probenecidin (Sigma) for 30-45 min at 37 °C. After washing, cells were diluted (250,000 cells/ml) and warmed prior to use (37 °C for 15 min). Lymphocyte stimulation was carried out with anti-CD3 mAb OKT3 (t = 80 s) plus GAM H+L mAb (t = 280 s). Analyses were performed by flow cytometry and MFI 3.4J2 software, a kind gift of Eric Martz (Scripps Institute), for mean fluorescence intensity data. IL-12 Enhances PM CD26-CD45R0 Interaction—Previously we reported a strong IL-12-dependent surface CD26 up-regulation on activated human T cells, including the effector/memory CD45R0 subset (25Ilangumaran S. Briol A. Hoessli D.C. Biochim. Biophys. Acta. 1997; 1328: 227-236Google Scholar, 30Salgado F.J. Vela E. Martín M. Franco R. Nogueira M. Cordero O.J. Cytokine. 2000; 12: 1136-1141Google Scholar). A weaker staining of these IL-12 blasts with anti-CD45R0 UCHL-1 mAb, previously observed (20Cordero O.J. Salgado F.J. Viñuela J.E. Nogueira M. Immunol. Lett. 1998; 61: 7-13Google Scholar), is shown on the same cells (Fig. 1A). Explanations such as loss of sialylation, isoform switching, or CD45 internalization in the presence of IL-12 were ruled out (data not shown). Down-regulation was discarded, because IL-12 enhanced the levels of PM PTP enzymatic activity, which is ∼90% CD45-specific (31Mustelin T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6302-6306Google Scholar) (Fig. 1B). Because CD26 is known to associate with CD45 (6Penninger J.M. Irie-Sasaki J. Sasaki T. Oliveira-dos-Santos A.J. Nat. Immunol. 2001; 2: 389-396Google Scholar, 7Johnson P. Maiti A. Ng D.H.W. Herzenberg L.A. Blackwell C. Weir's Handbook of Experimental Immunology: Cell Surface and Messenger Molecules of the Immune System. 2. Blackwell Science, Oxford, UK1996: 62.1-62.16Google Scholar, 8Dustin M.L. Bromley S.K. Davis M.M. Zhu C. Annu. Rev. Cell Dev. Biol. 2001; 17: 133-157Google Scholar, 9Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Google Scholar), we performed immunoprecipitation studies. Fig. 1C shows that, in the presence of IL-12 together with the expected CD26 up-regulation, anti-CD26 TP1/16 mAb also coprecipitates more CD45 (the two low Mr isoforms). In consonance with the above result, neither CD45 nor R0 Ags (which were surface biotinylated) were down-regulated by IL-12. The nature of the coprecipitated bands is confirmed later on in this article. An attractive model to explain these results is that IL-12 is up-regulating a certain type of CD26 expression (already existing; see Fig. 1C) that induces interaction with CD45R0 and/or other molecules and masks the anti-R0 Ab epitope. Thus, as observed in Fig. 1, UCHL-1 could precipitate only the free CD45. CD26 and CD45 Are Present in PM Microdomains, and Their Distribution Changes with T Cell Activation in the Presence or Absence of IL-12—CD26 was found in GEMs (4Horejsi V. Drbal K. Cebecauer M. Cerny J. Brdicka T. Angelisova P. Stockinger H. Immunol. Today. 1999; 20: 356-361Google Scholar) of mouse T cell lines (25Ilangumaran S. Briol A. Hoessli D.C. Biochim. Biophys. Acta. 1997; 1328: 227-236Google Scholar, 26Ilangumaran S. Arni S. van Echten-Deckert G. Borisch B. Hoessli D.C. Mol. Biol. Cell. 1999; 10: 891-905Google Scholar) and porcine lung (32Parkin E.T. Turner A.J. Hooper N.M. Biochem. J. 1996; 319: 887-896Google Scholar). We considered a possible relationship between the CD26-CD45R0 association and GEMs/PM rafts. The well known GPI-anchored (32Parkin E.T. Turner A.J. Hooper N.M. Biochem. J. 1996; 319: 887-896Google Scholar) AP was used as a control for microdomain purification by equilibrium density gradient centrifugation. AP was detected in the light density fractions (LDF) (Fig. 2A, lanes 4–6). Transferrin R (CD71) was used as control for soluble, non-raft protein (data not shown). The dual distribution of CD26, with ∼28% of the material detected in the LDF and 51% in the heavy density fractions (HDF) (Fig. 2A, lanes 10 and 11) of the sucrose gradient, is shown in human T cells (because monocytes and B and NK cells from PBMCs are essentially CD26–) (Fig. 2C). In activated T cells, in addition to a higher intensity, CD26 was redistributed to the GEMs (37% in LDF and 30% in HDF, percentages that are in agreement with data from mouse T cell line (25Ilangumaran S. Briol A. Hoessli D.C. Biochim. Biophys. Acta. 1997; 1328: 227-236Google Scholar), indicating the activated status of T cells). IL-12 enhanced CD26 intensity and enriched the intermediate fractions (33 and 26%, respectively in LDF and HDF, n = 4; Fig. 2, C and F). Note that there is more CD26 than the well described CD4 glycoprotein (which shows a bipolar pattern) in the rafts. In fact, CD26 is present in almost all sucrose gradient fractions, including fraction 3 (AP and CD4 are not detected in this fraction) (Fig. 2B). As described previously in a murine T cell lymphoma (25Ilangumaran S. Briol A. Hoessli D.C. Biochim. Biophys. Acta. 1997; 1328: 227-236Google Scholar) but not in Jurkat or B cells (22Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Google Scholar, 28Cheng P.C. Dykstra M.L. Mitchell R.N. Pierce S.K. J. Exp. Med. 1999; 190: 1549-1560Google Scholar, 33Rodgers W. Rose J.K. J." @default.
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• W2079804706 title "A Role for Interleukin-12 in the Regulation of T Cell Plasma Membrane Compartmentation" @default.
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