Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes.
OBJECTIVE
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 beta(2)-microglobulin (beta(2)m) 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 beta(2)m 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 beta(2)m 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 beta(2)m 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 beta(2)m and peptides loaded with either human cytomegalovirus pp65(495-503) 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.


CONCLUSIONS
The procedure is simple and efficient for generating peptide-MHC tetramers.
He
Xian-Hui
XH
Key Laboratory of Ministry of Education of China for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, Guangdong Province, China. thexh@jnu.edu.cn
Xu
Li-Hui
LH
Liu
Yi
Y
D003001:Cloning, Molecular / Q000379:methods*; D015234:HLA-A Antigens / Q000235:genetics*; D015789:HLA-A2 Antigen / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000502:physiology*; D001613:beta 2-Microglobulin / Q000235:genetics
D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin