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• W2073870582 abstract "Siglec-7 (p70/AIRM) and Siglec-9 are “CD33”-related siglecs expressed on natural killer (NK) cells and subsets of peripheral T cells. Like other inhibitory NK cell receptors, they contain immunoglobulin receptor family tyrosine-based inhibitory motifs in their cytoplasmic domains, and Siglec-7 has been demonstrated to negatively regulate NK cell activation. Based on reports of the presence of these siglecs on T cells, we sought to determine if they are capable of modulating T cell receptor (TCR) signaling using Jurkat T cells stably and transiently transfected with Siglec-7 or Siglec-9. Following either pervanadate stimulation or TCR engagement, both Siglecs exhibited increased tyrosine phosphorylation and recruitment of SHP-1. Effects of Siglec-7 and -9 were also evident in downstream events in the signaling pathway. Both siglecs reduced phosphorylation of Tyr319 on ZAP-70, known to play a pivotal role in up-regulation of gene transcription following TCR stimulation. There was also a corresponding decreased transcriptional activity of nuclear factor of activated T cells (NFAT) as determined using a luciferase reporter gene. Like all siglecs, Siglec-7 and -9 recognize sialic acid-containing glycans of glycoproteins and glycolipids as ligands. Mutation of the conserved Arg in the ligand binding site of Siglec-7 (Arg124) or Siglec-9 (Arg120) resulted in reduced inhibitory function in the NFAT/luciferase transcription assay, suggesting that ligand binding is required for optimal inhibition of TCR signaling. The combined results demonstrate that both Siglec-7 and Siglec-9 are capable of negative regulation of TCR signaling and that ligand binding is required for optimal activity. Siglec-7 (p70/AIRM) and Siglec-9 are “CD33”-related siglecs expressed on natural killer (NK) cells and subsets of peripheral T cells. Like other inhibitory NK cell receptors, they contain immunoglobulin receptor family tyrosine-based inhibitory motifs in their cytoplasmic domains, and Siglec-7 has been demonstrated to negatively regulate NK cell activation. Based on reports of the presence of these siglecs on T cells, we sought to determine if they are capable of modulating T cell receptor (TCR) signaling using Jurkat T cells stably and transiently transfected with Siglec-7 or Siglec-9. Following either pervanadate stimulation or TCR engagement, both Siglecs exhibited increased tyrosine phosphorylation and recruitment of SHP-1. Effects of Siglec-7 and -9 were also evident in downstream events in the signaling pathway. Both siglecs reduced phosphorylation of Tyr319 on ZAP-70, known to play a pivotal role in up-regulation of gene transcription following TCR stimulation. There was also a corresponding decreased transcriptional activity of nuclear factor of activated T cells (NFAT) as determined using a luciferase reporter gene. Like all siglecs, Siglec-7 and -9 recognize sialic acid-containing glycans of glycoproteins and glycolipids as ligands. Mutation of the conserved Arg in the ligand binding site of Siglec-7 (Arg124) or Siglec-9 (Arg120) resulted in reduced inhibitory function in the NFAT/luciferase transcription assay, suggesting that ligand binding is required for optimal inhibition of TCR signaling. The combined results demonstrate that both Siglec-7 and Siglec-9 are capable of negative regulation of TCR signaling and that ligand binding is required for optimal activity. The human siglec family of cell receptors is composed of eleven members of the Ig superfamily, which are functionally related by their ability to bind sialic acid-containing carbohydrates of glycoproteins and glycolipids as ligands (1Angata T. Brinkman-Van der Linden E. Biochim. Biophys. Acta. 2002; 1572: 294-316Crossref PubMed Scopus (95) Google Scholar, 2Crocker P.R. Varki A. Immunology. 