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• W2069349270 abstract "The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4+ T lymphocytes and T cell lines express CD3ɛ chain isoforms with different isoelectric points (pI), with important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3ɛ chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3ɛ chain isoforms. At a high pI, degraded CD3ɛ isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3ɛ. The abundance of CD3ɛ isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3ɛ isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3ɛ isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3ɛ isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3ɛ chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3ɛ N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection. The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4+ T lymphocytes and T cell lines express CD3ɛ chain isoforms with different isoelectric points (pI), with important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3ɛ chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3ɛ chain isoforms. At a high pI, degraded CD3ɛ isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3ɛ. The abundance of CD3ɛ isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3ɛ isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3ɛ isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3ɛ isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3ɛ chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3ɛ N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection. The antigen T cell receptor (TCR) 4The abbreviations used are: TCR, T cell receptor; IFN-γ, interferon-γ; ERK, extracellular signal-regulated kinase; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; IEF, isoelectric focusing; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EGFP, enhanced green fluorescent protein; DP, double-positive; SP, single-positive; pMHC, peptide-major histocompatibility complex. complex is responsible for antigen recognition through TCRαβ (or TCRγδ) variable heterodimers. These are noncovalently associated with CD3γ, CD3δ, CD3ɛ, and ζ (CD247) polypeptides involved in the initiation of signal transduction and the control of complex expression (reviewed in Refs. 1Pitcher L.A. van Oers N.S.C. Trends Immunol. 2003; 24: 554-560Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar and 2Alarcón B. Gil D. Delgado P. Schamel W.W.A. Immunol. Rev. 2003; 191: 38-46Crossref PubMed Scopus (111) Google Scholar). Current structural models of the TCR-CD3 complex support the idea that each minimal subunit contains one TCRαβ (or TCRγδ) heterodimer and two CD3ɛ polypeptides per TCR heterodimer (3Call M. Pyrdol J. Wucherpfennig K. EMBO J. 2004; 23: 2348-2357Crossref PubMed Scopus (86) Google Scholar, 4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 5Blumberg R.S. Ley S. Sancho J. Lonberg N. Lacy E. McDermott F. Schad V. Greenstein J.L. Terhorst C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7220-7224Crossref PubMed Scopus (101) Google Scholar, 6de la Hera A. Muöller U. Olsson C. Isaaz S. Tunnacliffe A. J. Exp. Med. 1991; 173: 7-17Crossref PubMed Scopus (116) Google Scholar, 7Schamel W.W.A. Arechaga I. Risueño R.M. van Santen H.M. Cabezas P. Risco C. Valpuesta J.M. Alarcón B. J. Exp. Med. 2005; 202: 493-503Crossref PubMed Scopus (247) Google Scholar, 8Hayes S.M. Love P.E. J. Exp. Med. 2006; 203: 47-52Crossref PubMed Scopus (30) Google Scholar). CD3ɛ ectodomains pair noncovalently with one CD3γ or CD3δ chain through unique excluding sites in their G strands and membrane-proximal stalk sequences, and it is thus assumed that there is one CD3ɛδ and one CD3ɛγ dimer per complex (9Alarcón B. Ley S.C. Sánchez-Madrid F. Blumberg R.S. Ju S.T. Fresno M. Terhorst C. EMBO J. 1991; 10: 903-912Crossref PubMed Scopus (92) Google Scholar, 10Borroto A. Mallabiabarrena A. Albar J.P. Martiínez A.C. Alarcón B. J. Biol. Chem. 1998; 273: 12807-12816Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Sun Z-Y. J. Kim K.S Wagner G. Reinherz E.L Cell. 2001; 105: 913-923Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 12Sun Z-Y. J. Kim S.T Kim I.C Fahmy A. Reinherz E.L. Wagner G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16867-16872Crossref PubMed Scopus (95) Google Scholar, 13Kjer-Nielsen L. Dunstone M.A. Kostenko L. Ely L.K. Beddoe T. Mifsud N.A. Purcell A.W. Brooks A.G. McCluskey J. Rossjohn J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7675-7680Crossref PubMed Scopus (124) Google Scholar, 14Arnett K.L. Harrison S.C. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16268-16273Crossref PubMed Scopus (115) Google Scholar). Finally, it has been shown that covalently linked ζ chain homodimers are needed for efficient transport of complete TCR-CD3 complexes from the endoplasmic reticulum to the cell surface (reviewed in Ref. 2Alarcón B. Gil D. Delgado P. Schamel W.W.A. Immunol. Rev. 2003; 191: 38-46Crossref PubMed Scopus (111) Google Scholar), and data from in vitro assembly of TCR-CD3 chains suggest the association of one ζ dimer per complex (3Call M. Pyrdol J. Wucherpfennig K. EMBO J. 2004; 23: 2348-2357Crossref PubMed Scopus (86) Google Scholar, 4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). From these data, it follows that the minimal TCR-CD3 complex unit contains eight polypeptides (αβ·ɛδ·ɛγ·ζ2). The data summarized above have been generated using many different cells and cell lines in diverse experimental approaches, and it would be reasonable to conclude that the TCR-CD3 complex is a constant structure, the components of which (beyond those differences arising from V region variability) are equal in all T cells. However, the existing data also indicate that there are profound quantitative and qualitative changes during the development of not only T lymphocytes (15von Boehmer H. Fehling H.J. Annu. Rev. Immunol. 1997; 13: 93-126Google Scholar), but also among different mature T cell subsets or T cell lines. Concerning CD3, different ratios of CD3γ and CD3δ polypeptides in TCR-CD3 complexes from different T cell lines have been reported (9Alarcón B. Ley S.C. Sánchez-Madrid F. Blumberg R.S. Ju S.T. Fresno M. Terhorst C. EMBO J. 1991; 10: 903-912Crossref PubMed Scopus (92) Google Scholar). Furthermore, CD3δ chains are absent, and there are wide differences in CD3γ chain glycosylation in γδ T cells (16Hayes S.M. Laky K. El-Khoury D. Kappes D.J. Fowlkes B.J. Love P.E. J. Exp. Med. 2002; 196 (Correction (2002) J. Exp. Med.196, 1653): 1355-1361Crossref PubMed Scopus (22) Google Scholar). Even so, TCRγδ-CD3 complexes contain two CD3ɛγ dimers per complex (8Hayes S.M. Love P.E. J. Exp. Med. 2006; 203: 47-52Crossref PubMed Scopus (30) Google Scholar). TCR-CD3 complexes naturally form aggregates, the degree of aggregation of which within one cell or among T cells cannot be easily ascribed to the nature of the TCR antigen recognition unit (7Schamel W.W.A. Arechaga I. Risueño R.M. van Santen H.M. Cabezas P. Risco C. Valpuesta J.M. Alarcón B. J. Exp. Med. 2005; 202: 493-503Crossref PubMed Scopus (247) Google Scholar, 17Hellwig S. Schamel W.W.A. Pflugfelder U. Gerlich B. Weltzien H.U. Immunobiology. 