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• W1888682760 abstract "In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis. In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the αβ-T cell receptor (αβ-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the γδ-TCR (γδ-T cell-mediated myositis). We investigated the antigen specificity of a human γδ-TCR previously identified in an autoimmune tissue lesion of γδ-T cell-mediated myositis. We show that this Vγ1.3Vδ2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short α-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both γδ-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized γδ-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a γδ-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis. γδ-T cells are believed to act at the intersection between innate and adoptive immunity, and to be in the first line of defense against pathogens (1Chien Y.H. Konigshofer Y. Antigen recognition by γδ T cells.Immunol. Rev. 2007; 215: 46-58Crossref PubMed Scopus (140) Google Scholar, 2Beetz S. Wesch D. Marischen L. Welte S. Oberg H.H. Kabelitz D. Innate immune functions of human γδ T cells.Immunobiology. 2008; 213: 173-182Crossref PubMed Scopus (125) Google Scholar, 3Hayday A.C. γδ T cells and the lymphoid stress-surveillance response.Immunity. 2009; 31: 184-196Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 4Bonneville M. O'Brien R.L. Born W.K. γδ T cell effector functions: A blend of innate programming and acquired plasticity.Nat. Rev. Immunol. 2010; 10: 467-478Crossref PubMed Scopus (694) Google Scholar). Although γδ-T cells serve a wide range of physiological functions, they may also be involved in autoimmunity (5Wucherpfennig K.W. Newcombe J. Li H. Keddy C. Cuzner M.L. Hafler D.A. γδ T-cell receptor repertoire in acute multiple sclerosis lesions.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 4588-4592Crossref PubMed Scopus (267) Google Scholar, 6Blink S.E. Miller S.D. The contribution of γδ T cells to the pathogenesis of EAE and MS.Curr. Mol. Med. 2009; 9: 15-22Crossref PubMed Scopus (47) Google Scholar, 7Zhang L. Jin N. Nakayama M. O'Brien R.L. Eisenbarth G.S. Born W.K. γδ T cell receptors confer autonomous responsiveness to the insulin-peptide B:9–23.J. Autoimmun. 2010; 34: 478-484Crossref PubMed Scopus (40) Google Scholar). A striking example of a putatively autoimmune human γδ-TCR 2The abbreviations used are:TCRT cell receptorAA-RSaminoacyl-tRNA synthetasesEcIF1translation initiation factor-1 from E. coliIMACimmobilized metal affinity chromatographyhPCNAhuman proliferating cell nuclear antigenCDRcomplementarity determining region. was described in 1991 (8Hohlfeld R. Engel A.G. Ii K. Harper M.C. Polymyositis mediated by T lymphocytes that express the γ/δ receptor.N. Engl. J. Med. 1991; 324: 877-881Crossref PubMed Scopus (139) Google Scholar). This γδ-TCR was isolated from muscle of a unique but well characterized patient with γδ-T cell-mediated myositis. In contrast to other forms of myositis where oligoclonally expanded CD8+ αβ-T cells attack muscle fibers (9Dalakas M.C. Inflammatory disorders of muscle: Progress in polymyositis, dermatomyositis and inclusion body myositis.Curr. Opin. Neurol. 2004; 17: 561-567Crossref PubMed Scopus (105) Google Scholar), in γδ-T cell-mediated myositis, muscle fibers are destroyed by monoclonally expanded T cells expressing a Vγ1.3Vδ2+-TCR (8Hohlfeld R. Engel A.G. Ii K. Harper M.C. Polymyositis mediated by T lymphocytes that express the γ/δ receptor.N. Engl. J. Med. 1991; 324: 877-881Crossref PubMed Scopus (139) Google Scholar, 10Pluschke G. Rüegg D. Hohlfeld R. Engel A.G. Autoaggressive myocytotoxic T lymphocytes expressing an unusual γ/δ T cell receptor.J. Exp. Med. 1992; 176: 1785-1789Crossref PubMed Scopus (54) Google Scholar). The monoclonal character of the T cell expansion allowed the cloning of both chains of this interesting γδ-TCR, subsequently referred to as M88. T cell receptor aminoacyl-tRNA synthetases translation initiation factor-1 from E. coli immobilized metal affinity chromatography human proliferating cell nuclear antigen complementarity determining region. To search for the target antigen(s), we expressed M88 on the surface of a T hybridoma cell line and as soluble single-chain Fv fragment excreted from COS-7 cells (11Wiendl H. Malotka J. Holzwarth B. Weltzien H.U. Wekerle H. Hohlfeld R. Dornmair K. An autoreactive γδ TCR derived from a polymyositis lesion.J. Immunol. 