2001; 103: 137-145Crossref PubMed Scopus (221) Google Scholar, 3Crocker P.R. Curr. Opin. Struct. Biol. 2002; 12: 609-615Crossref PubMed Scopus (278) Google Scholar). The Siglecs are predominately and differentially expressed on a wide variety of white blood cells (2Crocker P.R. Varki A. Immunology. 2001; 103: 137-145Crossref PubMed Scopus (221) Google Scholar, 3Crocker P.R. Curr. Opin. Struct. Biol. 2002; 12: 609-615Crossref PubMed Scopus (278) Google Scholar), the notable exceptions being myelin-associated glycoprotein expressed in glial cells (4Filbin M.T. Curr. Opin. Neurobiol. 1995; 5: 588-595Crossref PubMed Scopus (80) Google Scholar, 5Spencer T. Domeniconi M. Cao Z. Filbin M.T. Curr. Opin. Neurobiol. 2003; 13: 133-139Crossref PubMed Scopus (60) Google Scholar) and Siglec-6 expressed in placenta (2Crocker P.R. Varki A. Immunology. 2001; 103: 137-145Crossref PubMed Scopus (221) Google Scholar, 6Patel N. Brinkman-Van der Linden E.C. Altmann S.W. Gish K. Balasubramanian S. Timans J.C. Peterson D. Bell M.P. Bazan J.F. Varki A. Kastelein R.A. J. Biol. Chem. 1999; 274: 22729-22738Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The extracellular region has a variable number of C2-set Ig domains and a single homologous N-terminal “V-set” domain that binds to sialic acids (2Crocker P.R. Varki A. Immunology. 2001; 103: 137-145Crossref PubMed Scopus (221) Google Scholar). Crystal structure analysis of two Siglecs, sialoadhesin (Siglec-1) and Siglec-7, have revealed a shallow sialic acid binding pocket with a conserved sequence of six amino acids in the tip of the C-C′ loop that influences the specificity for binding various sialoside sequences found in nature (7Alphey M.S. Attrill H. Crocker P.R. Van Aalten D.M. J. Biol. Chem. 2003; 278: 3372-3377Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 8Yamaji T. Teranishi T. Alphey M.S. Crocker P.R. Hashimoto Y. J. Biol. Chem. 2002; 277: 6324-6332Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 9May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 10Zaccai N.R. Maenaka K. Maenaka T. Crocker P.R. Brossmer R. Kelm S. Jones E.Y. Structure (Camb.). 2003; 11: 557-567Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Within this sequence, a conserved arginine residue coordinates with the C-1 hydroxyl group of the sialic acid and is required for binding as evidenced by Arg to Ala mutations that abrogate binding to sialic acid-containing ligands (11Angata T. Varki A. J. Biol. Chem. 2000; 275: 22127-22135Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 12Angata T. Varki A. Glycobiology. 2000; 10: 431-438Crossref PubMed Scopus (98) Google Scholar). Another characteristic feature of the siglecs is the presence of consensus immunoglobulin receptor family tyrosine-based inhibitory motifs (ITIM) 1The abbreviations used are: ITIM, immune-globulin receptor family tyrosine-based inhibitory motifs; IRS, inhibitory-receptor superfamily; NKR, natural killer cell receptor; KOD polymerase, polymerase from Thermococcus kodakaraensis; Sig-7, Siglec-7; Sig-9, Siglec-9; TCR, T cell receptor; NFAT, nuclear factor of activated T cell; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PHA, P. vulgaris red kidney bean lectin-L; PBL, peripheral blood leukocyte; FACS, fluorescence-activated cell sorting; CMV, cytomegalovirus; FCS, fetal calf serum; SHP-1, SH2 domain-containing phosphatase 1; SHIP-1, SH2 inositol phosphatase-1. 1The abbreviations used are: ITIM, immune-globulin receptor family tyrosine-based inhibitory motifs; IRS, inhibitory-receptor superfamily; NKR, natural killer cell receptor; KOD polymerase, polymerase from Thermococcus kodakaraensis; Sig-7, Siglec-7; Sig-9, Siglec-9; TCR, T cell receptor; NFAT, nuclear factor of activated T cell; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PHA, P. vulgaris red kidney bean lectin-L; PBL, peripheral blood leukocyte; FACS, fluorescence-activated cell sorting; CMV, cytomegalovirus; FCS, fetal calf serum; SHP-1, SH2 domain-containing phosphatase 1; SHIP-1, SH2 inositol phosphatase-1. in the cytoplasmic domains of all but sialoadhesin (Siglec-1) and myelin-associated glycoprotein (Siglec-4) (2Crocker P.R. Varki A. Immunology. 