2005; 210: 685-694Crossref PubMed Scopus (14) Google Scholar). Differences in TCR-CD3 structure among human or mouse T cell lines have been also detected biochemically and by differences in their relative recognition by anti-TCR or anti-CD3 antibodies (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar, 19Zapata D.A. Pacheco-Castro A. Torres P.S. Ramiro A.R. San José E. Alarcón B. Alibaud L. Rubin B. Toribio M.L. Regueiro J.R. J. Biol. Chem. 1999; 274: 35119-35128Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 20Zapata D.A. Schamel W.W.A. Torres P.S. Alarcón B. Rossi N.E. Navarro M.N. Toribio M.L. Regueiro J.R. J. Biol. Chem. 2004; 279: 24485-24492Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). All these differences might have important functional consequences if they can alter the factors determining the efficiency of TCR-mediated signals. These include the efficiency of spontaneous (7Schamel W.W.A. Arechaga I. Risueño R.M. van Santen H.M. Cabezas P. Risco C. Valpuesta J.M. Alarcón B. J. Exp. Med. 2005; 202: 493-503Crossref PubMed Scopus (247) Google Scholar, 17Hellwig S. Schamel W.W.A. Pflugfelder U. Gerlich B. Weltzien H.U. Immunobiology. 2005; 210: 685-694Crossref PubMed Scopus (14) Google Scholar) or ligand-induced oligomerization/polymerization of TCR-CD3 complexes, the sensitivity to induction of conformational changes upon ligand binding (21Gil D. Schamel W.W.A. Montoya M. Sánchez-Madrid F. Alarcón B. Cell. 2002; 109: 901-912Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), and the efficiency of coreceptor association with the TCR-CD3 complex (22Suzuki S. Kupsch J. Eichmann K. Saizawa M.K. Eur. J. Immunol. 1992; 22: 2475-2479Crossref PubMed Scopus (39) Google Scholar, 23Doucey M-A. Goffin L. Naeher D. Michielin O. Baumgaörtner P. Guillaume P. Palmer E. Luescher I.F. J. Biol. Chem. 2003; 278: 3257-3264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In turn, these differences could correlate with the amount and distribution of molecules involved in the activation of distinct pathways and/or the threshold and kinetic patterns of T cell activation. In a previous study (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar), we detected differences in the recognition of mouse CD3 by monoclonal antibodies that were linked to differences in the N-terminal sequence of CD3ɛ as determined by recognition with an N-terminal peptide-specific antibody. These differences were not due to alternative splicing of the mini-exons coding for the N-terminal sequence of CD3ɛ, but were due to degradation by proteinases (including metalloproteinases) sensitive to phenanthroline. Interestingly, the association of CD3 with the TCR is weaker when CD3ɛ N-terminal sequences are degraded (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). Here, we have taken advantage of the presence of negatively charged amino acid residues in the N-terminal sequence of CD3ɛ to further analyze this phenomenon. Removal of these charged residues should affect the isoelectric point of CD3ɛ, rendering isoforms with distinct pI values that could be distinguished by two-dimensional PAGE. Furthermore, we have generated mutant CD3ɛ chains lacking charged amino acids and showed that their loss can enhance the response to TCR-mediated activation. Antibodies and Other Reagents—The following antibodies were used: 3D3 (clonotypic anti-D10 TCR, mouse IgG1) (24Kaye J. Porcelli S. Tite J. Jones B. Janeway Jr., C.A. J. Exp. Med. 1983; 158: 836-856Crossref PubMed Scopus (558) Google Scholar), F23.1 (anti-mouse TCR Vβ8, mouse IgG2a) (25Staerz U.D. Karasuyama H. Garner A.M. Nature. 1987; 329: 449-451Crossref PubMed Scopus (136) Google Scholar), YCD3-1 (anti-mouse CD3, rat IgG2b) (26Portolés P. Rojo J. Golby A. Bonneville M. Gromkowski S. Greenbaum L. Janeway Jr., C.A. Murphy D.B. Bottomly K. J. Immunol. 1989; 142: 4169-4175PubMed Google Scholar), 500A2 (anti-mouse CD3, hamster IgG2b) (27Havran W.L. Poenie M. Kimura J. Tsien R. Weiss A. Allinson J.P. Nature. 1987; 330: 170-173Crossref PubMed Scopus (233) Google Scholar), GK1.5 (anti-mouse CD4, rat IgG2b) (28Dialynas D.P. Wilde D.B. Marrack P. Pierres A. Wall K. Harran W. Otten G. Liken M. Pierres M. Kappler J. Fitch F. Immunol. Rev. 1983; 74: 29-56Crossref PubMed Scopus (905) Google Scholar), M1/70 (rat anti-mouse CD11b) (29Springer T. Galfré G. Secher D.S. Milstein C. Eur. J. Immunol. 