2002; 169: 515-521Crossref PubMed Scopus (37) Google Scholar, 12Dornmair K. Schneider C.K. Malotka J. Dechant G. Wiendl H. Hohlfeld R. Antigen recognition properties of a Vγ1.3Vδ2-T-cell receptor from a rare variant of polymyositis.J. Neuroimmunol. 2004; 152: 168-175Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In earlier experiments, we found that presumably several target antigens may be recognized. All contain a protein component and a conformational rather than a linear epitope. Antigens were present in several species ranging from human muscle cells to bacteria. Moreover, we were unable to express M88 in the cytosol of any cell line, and we could not generate transgenic mice. Even bacteria stopped growing when M88 was induced in the cytosol. We therefore reasoned that M88 might bind to an intracellular proteinaceous motif that is essential for cell growth. Here, we show that the target epitope of M88 is present on several structurally and functionally diverse proteins, including human and bacterial proteins of the translational apparatus. By extensive site-specific mutagenesis of a small paradigmatic protein, EcIF-1, we obtained evidence that part of the epitope is exposed in a short α-helix on the protein surface. Furthermore, we found that several aminoacyl-tRNA synthetases (AA-RS) were recognized. Although their structures are unknown, and they are quite large proteins, which impedes mutagenesis experiments, they are of particular interest because some of these are known targets of autoantibodies in several forms of human myositis (13Plotz P.H. The autoantibody repertoire: Searching for order.Nat. Rev. Immunol. 2003; 3: 73-78Crossref PubMed Scopus (166) Google Scholar, 14Levine S.M. Rosen A. Casciola-Rosen L.A. Anti-aminoacyl tRNA synthetase immune responses: Insights into the pathogenesis of the idiopathic inflammatory myopathies.Curr. Opin. Rheumatol. 2003; 15: 708-713Crossref PubMed Scopus (36) Google Scholar). Although it is still unknown how the B cell responses to AA-RS evolve during pathogenesis, these autoantibodies are used as diagnostic markers of myositis. Our observation that AA-RS are targeted by autoaggressive γδ-T cells therefore reveals a surprising link between early, semi-innate immune responses mediated by γδ-T cells, and late responses mediated by mature, complement-activating autoantibodies. The host strain E. coli BL21-Star-DE3 (Invitrogen) was stably transfected with a described single chain Fv construct of our Vγ1.3Vδ2+-TCR M88 (12Dornmair K. Schneider C.K. Malotka J. Dechant G. Wiendl H. Hohlfeld R. Antigen recognition properties of a Vγ1.3Vδ2-T-cell receptor from a rare variant of polymyositis.J. Neuroimmunol. 2004; 152: 168-175Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) in the expression plasmid pET33b(+) (Novagene). The VN(D)NJ regions of the γ- and δ-chains were connected by a 15-amino acid linker, but the construct used here did not contain a signal sequence. Bacteria were grown in the presence of 2 mm glucose and 50 μg/ml kanamycin unless stated otherwise to suppress γδ-TCR expression. After washing the bacteria by centrifugation, they were resuspended in medium without glucose, and expression of M88 was induced by adding 2 mm isopropylthiogalactoside (Merck). Growth curves were recorded by determining the optical density of the bacterial suspension