2001; 103: 137-145Crossref PubMed Scopus (221) Google Scholar). ITIM motifs are found in an expanding group of immunoglobulin family receptors that negatively regulate immune cell activation, which have been called the inhibitory-receptor superfamily (IRS) by Lanier (13Lanier L.L. Annu. Rev. Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1463) Google Scholar). Members of the IRS exhibit three characteristic properties: (i) they recruit SH2 domain-containing protein tyrosine-based phosphatases such as SHP-1 and/or SHP-2 via a cytoplasmic ITIM motif, (ii) they affect activation receptors in cis, and (iii) require co-ligation with the activation receptors to exert their effect (13Lanier L.L. Annu. Rev. Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1463) Google Scholar). One of the siglecs, CD22 (Siglec-2), is well established as a negative regulator of B cell receptor signaling and fulfills all the characteristics of a member of the IRS (13Lanier L.L. Annu. Rev. Immunol. 1998; 16: 359-393Crossref PubMed Scopus (1463) Google Scholar, 14Cornall R.J. Goodnow C.C. Cyster J.G. Curr. Top. Microbiol. Immunol. 1999; 244: 57-68PubMed Google Scholar). Based on the presence of ITIM motifs in the cytoplasmic domains of most siglecs, it is generally believed that others will also be shown to be inhibitory receptors (1Angata T. Brinkman-Van der Linden E. Biochim. Biophys. Acta. 2002; 1572: 294-316Crossref PubMed Scopus (95) Google Scholar, 2Crocker P.R. Varki A. Immunology. 2001; 103: 137-145Crossref PubMed Scopus (221) Google Scholar, 3Crocker P.R. Curr. Opin. Struct. Biol. 2002; 12: 609-615Crossref PubMed Scopus (278) Google Scholar). Direct evidence that this is the case for Siglec-7 has been obtained from several laboratories. Indeed, Siglec-7 was originally cloned as a negative regulator of NK cell cytolysis (15Falco M. Biassoni R. Bottino C. Vitale M. Sivori S. Augugliaro R. Moretta L. Moretta A. J. Exp. Med. 1999; 190: 793-802Crossref PubMed Scopus (192) Google Scholar). Recently, Crocker and colleagues (16Nicoll G. Avril T. Lock K. Furukawa K. Bovin N. Crocker P.R. Eur. J. Immunol. 2003; 33: 1642-1648Crossref PubMed Scopus (182) Google Scholar) demonstrated that NK cells were less able to kill target cells bearing a high affinity Siglec-7 ligand (ganglioside GD3), presumably a result of inhibition of NK activation by recruitment of Siglec-7 to the site of NK cell-target cell contact (16Nicoll G. Avril T. Lock K. Furukawa K. Bovin N. Crocker P.R. Eur. J. Immunol. 2003; 33: 1642-1648Crossref PubMed Scopus (182) Google Scholar, 17Ito A. Handa K. Withers D.A. Satoh M. Hakomori S. FEBS Lett. 2001; 498: 116-120Crossref PubMed Scopus (34) Google Scholar). Antibody cross-linking of Siglec-7 or CD33 (Siglec-3) has also been associated with negative regulation of cell growth by reducing proliferation and inducing apoptosis of CD34-positive hematopoietic precursors and chronic myeloid leukemia cells (18Vitale C. Romagnani C. Falco M. Ponte M. Vitale M. Moretta A. Bacigalupo A. Moretta L. Mingari M.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15091-15096Crossref PubMed Scopus (126) Google Scholar, 19Vitale C. Romagnani C. Puccetti A. Olive D. Costello R. Chiossone L. Pitto A. Bacigalupo A. Moretta L. Mingari M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5764-5769Crossref PubMed Scopus (93) Google Scholar). Similarly, Nutku et al. (20Nutku E. Aizawa H. Hudson S.A. Bochner B.S. Blood. 