Improved generation of HLA class I/peptide tetramers.

Abstract

As a tetrameric major histocompatibility complex (MHC) class I-peptide complex (tetramer) is capable of detecting antigen-specific cytotoxic T lymphocyte (CTL) by flow cytometry, significant information about the generation of in vivo immunity can be obtained. It is, however, difficult to make a soluble wild type of MHC class I heavy chain by the prokaryotic expression system. Therefore, we developed a new method for making soluble mutant HLA-A*2402 heavy chain. In this method, signal sequences were deleted, and the codon was changed to silent mutated nucleotide sequences that bacteria could use as preferable codon. When purified mutant HLA-A*2402 molecules were examined for the protein generation by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting using anti-HLA class I monoclonal antibody (mAb) as compared with wild type, a large amount of mutant heavy chain could be detected. In contrast, the expression of wild-type stable HLA-A*2402 heavy chain molecule was not detected in this system. Consequently, by using mutant HLA-A*2402/peptide tetramers, CTL precursors (CTLp) that specifically recognize antigenic peptide derived from the X;18 chromosomal translocations of synovial sarcoma were detected in patients' PBL.

• Authors: Yuriko Sato, Hiroeki Sahara, Tomohide Tsukahara, Masako Kondo, Yoshihiko Hirohashi, Yuki Nabeta, Satoshi Kawaguchi, Hideyuki Ikeda, Toshihiko Torigoe, Shingo Ichimiya, Yasuaki Tamura, Takuro Wada, Toshihiko Yamashita, Masashi Goto, Hideo Takasu, Noriyuki Sato
• Journal: Journal of immunological methods (J Immunol Methods) Vol. 271 Issue 1-2 Pg. 177-84 (Dec 20 2002) ISSN: 0022-1759 [Print] Netherlands
• PMID: 12445740 (Publication Type: Journal Article)

Chemical References

• Epitopes, T-Lymphocyte
• HLA-A Antigens
• Immunoglobulin Heavy Chains
• Oncogene Proteins, Fusion
• Recombinant Proteins
• SYT-SSX fusion protein
• beta 2-Microglobulin

Topics

• Base Sequence
• CD8-Positive T-Lymphocytes (immunology)
• Epitopes, T-Lymphocyte (genetics, immunology)
• Escherichia coli (genetics)
• HLA-A Antigens (biosynthesis, chemistry, genetics, immunology)
• Humans
• Immunoglobulin Heavy Chains (genetics, immunology)
• Molecular Sequence Data
• Mutation
• Oncogene Proteins, Fusion (genetics, immunology)
• Polymerase Chain Reaction
• Recombinant Proteins (biosynthesis, genetics, immunology)
• Sarcoma, Synovial (immunology)
• T-Lymphocytes, Cytotoxic (immunology)
• Transformation, Genetic (genetics, immunology)
• beta 2-Microglobulin (genetics, immunology)