culture at 600 nm. A cDNA expression library was constructed from mRNA of E. coli MG1655 (ATCC) and inserted into the expression plasmid pET21c(+) (Novagene). To this end, we first replaced the NdeI restriction site of pET21c(+) with a SmaI site by amplifying the sequence between the BglII and the NheI sites using the primer pair pET-BglII (5′-TAGAGGATCGAGATCTCGATCC-3′) and pET-Sma-Mut-rev (5′-AGTCATGCTAGCCCCGGGTATATCTCCTTC-3′) and pET21c(+) as template for PCR. Then, the parent BglII-NheI fragment was replaced by the new fragment, which contained the replacement NdeI to SmaI. This plasmid was digested with SmaI and NotI, and the former insert was removed. In parallel, a cDNA expression library from E. coli MG1655 was established. Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions. Contaminating DNA was removed by DNase I treatment. E. coli mRNA was amplified using the ExpressArt Bacterial mRNA amplification kit (AmpTec), which yields amplified cDNA. In the final synthesis step, cDNA was synthesized according to the protocol for second round amplification with the exception that primer C was replaced by a primer that contained a NotI restriction site (5′-ATAGTTTAgcggccgcGGGAGATTTTTTTTTTTT-3′. (The NotI site is underlined.) This product was finally digested with NotI. Due to the enzymes contained in the amplification kit, the library contains a blunt end on the other side. It was finally inserted into the plasmid pET21c(+) digested previously with SmaI and NotI. M88-transfected E. coli BL21-Star-DE3 cells were supertransfected by electroporation at 2.5 kV, 200 ohm, 25 μF in 2-mm electrode gap cuvettes with the cDNA library in pET21c(+) and grown for 30 min in LB medium with 2 mm glucose. Then ampicillin was added to a final concentration of 100 μg/ml. After 30 min, kanamycin was added to a final concentration of 50 μg/ml. After another 30 min, the bacteria were washed by centrifugation, resuspended in LB medium without glucose, and grown for a further 30 min. Then, expression was induced by adding isopropylthiogalactoside to a final concentration of 1 mm. After 30 min, bacteria were grown on agar plates that contained 100 μg/ml ampicillin, 50 μg/ml kanamycin, and 1 mm isopropyl thiogalactoside. After incubation for 40 h at 37 °C, the biggest colonies were picked and grown in suspension cultures in the presence of ampicillin and glucose, and plasmids were prepared and sequenced by standard methods. As a control experiment, we plated the bacteria on plates that contained 1% glucose, 100 μg/ml ampicillin, 50 μg/ml kanamycin, but without isopropyl thiogalactoside to shut down the promoter. Colonies of similar size became visible already after 18 h at 37 °C. The synthetic peptide EcIF1(33–46), which represents amino acids 33–46 of EcIF1, was synthesized by solid phase peptide synthesis and purified by reversed phase HPLC. Its correct sequence was verified by mass spectrometry. The purified recombinant human proteins His-, Thr-, and Ala-tRNA synthetases hH-RS (Jo-1), hT-RS (PL-7), hA-RS (PL-12), human formimidoyltransferase-cyclodeaminase (hLC1) and human proliferating cell nuclear antigen (hPCNA) were purchased from Diarect (Freiburg). All human proteins were produced identically in a baculovirus expression system and purified by immobilized metal affinity chromatography (IMAC). The polyclonal anti-Jo-1 human Ig fraction BP2040 was purchased from Acris, the monoclonal mouse α-Jo-1 IgG1 antibody HARSA6 was from GenWay, and the isotype control monoclonal mouse IgG1 Pure antibody X40 was from BD Biosciences. The anti-mouse CD3ϵ antibody 145–2C11 (BD Biosciences) was used as control for γδ-TCR activation. All bacterial proteins are contained in the PUREexpress in vitro translation kit (New England Biolabs). They were expressed in the cytosol of E. coli and purified by IMAC as described (15Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nature Biotech. 2001; 19: 751-755Crossref PubMed Scopus (1311) Google Scholar). In contrast to the kit, where the proteins are pooled, here, all proteins were expressed and tested individually. Total RNA from E. coli SG13009 (Qiagen) was isolated using the RNeasy mini kit and RNase-free DNase Set (Qiagen). cDNA was prepared using the clone-specific primer IF1wt-rev and SuperScript III reverse transcriptase (Invitrogen). E. coli IF1 wild-type cDNA was amplified using the primers IF1wt-for and IF1wt-rev. See supplemental Table S1 for all primer sequences. The PCR reaction was carried out for 40 cycles at 94 °C, 56 °C, and 72 °C for 1 min each. The PCR product was cloned into pCR2.1. TOPO (Invitrogen), which served as a template for all further experiments. Site-directed mutagenesis was performed using a PCR-based method: nucleotides coding for the amino acid of choice were introduced into forward (Mut-for) and reverse (Mut-rev) PCR primers (supplemental Table S1) that span the selected positions. In addition, these primers contained silent nucleotide exchanges to introduce unique restriction sites at positions that closely flanked the desired mutation. Two fragments were amplified. For the first fragment we used the primer IF1-wt-for-BamHI together with one of the reverse primers IF-X-rev-Y, where X denoted the amino acid to be exchanged, and Y denoted the restriction site to be inserted. For the second fragment, we used the primer IF1-wt-rev-HindIII together with one of the forward primers IF-X-for-Y. The conditions of the PCR reactions were as described above. The PCR fragments were digested with the restriction enzymes Y, purified, and ligated, and a second PCR was performed using primers IF1wt-for-BamHI and IF1wt-rev-HindIII only. The PCR products encoding the full-length sequences of IF1 wild-type and mutants were cloned into the plasmid pCR2.1.-TOPO, digested with the restriction enzymes BamHI and HindIII and cloned into the BamHI and HindIII sites of expression plasmid pQE30 (Qiagen), which carries a sequence coding for a His6 tag at the N terminus. Therefore, the expressed proteins are extended for the N-terminal sequence, MRGS-His6-GS. Plasmids were transfected into E. coli strain DH5α-F́IQ (Invitrogen). As negative control, we used E. coli DH5α-F́IQ transfected with myelin oligodendrocyte glycoprotein (16Amor S. Groome N. Linington C. Morris M.M. Dornmair K. Gardinier M.V. Matthieu J.M. Baker D. Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice.J. Immunol. 1994; 153: 4349-4356PubMed Google Scholar). Bacteria were grown in LB containing 100 μg/ml ampicillin (Sigma). Protein expression was induced at a cell density of 0.4–0.5 A600 nm by adding isopropylthiogalactoside to a final concentration of 2 mm. Bacteria were harvested after 4 h by centrifugation for 5 min at 15,000 × g. Bacterial pellets from a 250-ml culture were resuspended in 70 ml of lysis buffer (50 mm sodium phosphate buffer, 300 mm NaCl, 10 mm imidazole, 0.05 mg/ml DNase (Sigma) 10 mm MgCl2, 5 μg/ml aprotinin (Sigma), 0.1 mm PMSF, 1 mg/ml lysozyme (Sigma), pH 8.0) and lysed by sonication on ice for 15 min at 30 W using a Branson 450 sonifier. After centrifugation for 10 min at 15,000 × g the supernatants were purified by IMAC. The supernatants were loaded at a flow rate of 1 ml/min onto 5-ml nickel-nitrilotriacetic acid-agarose columns (Qiagen) equilibrated with lysis buffer. The columns were washed with 10–15 column volumes of 50 mm sodium phosphate buffer, 300 mm NaCl, 50 mm imidazole, pH 8.0, and eluted with 50 mm sodium phosphate buffer, 300 mm NaCl, 250 mm imidazole, pH 8.0. Purified proteins were dialyzed against 10 mm sodium phosphate buffer (pH 7.4), 150 mm NaCl, or 50 mm sodium phosphate buffer, 300 mm NaCl, pH 7.8, and stored at −80 °C. Bacteria expressing myelin oligodendrocyte glycoprotein were treated identically. Because this protein precipitates quantitatively in inclusion bodies, this mock preparation contains only co-purifying contaminating bacterial proteins. Protein concentrations were measured according to Peterson et al. (17Peterson G.L. A simplification of the protein assay method of Lowry et al. which is more generally applicable.Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7127) Google Scholar). Purity of the proteins was assessed by SDS-PAGE using 4–20% tris-glycine polyacrylamid Novex protein gels (Invitrogen). Gels were stained by Coomassie Blue (Fluka) or with silver (18Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7807) Google Scholar). We confirmed the native secondary structure of EcIF1 by measuring circular dichroism spectra at 25 °C in the range from 195 to 250 nm using a Jasco J-715 spectrometer. The protein concentration was 18.3 μm in 10 mm sodium phosphate buffer, pH 7.4, 150 mm NaCl. The spectrum of the buffer was subtracted prior to data analysis. The experimental curves were fitted to a set of standard proteins using the program Spectra Manager CDProAnalysis Contin SMP56 (Jasco). For some experiments, proteins were further purified by size exclusion chromatography. We used a 7.8 mm × 30 cm TSK-Gel G2000SWXL 5–150 kDa column with a particle size of 5.0 μm (Tosoh Bioscience) and HP1100 HPLC equipment (Agilent). Chromatography was performed in 200 mm sodium phosphate buffer, 200 mm NaCl, pH 6.8, at a flow rate of 0.8 ml/min. 27–30 μg of protein were injected, and >12 fractions in a time range of 5–21 min were collected. The cDNAs of M88 wild-type chains and chains carrying a set of mutated γ- and δ-chains with altered V-, N(D)N(J)-, or Cγ- regions were cloned into expression plasmids, and expressed on the surface of the T hybridoma cell line 58α−β− (19Blank U. Boitel B. Mège D. Ermonval M. Acuto O. Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma.Eur. J. Immunol. 1993; 23: 3057-3065Crossref PubMed Scopus (64) Google Scholar), which lacks endogenous TCR chains, as described (11Wiendl H. Malotka J. Holzwarth B. Weltzien H.U. Wekerle H. Hohlfeld R. Dornmair K. An autoreactive γδ TCR derived from a polymyositis lesion.J. Immunol. 2002; 169: 515-521Crossref PubMed Scopus (37) Google Scholar, 12Dornmair K. Schneider C.K. Malotka J. Dechant G. Wiendl H. Hohlfeld R. Antigen recognition properties of a Vγ1.3Vδ2-T-cell receptor from a rare variant of polymyositis.J. Neuroimmunol. 2004; 152: 168-175Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). 58α−β− cells express the CD3 complex and the downstream signaling machinery for secreting mouse interleukin-2 (IL-2) after TCR activation by antigens. Individual clones were picked and analyzed independently. All selected clones expressed the heterodimeric γδ-TCR on their surface and were capable of IL-2 secretion after TCR activation by antigens. For all T cell activation experiments, the candidate and control proteins were coated to flat-bottomed 96-well tissue culture plates (Costar). EcK-RS and the anti-CD3ϵ control antibody 145-2C11 were coated for 3 h at 37 °C at 20 μg/ml and 1 μg/ml, respectively, in 10 mm sodium phosphate buffer, pH 7.4, 150 mm NaCl. Samples from the HPLC columns were coated directly in HPLC buffer and incubated for 3 h at 37 °C. For testing antigen recognition of wild-type M88-transfectants or mutants with altered TCR γ-or δ-chains, 10 μg/well wild-type EcIF1 was coated for 3 h at 37 °C. To detect direct M88 activation by human recombinant proteins and for the antibody blocking assay, the candidate proteins were coated at 0.5 μg/well in 50 μl of 10 mm sodium phosphate buffer, 150 mm NaCl, pH 7.4, for 2 h at 37 °C. Then, the plates were washed with buffer. For the antibody blocking assay, 50 μl of antibody solution in RPMI 1640 medium (Invitrogen) supplemented with 5 to 10% heat-inactivated fetal bovine serum (lot 075K3398 (Sigma)), 1.5 mg/ml geneticin (Invitrogen) and 0.3 mg/ml hygromycin B (Invitrogen) were incubated for 30 min at 37 °C with the coated candidate proteins. The polyclonal anti-Jo-1 human Ig fraction BP2040 was used at dilutions of 1:10 and 1:100, and the monoclonal mouse α-Jo-1 IgG1 antibody HARSA6 at 10 μg/ml final concentration. The monoclonal mouse IgG1 Pure antibody X40 served as isotype control at 10 μg/ml. To test recognition of the synthetic peptide EcIF1(33–46), it was coated at concentrations between 5.0 × 102 and 5.0 × 10−5 μg/well for 3 h at 37 °C in 10 mm sodium phosphate buffer, pH 7.4, 150 mm NaCl. To test competition of EcIF1(33–46) with wild-type EcIF1, EcIF1 was coated at 0.5 μg/well for 3 h at 37 °C in 10 mm sodium phosphate buffer, pH 7.4, 150 mm NaCl. After washing the wells with buffer, EcIF1(33–46) was added at concentrations between 666.0 and 3.3 × 10−4 μg/ml together with M88 transfected hybridoma cells. For investigating recognition of denatured EcIF1, recombinant EcIF1 was coated at 0.5 μg/well for 3 h at 37 °C in 10 mm sodium phosphate buffer, pH 7.4, 150 mm NaCl. Then, the wells were washed with buffer, and the samples were incubated for 24 h at 37 °C with 6 m guandinium thiocyanate, 2 m HCl, 5 m NaOH, or 200 μg/ml proteinase K (Roche). EcIF1 was digested in solution with 1 μg/ml trypsin (Merck) in 25 mm Tris/HCl buffer, pH 8.5, for 3 h at 37 °C and coated thereafter. For the dose-response experiments, EcIF1 wild-type and mutant proteins were centrifuged before use in 30-kDa spin columns (Amicon, Millipore) for 20 min at 3,000 × g. After centrifugation, the protein concentration of the flow-through was measured. Proteins were coated at concentrations between 0.1 and 10 μg/well in 50 μl of 50 mm sodium phosphate buffer, 300 mm NaCl, pH 7.8, for 3 h at 37 °C. For all experiments, the coated plates were washed with RPMI 1640 medium (Invitrogen), except for the antibody-blocking experiments, where the plates were not washed. Then, 40,000 M88 transfectants were added to each well at a final volume of 150 μl in medium as described above and incubated for 17 h at 37 °C in a 5% CO2 atmosphere. Finally, 50 μl of supernatant were removed, and mouse IL-2 was measured by ELISA (eBioscience). Background signals from samples without antigens were subtracted, and absolute IL-2 concentrations were determined using a standard curve. As an initial step toward identification of the target antigen of the γδ-TCR M88, we “rescued” E. coli cells from the growth-inhibiting effects of M88 by supertransfecting the bacteria with a cDNA library. Growth of bacteria was halted when the expression of a single-chain Fv construct (12Dornmair K. Schneider C.K. Malotka J. Dechant G. Wiendl H. Hohlfeld R. Antigen recognition properties of a Vγ1.3Vδ2-T-cell receptor from a rare variant of polymyositis.J. Neuroimmunol. 2004; 152: 168-175Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) of M88 was induced in the cytosol of E. coli BL21 (Fig. 1A). Because there were no reasons to assume that M88 was toxic (the protein is not particularly hydrophobic or charged and presumably has no enzymatic activity), we surmised that M88 might bind and neutralize a bacterial compound that is essential for cell growth. We further knew from previous experiments that a protein moiety is at least part of the antigen (11Wiendl H. Malotka J. Holzwarth B. Weltzien H.U. Wekerle H. Hohlfeld R. Dornmair K. An autoreactive γδ TCR derived from a polymyositis lesion.J. Immunol. 