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J. Biol. Chem., Vol. 283, Issue 18, 12528-12537, May 2, 2008

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Identification of Human T Cell Receptor -recognized Epitopes/Proteins via CDR3 Peptide-based Immunobiochemical Strategy^*

From the Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Peking Union Medical College, National Key Laboratory of Medical Molecular Biology, Beijing 100005, China

Human T lymphocytes, bearing T cell receptor (TCR) , play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCR have been defined so far. To investigate antigenic epitopes/proteins recognized by T cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR chain (CDR3) peptide-mediated epitope/protein-binding assays. CDR3 peptides synthesized using the CDR3 region in TCR V2 chain were validated for their binding specificity to target cells or tissues. These CDR3 peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionization-tandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCR, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to T cells but also functionally activate T cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCR. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of V2 T cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCR. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by T cells.

Received for publication, September 27, 2007 , and in revised form, March 5, 2008.

^* This work was supported by Grants 2001CB510009, 2004CB518706, and 2007CB512405 from the National Program for Key Basic Research Projects; Grants 2006AA02Z480 and 2006AA02A245 from the National Program for Biomedical Hightech, the Ministry of Science and Technology of China; and Grant 30490244 from the National Natural Science Foundation of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1-5 and Figs. 1, 2 and 3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, National Key Laboratory of Medical Molecular Biology, 5 Dong Dan San Tiao, Beijing 100005, China. Tel.: 86-10-65136981; Fax: 86-10-65105909; E-mail: heweiimu{at}public.bta.net.cn.

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