Abstract
Despite progress in defining the nature of major histocompatibility complex products that are recognized by the T-cell antigen receptor, the binding properties and structure of the receptor have not been solved. The primary problem has been the difficulty in obtaining sufficient quantities of active receptor. In this report we show that a single-chain T-cell receptor gene can be expressed in Escherichia coli. The protein consists of the variable (V) regions of the alpha and beta chains (V alpha and V beta) encoded by the cytotoxic T-lymphocyte clone 2C (a H-2b anti-H-2d alloreactive cell line) linked by a 25-amino acid flexible peptide. Solubilized extracts that contain the 27-kDa V alpha 3V beta 8 protein are positive in solid-phase immunoassays with the anti-V beta 8 antibody KJ16 and the anti-clonotypic antibody 1B2. Approximately 1% of the protein can be specifically purified on a 1B2-conjugated column. These results indicate that a fraction of the protein is able to fold into a native conformation and that single-chain proteins should be useful not only as immunogens for eliciting anti-T-cell receptor antibodies but in the study of T-cell receptor structure and function.
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Selected References
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- Ajitkumar P., Geier S. S., Kesari K. V., Borriello F., Nakagawa M., Bluestone J. A., Saper M. A., Wiley D. C., Nathenson S. G. Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor. Cell. 1988 Jul 1;54(1):47–56. doi: 10.1016/0092-8674(88)90178-x. [DOI] [PubMed] [Google Scholar]
- Bates R. M., Ballard D. W., Voss E. W., Jr Comparative properties of monoclonal antibodies comprising a high-affinity anti-fluorescyl idiotype family. Mol Immunol. 1985 Aug;22(8):871–877. doi: 10.1016/0161-5890(85)90072-0. [DOI] [PubMed] [Google Scholar]
- Bedzyk W. D., Weidner K. M., Denzin L. K., Johnson L. S., Hardman K. D., Pantoliano M. W., Asel E. D., Voss E. W., Jr Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody. J Biol Chem. 1990 Oct 25;265(30):18615–18620. [PubMed] [Google Scholar]
- Berg L. J., Pullen A. M., Fazekas de St Groth B., Mathis D., Benoist C., Davis M. M. Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand. Cell. 1989 Sep 22;58(6):1035–1046. doi: 10.1016/0092-8674(89)90502-3. [DOI] [PubMed] [Google Scholar]
- Bill J., Palmer E. Positive selection of CD4+ T cells mediated by MHC class II-bearing stromal cell in the thymic cortex. Nature. 1989 Oct 19;341(6243):649–651. doi: 10.1038/341649a0. [DOI] [PubMed] [Google Scholar]
- Bird R. E., Hardman K. D., Jacobson J. W., Johnson S., Kaufman B. M., Lee S. M., Lee T., Pope S. H., Riordan G. S., Whitlow M. Single-chain antigen-binding proteins. Science. 1988 Oct 21;242(4877):423–426. doi: 10.1126/science.3140379. [DOI] [PubMed] [Google Scholar]
- Brändle D., Bürki K., Wallace V. A., Rohrer U. H., Mak T. W., Malissen B., Hengartner H., Pircher H. Involvement of both T cell receptor V alpha and V beta variable region domains and alpha chain junctional region in viral antigen recognition. Eur J Immunol. 1991 Sep;21(9):2195–2202. doi: 10.1002/eji.1830210930. [DOI] [PubMed] [Google Scholar]
- Chothia C., Boswell D. R., Lesk A. M. The outline structure of the T-cell alpha beta receptor. EMBO J. 1988 Dec 1;7(12):3745–3755. doi: 10.1002/j.1460-2075.1988.tb03258.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis M. M., Bjorkman P. J. T-cell antigen receptor genes and T-cell recognition. Nature. 1988 Aug 4;334(6181):395–402. doi: 10.1038/334395a0. [DOI] [PubMed] [Google Scholar]
- Denzin L. K., Whitlow M., Voss E. W., Jr Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20. J Biol Chem. 1991 Jul 25;266(21):14095–14103. [PubMed] [Google Scholar]