1979; 9: 301-306Crossref PubMed Scopus (870) Google Scholar), and Y-19 (rat anti-mouse CD90) (30Jones B. Janeway Jr., C.A. Eur. J. Immunol. 1981; 11: 584-592Crossref PubMed Scopus (34) Google Scholar). They were purified from culture supernatants on protein A or G affinity columns. Where indicated, the purified antibodies were conjugated with biotin, fluorescein, or DyLight 649 using N-hydroxysuccinimidobiotin or fluorescein isothiocyanate (Sigma) or with DyLight 649 N-hydroxysuccinimidoester (Pierce) by standard procedures or as indicated by the manufacturer. Hamster anti-mouse CD28 antibody 37.51, anti-phosphotyrosine antibody PY-20, and phycoerythrin-conjugated anti-mouse interferon-γ (IFN-γ) antibody XMG1.2 were from Pharmingen. Rabbit polyclonal antibodies against the mouse CD3ɛ extracellular domain and ZAP-70 have been described (31Jiménez-Periañez A. Ojeda G. Criado G. Sánchez A. Pini E. Madrenas J. Rojo J.M. Portolés P. J. Leukocyte Biol. 2005; 78: 1386-1396Crossref PubMed Scopus (23) Google Scholar, 32Feito M.J. Vaschetto R. Criado G. Sánchez A. Chiocchetti A. Jiménez-Periañez A. Dianzani U. Portolés M.P. Rojo J.M. Eur. J. Immunol. 2003; 33: 204-214Crossref PubMed Scopus (34) Google Scholar). Rabbit antiserum against a peptide comprising Gly50– Gly63 of mouse CD4 was raised by immunizing with the peptide coupled to ovalbumin. All these rabbit antibodies were affinity-purified over columns of the immunizing antigen coupled to CNBr-Sepharose. Rabbit anti-phosphothreonine/phosphotyrosine ERK antibody was from Promega Corp. Rabbit anti-ERK2 antibody was from Santa Cruz Biotechnology, Inc. Horseradish peroxidase-coupled anti-mouse IgG, anti-rabbit IgG, protein A, and ExtrAvidin were from Sigma. Phenanthroline, phorbol 12-myristate 13-acetate, and brefeldin were from Sigma; ionomycin was from Calbiochem. Cells and Cell Lines—The mouse Th2 T cell lines SR.D10 (D10) (33Ojeda G. Ronda M. Ballester S. Diíez-Orejas R. Feito M.J. Garciía-Albert L. Rojo J.M. Portolés P. Cell. Immunol. 1995; 164: 265-278Crossref PubMed Scopus (21) Google Scholar), D10.TCR2.3 (34Dittel B.N. Sant'Angelo D.B. Janeway Jr., C.A. J. Immunol. 1997; 158: 4065-4073PubMed Google Scholar), and AK-8 (35Rojo J.M. Kerner J.D. Janeway Jr., C.A. Eur. J. Immunol. 1989; 19: 2061-2067Crossref PubMed Scopus (19) Google Scholar, 36Hong S-C. Sant'Angelo D.B. Dittel B.N. Medzhitov R. Yoon S.T. Waterbury P.G. Janeway Jr., C.A. J. Immunol. 1997; 159: 4395-4402PubMed Google Scholar) are specific for a peptide comprising residues 134–146 of chicken conalbumin and major histocompatibility complex (MHC) class II of the κ haplotype. AE103 is an I-Ak-specific Th1 clone (37Yagi J. Baron J. Buxser S. Janeway Jr., C.A. J. Immunol. 1990; 144: 892-901PubMed Google Scholar). They were cultured and grown as described previously in detail (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). Spleen and thymus cell suspensions were obtained from BALB/c mice. Plasmids and Transfectants—A mouse CD3ɛ cDNA (nucleotides 69–650 of GenBank™/EBI Data Bank accession number NM007648, including the mouse CD3ɛ open reading frame between nucleotides 80 and 649) was amplified by reverse transcription-PCR from total RNA extracted from SR.D10 cells using oligonucleotides 5′-GAGAGAGAATTCTGAGAGGATGCGG-3′ (sense) and 5′-GTCAGACTGCTCTCTGATTCAGGCC-3′ (antisense). The amplified cDNA was ligated into the pTA-TOPO plasmid vector (Invitrogen). A C-terminal sequence tag for five residues of the vesicular stomatitis virus glycoprotein was introduced by PCR amplification using the CD3ɛ cDNA cloned in pTA-TOPO as a template and primers 5′-GAGAGAGAATTCTGAGAGGATGCGG-3′ (sense) and 5′-CGGAATTCTCATTTGCCAAGCCGGTTGACTGCTCTCTGATTCAGGCC-3′ (antisense). The PCR product was digested with the restriction enzyme EcoRI and ligated into the pSRα vector (pSRα/CD3ɛ-VSV). The