2003; 101: 5014-5020Crossref PubMed Scopus (262) Google Scholar) have shown that antibody ligation of Siglec-8 induces capsase-3-like-dependent apoptosis in eosinophils. Recently, ligation of Siglec-5 on neutrophils has been shown to augment oxidative burst induced by formylmethionylleucylphenylalanine (21Erickson-Miller C.L. Freeman S.D. Hopson C.B. D'Alessio K.J. Fischer E.I. Kikly K.K. Abrahamson J.A. Holmes S.D. King A.G. Exp. Hematol. 2003; 31: 382-388Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, in contrast to the case of CD22 regulation of B cell receptor signaling, the activation receptor modulated by these siglecs in NK cells, eosinophils, and neutrophils has not been identified. Siglec-7 (p75/AIRM) has been classified as an inhibitory NK cell receptor (NKR) based on its structural homology to other immunoglobulin-like NKRs (22Farag S.S. Fehniger T.A. Ruggeri L. Velardi A. Caligiuri M.A. Blood. 2002; 100: 1935-1947Crossref PubMed Scopus (420) Google Scholar, 23McQueen K.L. Parham P. Curr. Opin. Immunol. 2002; 14: 615-621Crossref PubMed Scopus (160) Google Scholar, 24Ugolini S. Vivier E. Curr. Opin. Immunol. 2000; 12: 295-300Crossref PubMed Scopus (65) Google Scholar), its gene locus (19q13.3-13.4) (25Yousef G.M. Ordon M.H. Foussias G. Diamandis E.P. Gene (Amst.). 2002; 286: 259-270Crossref PubMed Scopus (26) Google Scholar) proximal to other NKR gene families (23McQueen K.L. Parham P. Curr. Opin. Immunol. 2002; 14: 615-621Crossref PubMed Scopus (160) Google Scholar), and the presence of ITIM motifs in its cytoplasmic domain (24Ugolini S. Vivier E. Curr. Opin. Immunol. 2000; 12: 295-300Crossref PubMed Scopus (65) Google Scholar). Although classic NKRs bind major histocompatibility complex class 1 molecules as ligands, there are a growing number of NKRs like Siglec-7 that bind other ligands or whose ligands are unknown (22Farag S.S. Fehniger T.A. Ruggeri L. Velardi A. Caligiuri M.A. Blood. 2002; 100: 1935-1947Crossref PubMed Scopus (420) Google Scholar). It is noteworthy that Siglec-9 is also expressed on NK cells, although it has not yet been classified as an NKR. Several NKRs are expressed on subsets of CD8+ T cells with a memory phenotype (26Uhrberg M. Valiante N.M. Young N.T. Lanier L.L. Phillips J.H. Parham P. J. Immunol. 2001; 166: 3923-3932Crossref PubMed Scopus (121) Google Scholar, 27Phillips J.H. Gumperz J.E. Parham P. Lanier L.L. Science. 1995; 268: 403-405Crossref PubMed Scopus (302) Google Scholar, 28Mingari M.C. Vitale C. Cambiaggi A. Schiavetti F. Melioli G. Ferrini S. Poggi A. Int. Immunol. 1995; 7: 697-703Crossref PubMed Scopus (212) Google Scholar, 29Ferrini S. Cambiaggi A. Meazza R. Sforzini S. Marciano S. Mingari M.C. Moretta L. Eur. J. Immunol. 1994; 24: 2294-2298Crossref PubMed Scopus (137) Google Scholar) and have been implicated in regulation of T cell receptor (TCR) signaling (30Braud V.M. Aldemir H. Breart B. Ferlin W.G. Trends Immunol. 2003; 24: 162-164Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 31Mingari M.C. Schiavetti F. Ponte M. Vitale C. Maggi E. Romagnani S. Demarest J. Pantaleo G. Fauci A.S. Moretta L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12433-12438Crossref PubMed Scopus (225) Google Scholar, 32Ugolini S. Arpin C. Anfossi N. Walzer T. Cambiaggi A. Forster R. Lipp M. Toes R.E. Melief C.J. Marvel J. Vivier E. Nat. Immunol. 2001; 2: 430-435Crossref PubMed Scopus (21) Google Scholar, 33Vivier E. Anfossi N. Nat. Rev. Immunol. 2004; 4: 190-198Crossref PubMed Scopus (186) Google Scholar). In particular, the immunoglobulin-like NK cell receptor, LIR1/ILT2, has been demonstrated to regulate cytolysis, cytokine secretion, proliferation, and actin cytoskeleton reorganization through negative regulation of the TCR complex (34Colonna M. Navarro F. Bellon T. Llano M. Garcia P. Samaridis J. Angman L. Cella M. Lopez-Botet M. J. Exp. Med. 1997; 186: 1809-1818Crossref PubMed Scopus (773) Google Scholar, 35Dietrich J. Cella M. Colonna M. J. Immunol. 