Basic Research
Copyright ©The Author(s) 2005. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 21, 2005; 11(27): 4180-4187
Published online Jul 21, 2005. doi: 10.3748/wjg.v11.i27.4180
Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes
Xian-Hui He, Li-Hui Xu, Yi Liu
Xian-Hui He, Key Laboratory of Ministry of Education of China for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, Guangdong Province, China
Li-Hui Xu, Institute of Bioengineering, Jinan University, Guangzhou 510632, Guangdong Province, China
Yi Liu, Department of Dermatology, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30230350 and No. 30371651, and Major State Basic Research Development Program of China, 973 Program, No. G2000057006
Correspondence to: Dr. Xian-Hui He, Key Laboratory of Ministry of Education of China for Tissue Transplantation and Immunology, Jinan University, 601 Huangpu Road West, Guangzhou 510632, Guangdong Province, China. thexh@jnu.edu.cn
Telephone: +86-20-85220679 Fax: +86-20-85221337
Received: August 18, 2004
Revised: November 14, 2004
Accepted: November 19, 2004
Published online: July 21, 2005
Abstract

AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.

METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were cloned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.

RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully cloned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV, NLV; designated as A2-NLV) or influenza virus matrix protein Mp58–66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor, but failed to bind to those from a HLA-A2- donor.

CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.

Keywords: Major histocompatibility complex; HLA-A2; Tetramers; Cytomegalovirus; Immune responses; Cytotoxic T lymphocytes