insert was extracted using EcoRI and ligated into the bicistronic pIRES2-EGFP expression vector (Clontech). This plasmid (pIRES2-EGFP/CD3ɛ) was grown in Escherichia coli DH5α, purified, and sequenced. A cDNA coding for CD3ɛ with three acidic N-terminal residues (Asp2, Asp3, Glu5) mutated to Gly by A-to-G substitutions in the relevant codons was obtained using the QuikChange site-directed mutagenesis kit (Stratagene) using pSRα/CD3ɛ-VSV as template and primers 5′-GGCACTTGCCAGGGCGGTGCCGGGAACATTGGATACAAAGTCTCC-3′ (sense) and 5′-GGAGACTTTGTATCCAATGTTCCCGGCACCGCCCTGGCAAGTGCC-3′ (antisense). The plasmid containing the mutant CD3ɛ DNA was grown and sequenced as described above to confirm the mutations. After digestion with EcoRI, the insert was subcloned into the pIRES2-EGFP vector to generate the pIRES2-EGFP/CD3ɛMUT expression vector. AE103 cells (2 × 106/100 μl) were transfected with 2 μg of DNA of the bicistronic expression vectors pIRES2-EGFP, pIRES2-EGFP/CD3ɛ, and pIRES2-EGFP/CD3ɛMUT using program O-17 of Nucleofector (Amaxa Biosystems). Stable transfectants were selected with Geneticin, and the transfectants were checked for green fluorescent protein and TCR expression. Cell Activation—For antigen activation, 104 T cells were cultured in Click’s medium supplemented with 10% inactivated fetal calf serum (culture medium) in 96-well plates (Costar) with mitomycin C-treated 105 B10.BR spleen cells as antigen-presenting cells plus the indicated concentrations of conalbumin-(134–146) peptide. Antigen-presenting cells were previously depleted of T cells by incubation with anti-CD90 antibody and rabbit complement. After 72 h of incubation at 37 °C and 5% CO2, proliferation was determined by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described (38Sánchez A. Feito M.J. Rojo J.M. Eur. J. Immunol. 2004; 34: 2439-2448Crossref PubMed Scopus (38) Google Scholar). For activation with pervanadate, a pervanadate solution was prepared by adding 5 μl of 1 m H2O2 to 1 ml of 5 mm NaVO4 (pH 7.0) in culture medium that was left for 20 min at room temperature. Pervanadate was then added to cells in culture medium (10 μl of pervanadate solution/100 μl of cells at 108 cells/ml). After 5 min of incubation at 37 °C in a water bath, the reaction was stopped with ice-cold phosphate-buffered saline (PBS), 0.5 mm EDTA, and 1 mm NaVO4. To determine early activation events in SR.D10 cells, anti-TCR antibody 3D3 or a control antibody was adsorbed (5 μg/ml) to polystyrene microbeads. These beads were mixed with cells (107 in 100 μl of culture medium/sample) at a 1:1 ratio as described (32Feito M.J. Vaschetto R. Criado G. Sánchez A. Chiocchetti A. Jiménez-Periañez A. Dianzani U. Portolés M.P. Rojo J.M. Eur. J. Immunol. 2003; 33: 204-214Crossref PubMed Scopus (34) Google Scholar). Cells were incubated for 5 min at 37 °C in a water bath, and the reaction was stopped with ice-cold PBS, 0.5 mm EDTA, and 1 mm NaVO4. For intracellular detection of IFN-γ in AE103 transfectants, cells were incubated at 37 °C in 24-well culture plates precoated with anti-TCR antibody F23.1 (10 μg/ml, 106 cells/ml of culture medium, 1 ml/well, in the presence of 10 μg/ml anti-CD28 antibody). After 90 min of incubation at 37 °C, brefeldin (10 μg/ml) was added. For TCR-independent activation, the cells were stimulated with phorbol 12-myristate 13-acetate (20 ng/ml) plus 1 μm ionomycin. Surface Biotinylation—Cells (10–20 × 106 cells/ml, 1 ml/sample) were washed with cold PBS and resuspended in 20 mm HEPES and 150 mm NaCl (pH 8.8). The cells were then vectorially biotinylated with N-hydroxysulfosuccinimidobiotin (Pierce) as described in detail (39Portolés P. Rojo J.M. Janeway Jr., C.A. J. Immunol. Methods. 