2001; 166: 2514-2521Crossref PubMed Scopus (109) Google Scholar, 36Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar, 37Nikolova M. Musette P. Bagot M. Boumsell L. Bensussan A. Blood. 2002; 100: 1019-1025Crossref PubMed Scopus (25) Google Scholar). Because Siglec-7 and Siglec-9 were both detected on subsets of T cells (38Zhang J.Q. Nicoll G. Jones C. Crocker P.R. J. Biol. Chem. 2000; 275: 22121-22126Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), we hypothesized that they may participate in regulation of T cell signaling through the T cell receptor complex as observed for other NKRs. Accordingly, we investigated their ability to regulate TCR activation using Jurkat cell lines expressing Siglec-7 or Siglec-9 and their receptor-binding mutants with Arg to Ala substitutions at residues 124 and 120, respectively. The results show that both Siglec-7 and -9 can negatively regulate TCR activation by recruitment of SHP-1, resulting in reduced transcription of NFAT-mediated gene transcription. Equivalent expression of the receptor-binding mutants had no effect on TCR activation indicating that a functional ligand binding domain is required for optimal activity. The results suggest that these two siglecs may participate in modulating the activation threshold of T cells expressing them. Cells, Antibodies, and Reagents—Jurkat cells were routinely cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (HyClone, Logan, UT), 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 0.1 mm nonessential amino acids (Invitrogen), and 1 mm sodium pyruvate (Invitrogen). FITC-labeled anti-CD3 (UCHT1), anti-human γδ TCR, and anti-human αβ TCR mAbs, were purchased from BD Biosciences (San Diego, CA). Red phycoerythrin-conjugated goat anti-mouse IgG mAb was purchased from Jackson ImmunoResearch (West Grove, PA). Anti-Siglec-7 (S7) and anti-Siglec-9 (K8) mAbs were kind gifts from Dr. Paul R. Crocker, University of Dundee (38Zhang J.Q. Nicoll G. Jones C. Crocker P.R. J. Biol. Chem. 2000; 275: 22121-22126Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Anti-ZAP70, and anti-phosphoZAP70 Abs were purchased from Cell Signaling (Beverly, MA). Anti-phospho tyrosine (4G10), anti-SHP-1 polyclonal Abs, and anti-mouse IgG horseradish peroxidase-labeled Abs were from Upstate Biotechnology (Lake Placid, NY). Phaseolus vulgaris red kidney bean lectin-L (PHA), anti-FLAG M2 mAb, anti-FLAG mAb-conjugated beads, anti-FLAG M2 polyclonal antibody, and Triton X-100 were purchased from Sigma-Aldrich. Orthovanadate was from Calbiochem. Blood was collected from three healthy adult volunteers in 10 mm sodium citrate, from which resting human peripheral blood lymphocytes (PBLs) were isolated by density-gradient centrifugation using Lymphoprep (Invitrogen). The interface containing a mixed white cell population was washed twice with phosphate-buffered saline, and monocytes and macrophages were removed by adherence to plastic in complete RPMI medium. The nonadherent cells (PBLs) were re-suspended in complete RPMI medium and used within 12 h of isolation. Two to five analyses were performed on each donor spanning at least 1 month. Flow Cytometry and Fluorescence Microscopy—Surface expression of CD3ϵ chain, γδ TCR, and αβ TCR were routinely evaluated by FACS using direct staining with immunofluorescent antibodies. Cultured cells and PBLs were washed twice with FACS buffer (5 mm EDTA, 5 mg/ml bovine serum albumin containing phosphate-buffered saline), and incubated on ice for 30 min with anti-Siglec antibodies, S7, or K8 (38Zhang J.Q. Nicoll G. Jones C. Crocker P.R. J. Biol. Chem. 2000; 275: 22121-22126Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). For FACS analysis, after washing twice with FACS buffer, cells were first incubated with red phycoerythrin-conjugated goat anti-mouse IgG mAb on ice for an additional 30 min and then with FITC-labeled