2002; 169: 515-521Crossref PubMed Scopus (37) Google Scholar). Therefore, we supertransfected M88-expressing bacteria with a cDNA library from E. coli to rescue them from growth inhibition by M88. Clones expressing library coded proteins that contain the antigen would be expected to bind to M88 and neutralize it. Such bacteria would grow in big colonies, whereas clones that contain irrelevant transcripts would yield small colonies or would not grow at all. Indeed, after supertransfection with the Fv-M88 and the library and induction of M88 and library expression, we observed many very small bacterial clones but also a few clones that grew to very big colonies (Fig. 1B). Under conditions where M88 expression was prevented, all colonies were of the same size (Fig. 1C). We picked some of the biggest colonies and sequenced the inserts of the plasmids coding for the library. In two clones, we identified E. coli lysyl-tRNA synthetase (EcK-RS) as a candidate antigen. To confirm that EcK-RS contains the antigenic epitope, we coated purified, recombinant EcK-RS (15Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nature Biotech. 2001; 19: 751-755Crossref PubMed Scopus (1311) Google Scholar) to a microtiter plate, added T hybridoma cells that expressed M88 (11Wiendl H. Malotka J. Holzwarth B. Weltzien H.U. Wekerle H. Hohlfeld R. Dornmair K. An autoreactive γδ TCR derived from a polymyositis lesion.J. Immunol. 2002; 169: 515-521Crossref PubMed Scopus (37) Google Scholar), and measured secreted IL-2 in the supernatant. We found that M88 indeed recognizes EcK-RS (Fig. 1D). Because we surmised that M88 might recognize an epitope present on several proteins, we tested other proteins from the translation apparatus of E. coli (15Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nature Biotech. 2001; 19: 751-755Crossref PubMed Scopus (1311) Google Scholar). Several proteins were indeed recognized. Although all proteins were highly purified, some minor contaminations were detectable in some of the preparations (supplemental Fig. S1A). To exclude that such contaminations were recognized, we subjected some of the samples to size-exclusion HPLC and tested all eluted fractions for M88-activating capacity (Fig. 2). We found that in addition to EcK-RS, the E. coli AA-RS for asparagine (EcN-RS) and the EcIF1 1 (E. coli translation initiation factor) specifically activated M88 because M88 activation was observed only with fractions that contained the respective proteins. By contrast, AA-RS for aspartic acid (EcD-RS) was not recognized (Fig. 2). Together, these data show that M88 recognizes several functionally and structurally different bacterial proteins. Because we knew that M88 may also recognize proteins from human muscle cell extracts (11Wiendl H. Malotka J. Holzwarth B. Weltzien H.U. Wekerle H. Hohlfeld R. Dornmair K. An autoreactive γδ TCR derived from a polymyositis lesion.J. Immunol. 2002; 169: 515-521Crossref PubMed Scopus (37) Google Scholar), we tested recombinant human AA-RS for histidine and alanine (hH-RS, hA-RS), which are also known as myositis antigens “Jo-1” and “PL-12”. As above, we performed size-exclusion HPLC to exclude that contaminations were recognized (Fig. 3 and supplemental Fig. S1B). M88 activation was observed only in fractions containing hH-RS and hA-RS, providing evidence that these proteins were recognized directly. M88 activation was not limited to AA-RS because also hLC1 (20Mao Y. Vyas N.K. Vyas M.N. Chen D.H. Ludtke S.J. Chiu W. Quiocho F.A. Structure of the bifunctional and Golgi-associated formiminotransferase cyclodeaminase octamer.EMBO J. 2004; 23: 2963-2971Crossref PubMed Scopus (24) Google Scholar), a hepatitis antigen, was recognized. The control protein hPCNA, which was identically produced and purified, was not recognized. Activation of hH-RS was specific, as it was blocked completely by a polyclonal anti-serum and by a monoclonal antibody against hH-RS (Fig. 4). Recognition of hA-RS and of the hu" @default.
• W1888682760 created "2016-06-24" @default.
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• W1888682760 creator A5025633573 @default.
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• W1888682760 date "2012-06-01" @default.
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• W1888682760 title "Target Specificity of an Autoreactive Pathogenic Human γδ-T Cell Receptor in Myositis" @default.
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