1990; 129: 105-109Crossref PubMed Scopus (10) Google Scholar). Immunoprecipitation, Isoelectric Focusing (IEF), and Immunoblotting—Immunoprecipitations of cell-surface TCR or CD3 were performed with 2 × 107 cells/determination as described (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). Briefly, cells were incubated with antibodies in cold PBS containing 2% inactivated fetal calf serum and 0.1% sodium azide (staining buffer). Unbound antibodies were washed, and the cells were lysed on ice for 30 min with lysis buffer (1 ml/107 cells; 10 mm CHAPS in 50 mm Tris-HCl and 150 mm NaCl (pH 7.6) containing 1 mm MgCl2, 1 mm EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm NaVO4). After centrifugation, post-nuclear lysates were precleared by rotation with an irrelevant antibody coupled to Sepharose and centrifuged, and immunoprecipitation was carried out for 2 h at 4 °C by rotation of the precleared supernatants with affinity-purified rabbit anti-mouse or anti-rat antibodies coupled to Sepharose as appropriate. The immunoprecipitates were eventually washed five times with cold wash buffer (2 mm CHAPS in 50 mm Tris-HCl and 150 mm NaCl (pH 7.6) containing 1 mm MgCl2, 1 mm EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm NaVO4) and extracted with SDS-PAGE sample buffer. Immunoprecipitations from cell lysates were performed as above, except that precipitation was performed with anti-rat antibodies coupled to Sepharose beads previously incubated with rat anti-CD3 antibody YCD3-1 (3 μg/sample) and washed. For IEF, the last immunoprecipitation wash was done with distilled water, and the immunoprecipitates were extracted with 7 m urea, 2 m thiourea, 4% Triton X-100, and 100 mm dithiothreitol plus ampholytes (Bio-Lyte 3–10, Bio-Rad) and bromphenol blue. The extracted samples were used to rehydrate polyacrylamide IEF strips (7-cm ReadyStrip™ immobilized pH gradient strips (pH 3–10), Bio-Rad). After active rehydration of the strips (12 h, 500 V), IEF was performed for 15 min at 250 V, for 1 h at 1000 V, for 1 h at 8000 V, and at 500 V for the time needed to achieve 8000–13,000 V-h. The strips were then incubated at room temperature for 10 min each with 6 m urea, 2% SDS, 375 mm Tris-HCl (pH 8.8), 20% glycerol, 130 mm dithiothreitol, and bromphenol blue and then with 6 m urea, 2% SDS, 375 mm Tris-HCl (pH 8.8), 20% glycerol, 135 mm iodoacetamide, and bromphenol blue. Immunoblotting of immunoprecipitates and lysates was performed as described (32Feito M.J. Vaschetto R. Criado G. Sánchez A. Chiocchetti A. Jiménez-Periañez A. Dianzani U. Portolés M.P. Rojo J.M. Eur. J. Immunol. 2003; 33: 204-214Crossref PubMed Scopus (34) Google Scholar) using horseradish peroxidase-conjugated protein A for immunoprecipitates, horseradish peroxidase-conjugated ExtrAvidin for biotinylated samples, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG for cell lysates. Surface and Intracellular Staining and Flow Cytometry—For surface staining, cells (0.5 × 106/sample in 0.05 ml of cold staining buffer) were incubated for 30 min on ice with biotinylated antibodies. After washing with staining buffer, the cells were incubated for an additional 30 min on ice with phycoerythrin-conjugated streptavidin in staining buffer plus fluorescein isothiocyanate- or DyLight 649-coupled antibodies as indicated. The cells were washed and analyzed with a FACScan flow cytometer (Coulter Electronics). Three-color analysis of thymus populations was performed with a FACSCalibur flow cytometer (BD Biosciences) with CellQuest acquisition and analysis software. For intracellular staining, cells (2 × 106) were washed with cold PBS and fixed for 5 min with 0.25 ml of 4% paraformaldehyde in PBS at room temperature. After adding 1 ml of PBS, 0.1% bovine serum albumin, 0.05% sodium azide, 1 mm MgSO4, 1 mm CaCl2, and 10 mm HEPES (pH 7.2) (PBS/bovine serum albumin), the cells were spun and frozen at –70 °C in PBS and 10% Me2SO until used. The cells were washed with 0.1% saponin (Sigma) in PBS/bovine