anti-CD3ϵ chain, γδ TCR, or αβ TCR. For immunofluorescence microscopy, cells were first incubated with anti-Siglec antibodies, and after washing twice with FACS buffer, were incubated for 30 min on ice with Alexa fluor 488-conjugated goat anti-mouse IgG mAb (Molecular Probes, Eugene, OR). Cells were then incubated for an additional 30 min with anti-CD3ϵ antibodies labeled with Texas Red X using Zenon Mouse IgG-labeling kits (Molecular Probes) and then washed twice with cold phosphate-buffered saline. Stained cells were mounted on glass slides with Vectashield Hard Set Mounting Medium (Vector Laboratories, Burlingame, CA) to reduce photobleaching during observation. In all cases, isotype-matched monoclonal antibodies were used as a negative control. Expression Constructs—Expression constructs of Siglec-7 and -9 with a FLAG tag epitope at the C terminus were constructed as follows. The entire coding sequences of Siglec-7 and -9 were amplified by PCR using cDNA from peripheral blood as a template, with respective sets of specific primers, and KOD polymerase HiFi (Novagen, Milwaukee, WI). Primers used were 5′-AAAAGGAAAAGCGGCCGCACCTCCAACCCCAGATATGC-3′ and 5′-CGCGGATCCTTGGGGATCTTGATCTC-3′ for Siglec-7 and 5′-AAAAGGAAAAGCGGCCGCACCTCTAACCCCAGACATGC-3′ and 5′-CGCGGATCCCTGTGGATCTTGATCTCCGAG-3′ for Siglec-9. The PCR products were inserted into the NotI/BamHI site of the pCMV Tag 4B vector (Stratagene, San Diego, CA) to produce FLAG tag epitope fusion proteins. Mutant constructs with Arg124 to Ala substitution on Siglec-7 and Arg120 to Ala substitution on Siglec-9 were also prepared, because these substitutions have already been shown to eliminate sialic acid binding (11Angata T. Varki A. J. Biol. Chem. 2000; 275: 22127-22135Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 12Angata T. Varki A. Glycobiology. 2000; 10: 431-438Crossref PubMed Scopus (98) Google Scholar). The respective nucleotide substitutions for the Arg to Ala mutation in Siglec-7 and -9 were generated by crossover PCR, and the corresponding expression vectors were constructed as described above. The sequences of all expression constructs were verified by DNA sequencing. Generation of Stable Expressing Cells—Jurkat cells were washed twice with RPMI medium, and added at a final density of 3 × 107/ml cells (0.4 ml) to a 0.4-cm gap cuvette (Invitrogen) containing 30 μg of the desired plasmid DNA. Electroporation was then carried out at 250 V, 950 microfarads with the use of a Gene Pulser (Bio-Rad). Cells were immediately transferred to a small tissue culture plate with RPMI containing 5% FCS, and cultured for 16-18 h at 37 °C, 5% CO2. To establish three independent clones, we performed transfection more than three times on each expression construct. Cells were plated at various dilutions in 12-well plates with complete RPMI medium containing 1 mg/ml G418 to generate stable cell lines. Three weeks later, clones were transferred and expanded. Expression of Siglec-7 or -9 was confirmed by FACS analysis. Transient Expression of Siglecs and NFAT Luciferase Assays—Jurkat cells, prepared as described above, were electroporated (250 V, 950 microfarads) with a total of 30 μg of plasmid DNA comprising the siglec expression vector and/or empty vector as a control. After 16-18 h of incubation at 37 °C with RPMI containing 5% FCS, viable cells were purified with Histoplaque (Sigma-Aldrich). Following TCR stimulation and incubation for 48 h in RPMI containing 10% FCS, the cells were subjected to FACS analysis, and phosphorylation status was determined by immunoblot. For NFAT luciferase assays involving transient expression of Siglec-7 or -9, Jurkat cells (1.2 × 107) were electroporated with a total of 30 μg of empty vector or Siglec-7 or Siglec-9 expression constructs, 20 μg of NFAT-luciferase reporter constructs (BD Biosciences), and 5 μg of pR-TK control constructs (Promega). For NFAT-luciferase assay using Jurkat cells stably expressing Siglec-7 or Siglec-9, 20 μg of NFAT-luciferase reporter constructs and 5 μg of pR-TK control constructs were electroporated (250 V, 950 microfarads) into 3 × 107 cells/ml (0.4 ml) in RPMI media of the desired cell line. Assays under each condition were performed in triplicate. After 18-h incubation in RPMI containing 5% FCS, viable cells were purified with Histoplaque and cultured in complete RPMI media for 48 h at 37 °C, 5% CO2 to be tested for analysis of Siglec expression by FACS analysis and luciferase assay. NFAT luciferase assay was performed with a Dual Luciferase Assay System (Promega) and luminometer (Monolight 3010, BD Biosciences) according to the supplier's instructions. Each experiment was repeated three or more times. Cellular Stimulation, Immunoprecipitation, and Western Blot Analysis—Unless otherwise indicated, Jurkat-derived cell lines (1 × 107) were incubated for different time periods with either anti-CD3 (5 μg/ml), PHA (1.25 μg/ml), or vanadate (200 μm sodium orthovanadate 0.03% H2O2 at 37 °C in 1 ml of RPMI, which inhibits phosphatase activity and increases protein tyrosine phosphorylation). Cells were then lysed at 4 °C in lysis buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, and complete Protease Inhibitor Mixture from Roche Applied Science, Indianapolis, IN). After pre-clearing for 1 h at 4 °C with protein G-Sephadex beads (Amersham Biosciences), lysates were subjected to immunoprecipitation with anti-FLAG M2 beads, according to the Sigma manual. Alternatively, cell aliquots were directly lysed in Laemmli sample buffer for subsequent immunoblotting with anti-phospho ZAP70 and anti-ZAP70 antibodies. Precipitates or whole cell lysates were separated by 10% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes (Amersham Biosciences) and immunoblotted with indicated Abs. Bound Abs were visualized using Western Lightning Chemiluminescence Plus (PerkinElmer Life Sciences). Expression of Siglec-7 and Siglec-9 by αβ and γδ T Cells—In their initial reports Crocker and colleagues (38Zhang J.Q. Nicoll G. Jones C. Crocker P.R. J. Biol. Chem. 2000; 275: 22121-22126Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) had noted that a majority of cells expressing NK cell markers (CD56 or CD16), and a small subset of CD3-positive peripheral blood leukocytes, expressed Siglec-7 and Siglec-9. However, Vitale et al. (18Vitale C. Romagnani C. Falco M. Ponte M. Vitale M. Moretta A. Bacigalupo A. Moretta L. Mingari M.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15091-15096Crossref PubMed Scopus (126) Google Scholar) found no Siglec-7 expression in CD3-expressing T cells. Because our interest was to investigate the effects of Siglec-7 and -9 on TCR activation, we first confirmed their expression in peripheral blood leukocytes using antibodies to the CD3ϵ, αβ, and γδ TCR subunits. Representative results from three donors are shown in Fig. 1. As observed previously (38Zhang J.Q. Nicoll G. Jones C. Crocker P.R. J. Biol. Chem. 2000; 275: 22121-22126Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 39Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), there was clear surface expression of Siglec-7 and Siglec-9 on CD3ϵ chain-positive cells in all three donors. For the three donors, the fraction of CD3-positive cells expressing Siglec-7 and Siglec-9 varied from 3.8 to 10% and from 5.2 to 19%, respectively. Of the Siglec-positive CD3 cells, greater than 90% expressed the αβ TCR sub-units. The population of peripheral blood leukocytes expressing the γδ TCR was small and variable (0.15-1.8% of total cells) as pointed out previously (40Battistini L. Borsellino G. Sawicki G. Poccia F. Salvetti M. Ristori G. Brosnan C.F. J. Immunol. 