- DerSimonian H., Band H., Brenner M. B. Increased frequency of T cell receptor V alpha 12.1 expression on CD8+ T cells: evidence that V alpha participates in shaping the peripheral T cell repertoire. J Exp Med. 1991 Sep 1;174(3):639–648. doi: 10.1084/jem.174.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gascoigne N. R. Transport and secretion of truncated T cell receptor beta-chain occurs in the absence of association with CD3. J Biol Chem. 1990 Jun 5;265(16):9296–9301. [PubMed] [Google Scholar]
- Grégoire C., Rebaï N., Schweisguth F., Necker A., Mazza G., Auphan N., Millward A., Schmitt-Verhulst A. M., Malissen B. Engineered secreted T-cell receptor alpha beta heterodimers. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8077–8081. doi: 10.1073/pnas.88.18.8077. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haskins K., Hannum C., White J., Roehm N., Kubo R., Kappler J., Marrack P. The antigen-specific, major histocompatibility complex-restricted receptor on T cells. VI. An antibody to a receptor allotype. J Exp Med. 1984 Aug 1;160(2):452–471. doi: 10.1084/jem.160.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hedrick S. M., Engel I., McElligott D. L., Fink P. J., Hsu M. L., Hansburg D., Matis L. A. Selection of amino acid sequences in the beta chain of the T cell antigen receptor. Science. 1988 Mar 25;239(4847):1541–1544. doi: 10.1126/science.2832942. [DOI] [PubMed] [Google Scholar]
- Howell M. D., Winters S. T., Olee T., Powell H. C., Carlo D. J., Brostoff S. W. Vaccination against experimental allergic encephalomyelitis with T cell receptor peptides. Science. 1989 Nov 3;246(4930):668–670. doi: 10.1126/science.2814489. [DOI] [PubMed] [Google Scholar]
- Hubbard S. C., Kranz D. M., Longmore G. D., Sitkovsky M. V., Eisen H. N. Glycosylation of the T-cell antigen-specific receptor and its potential role in lectin-mediated cytotoxicity. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1852–1856. doi: 10.1073/pnas.83.6.1852. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huston J. S., Levinson D., Mudgett-Hunter M., Tai M. S., Novotný J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. doi: 10.1073/pnas.85.16.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kappler J. W., Roehm N., Marrack P. T cell tolerance by clonal elimination in the thymus. Cell. 1987 Apr 24;49(2):273–280. doi: 10.1016/0092-8674(87)90568-x. [DOI] [PubMed] [Google Scholar]
- Kappler J. W., Wade T., White J., Kushnir E., Blackman M., Bill J., Roehm N., Marrack P. A T cell receptor V beta segment that imparts reactivity to a class II major histocompatibility complex product. Cell. 1987 Apr 24;49(2):263–271. doi: 10.1016/0092-8674(87)90567-8. [DOI] [PubMed] [Google Scholar]
- Kisielow P., Blüthmann H., Staerz U. D., Steinmetz M., von Boehmer H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature. 1988 Jun 23;333(6175):742–746. doi: 10.1038/333742a0. [DOI] [PubMed] [Google Scholar]
- Kisielow P., Teh H. S., Blüthmann H., von Boehmer H. Positive selection of antigen-specific T cells in thymus by restricting MHC molecules. Nature. 1988 Oct 20;335(6192):730–733. doi: 10.1038/335730a0. [DOI] [PubMed] [Google Scholar]
- Kranz D. M., Sherman D. H., Sitkovsky M. V., Pasternack M. S., Eisen H. N. Immunoprecipitation of cell surface structures of cloned cytotoxic T lymphocytes by clone-specific antisera. Proc Natl Acad Sci U S A. 1984 Jan;81(2):573–577. doi: 10.1073/pnas.81.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kranz D. M., Tonegawa S., Eisen H. N. Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7922–7926. doi: 10.1073/pnas.81.24.7922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kranz D. M., Voss E. W., Jr Partial elucidation of an anti-hapten repertoire in BALB/c mice: comparative characterization of several monoclonal anti-fluorescyl antibodies. Mol Immunol. 1981 Oct;18(10):889–898. doi: 10.1016/0161-5890(81)90012-2. [DOI] [PubMed] [Google Scholar]
- Liao N. S., Maltzman J., Raulet D. H. Positive selection determines T cell receptor V beta 14 gene usage by CD8+ T cells. J Exp Med. 1989 Jul 1;170(1):135–143. doi: 10.1084/jem.170.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin A. Y., Devaux B., Green A., Sagerström C., Elliott J. F., Davis M. M. Expression of T cell antigen receptor heterodimers in a lipid-linked form. Science. 1990 Aug 10;249(4969):677–679. doi: 10.1126/science.1696397. [DOI] [PubMed] [Google Scholar]
- MacDonald H. R., Lees R. K., Schneider R., Zinkernagel R. M., Hengartner H. Positive selection of CD4+ thymocytes controlled by MHC class II gene products. Nature. 1988 Dec 1;336(6198):471–473. doi: 10.1038/336471a0. [DOI] [PubMed] [Google Scholar]
- McIntyre B. W., Allison J. P. Biosynthesis and processing of murine T-cell antigen receptor. Cell. 1984 Oct;38(3):659–665. doi: 10.1016/0092-8674(84)90260-5. [DOI] [PubMed] [Google Scholar]
- Novotny J., Ganju R. K., Smiley S. T., Hussey R. E., Luther M. A., Recny M. A., Siliciano R. F., Reinherz E. L. A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8646–8650. doi: 10.1073/pnas.88.19.8646. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novotný J., Tonegawa S., Saito H., Kranz D. M., Eisen H. N. Secondary, tertiary, and quaternary structure of T-cell-specific immunoglobulin-like polypeptide chains. Proc Natl Acad Sci U S A. 1986 Feb;83(3):742–746. doi: 10.1073/pnas.83.3.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pantoliano M. W., Bird R. E., Johnson S., Asel E. D., Dodd S. W., Wood J. F., Hardman K. D. Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli. Biochemistry. 1991 Oct 22;30(42):10117–10125. doi: 10.1021/bi00106a007. [DOI] [PubMed] [Google Scholar]
- Saito H., Kranz D. M., Takagaki Y., Hayday A. C., Eisen H. N., Tonegawa S. A third rearranged and expressed gene in a clone of cytotoxic T lymphocytes. Nature. 1984 Nov 1;312(5989):36–40. doi: 10.1038/312036a0. [DOI] [PubMed] [Google Scholar]
- Sha W. C., Nelson C. A., Newberry R. D., Kranz D. M., Russell J. H., Loh D. Y. Positive and negative selection of an antigen receptor on T cells in transgenic mice. Nature. 1988 Nov 3;336(6194):73–76. doi: 10.1038/336073a0. [DOI] [PubMed] [Google Scholar]
- Sha W. C., Nelson C. A., Newberry R. D., Kranz D. M., Russell J. H., Loh D. Y. Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature. 1988 Sep 15;335(6187):271–274. doi: 10.1038/335271a0. [DOI] [PubMed] [Google Scholar]
- Sha W. C., Nelson C. A., Newberry R. D., Pullen J. K., Pease L. R., Russell J. H., Loh D. Y. Positive selection of transgenic receptor-bearing thymocytes by Kb antigen is altered by Kb mutations that involve peptide binding. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6186–6190. doi: 10.1073/pnas.87.16.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Slanetz A. E., Bothwell A. L. Heterodimeric, disulfide-linked alpha/beta T cell receptors in solution. Eur J Immunol. 1991 Jan;21(1):179–183. doi: 10.1002/eji.1830210127. [DOI] [PubMed] [Google Scholar]
- Utsunomiya Y., Bill J., Palmer E., Gollob K., Takagaki Y., Kanagawa O. Analysis of a monoclonal rat antibody directed to the alpha-chain variable region (V alpha 3) of the mouse T cell antigen receptor. J Immunol. 1989 Oct 15;143(8):2602–2608. [PubMed] [Google Scholar]
- Vandenbark A. A., Hashim G., Offner H. Immunization with a synthetic T-cell receptor V-region peptide protects against experimental autoimmune encephalomyelitis. Nature. 1989 Oct 12;341(6242):541–544. doi: 10.1038/341541a0. [DOI] [PubMed] [Google Scholar]
- Wither J., Pawling J., Phillips L., Delovitch T., Hozumi N. Amino acid residues in the T cell receptor CDR3 determine the antigenic reactivity patterns of insulin-reactive hybridomas. J Immunol. 1991 May 15;146(10):3513–3522. [PubMed] [Google Scholar]