serum albumin (PBS/saponin) and blocked at 4 °C for 30 min with 5% nonfat milk in PBS/saponin. The cells were then stained with phycoerythrin-conjugated anti-mouse IFN-γ antibody XMG1.2 in 5% nonfat milk in PBS/saponin for 30 in the cold. After washing three times with PBS/saponin, the cells were analyzed as described above. CD3ɛ Isoforms with Different pI Values Are Detected in TCR-CD3 Complexes on the Surface of CD4+ T cells—Our previous data suggested that one anti-CD3 antibody (YCD3-1) has different avidity for CD3 expressed by different CD4+ T cells (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). Particularly, it binds with high avidity to CD3 expressed by the SR.D10 cell line, whereas its avidity is low for CD3 in AE103 cells. In contrast, other anti-CD3 antibodies (i.e. 500A2) or anti-TCR antibodies show no major differences in avidity for the same cells (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). High avidity of antibody YCD3-1 correlates with higher N-terminal degradation by metalloproteinases as determined using anti-CD3ɛ N-terminal peptide antibodies and proteinase inhibitors such as phenanthroline. CD3ɛ chains from different species, including mouse, possess negatively charged amino acid residues in their N-terminal sequence that affect their predicted pI values (Fig. 1A). We reasoned that CD3ɛ immunoprecipitates from different mouse T cell lines and/or using different anti-CD3 or anti-TCR antibodies should show distinct patterns of CD3ɛ chain isoforms, which could be distinguished by IEF. Because antibody 500A2 showed little variation among T cells in terms of avidity, immunoprecipitation of CD3 with antibody 500A2 was initially used as a mean to assess the CD3ɛ isoform distribution in AE103 and SR.D10 cells (Fig. 1, B and C, upper panels). Immunoprecipitates were separated by IEF (first dimension) and PAGE (second dimension) and immunoblotted using rabbit anti-mouse CD3ɛ extracellular domain antibodies. As predicted, different CD3ɛ isoforms were present within the pI range for complete chains or chains lacking one or two aspartic acid residues of the N-terminal sequence. The most abundant species in both cell lines had a pI close to 7, fitting the predicted pI for CD3ɛ chains lacking one aspartic acid. However, at a low pI, complete CD3ɛ chains were abundant in AE103 cells, but almost absent in SR.D10 precipitates (Fig. 1, B and C, upper panels). Immunoprecipitation of surface CD3ɛ using antibody YCD3-1 showed that this antibody bound better to isoforms with higher pI values, i.e. those lacking one or two N-terminal aspartic acids. Thus, these isoforms are over-represented in YCD3-1 immunoprecipitates from either AE103 or SR.D10 cells (Fig. 1, B and C; see also Fig. 2A), in agreement with the predicted behavior of this antibody based on our previous data (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). Our previous results also suggested that the less degraded CD3ɛ chains maintain strong ties with the TCR, whereas TCR–CD3 bonds are looser for degraded CD3ɛ chains (18Criado G. Feito M.J. Ojeda G. Sánchez A. Janeway Jr., C.A. Portolés P. Rojo J.M. Eur. J. Immunol. 2000; 30: 1469-1479Crossref PubMed Scopus (11) Google Scholar). This was confirmed in immunoprecipitates of surface TCR from SR.D10 cells. Indeed, as depicted in Fig. 1C (lower panel), despite the low proportion of complete low pI CD3ɛ chains among CD3 chains from SR.D10 cells observed in anti-CD3 precipitates (upper and middle panels), these low pI chains coprecipitated very efficiently w" @default.
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• W2069349270 title "Loss of N-terminal Charged Residues of Mouse CD3ɛ Chains Generates Isoforms Modulating Antigen T Cell Receptor-mediated Signals and T Cell Receptor-CD3 Interactions" @default.
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