1997; 159: 3723-3730PubMed Google Scholar). Although no expression of Siglec-9 was observed in the γδ T cells, one donor exhibited clear expression of Siglec-7 in ∼13% of the γδ T cells. The variation in the expression of the Siglecs in T cells was donor-specific variation, rather than assay-specific variation, because virtually identical results were seen for each donor in two to five separate analyses taken over a span of at least 1 month. Taken together, the results indicate that the majority of CD3-positive peripheral blood leukocytes expressing Siglec-7 or -9 are αβ T lymphocytes, and that the fraction of Siglec-expressing cells differs from donor to donor. Generation of Stable Siglec-7- and Siglec-9-expressing Jurkat Cells for Analysis of TCR Signaling—Jurkat cells are widely used to study TCR signal transduction. Flow cytometry analysis of Jurkat cells revealed that they do not express either Siglec-7 or Siglec-9 (not shown), making them ideal for the analysis of the affect of these receptors on TCR signaling. Accordingly, we adopted a strategy of comparing TCR signaling of native Jurkat cells with Jurkat cells that are stably or transiently expressing Siglec-7 or Siglec-9. As described in “Materials and Methods,” stable Jurkat cell lines were generated expressing FLAG-tagged Siglec-7 (Sig7) or Siglec-9 (Sig9) or neither siglec (mock-Jurkat; transfected with empty vector). Three independent clonal lines were prepared for each, with Siglec-7 and Siglec-9 expressing cells chosen to have similar levels of siglec expression by flow cytometry (see Fig. 2A for representative Sig-7 and Sig-9 clones). Based" @default.
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• W2073870582 title "Negative Regulation of T Cell Receptor Signaling by Siglec-7 (p70/AIRM) and Siglec-9" @default.
• W2073870582 cites W1541131175 @default.
• W2073870582 cites W1544881505 @default.
• W2073870582 cites W1573613966 @default.
• W2073870582 cites W1575885906 @default.
• W2073870582 cites W1821650315 @default.
• W2073870582 cites W1963962536 @default.
• W2073870582 cites W1966056248 @default.
• W2073870582 cites W1971426454 @default.
• W2073870582 cites W1975933816 @default.
• W2073870582 cites W1988621302 @default.
• W2073870582 cites W1991623174 @default.
• W2073870582 cites W1996222281 @default.
• W2073870582 cites W2000518846 @default.
• W2073870582 cites W2003676228 @default.
• W2073870582 cites W2013933972 @default.
• W2073870582 cites W2015001083 @default.
• W2073870582 cites W2020181553 @default.
• W2073870582 cites W2023279370 @default.
• W2073870582 cites W2037133606 @default.
• W2073870582 cites W2041196497 @default.
• W2073870582 cites W2052061247 @default.
• W2073870582 cites W2059450339 @default.
• W2073870582 cites W2062456152 @default.
• W2073870582 cites W2067591449 @default.
• W2073870582 cites W2067766312 @default.
• W2073870582 cites W2077946227 @default.
• W2073870582 cites W2082706780 @default.
• W2073870582 cites W2084178563 @default.
• W2073870582 cites W2093591730 @default.
• W2073870582 cites W2093822963 @default.
• W2073870582 cites W2096549417 @default.
• W2073870582 cites W2105257219 @default.
• W2073870582 cites W2110000889 @default.
• W2073870582 cites W2115806827 @default.
• W2073870582 cites W2122144067 @default.
• W2073870582 cites W2126450986 @default.
• W2073870582 cites W2127566892 @default.
• W2073870582 cites W2132268212 @default.
• W2073870582 cites W2133776873 @default.
• W2073870582 cites W2144387927 @default.
• W2073870582 cites W2146266775 @default.
• W2073870582 cites W2147026827 @default.
• W2073870582 cites W2147075715 @default.
• W2073870582 cites W2148050927 @default.
• W2073870582 cites W2148932927 @default.
• W2073870582 cites W2149425106 @default.
• W2073870582 cites W2159533632 @default.
• W2073870582 cites W2160520392 @default.
• W2073870582 cites W2164993579 @default.
• W2073870582 cites W3142951069 @default.
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