High Level Expression of HLA-A ~*0203-BSP Fusion Protein in Escherichia coli and Construction of Soluble HLA-A ~*0203 Monomer and Tetramer Loaded with Epstein-Barr Virus Peptide

Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with Epstein- Barr virus EBNA3596-604 peptide （SVRDRLARL, SVR）. Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A＊0203 fused with a BirA substrate peptide （HLA-A＊0203-BSP） was constructed and the expression conditions of the fusion protein in Escherichia coli （E. coli） were optimized. The fusion protein was highly expressed in inclusion bodies within E. coil It was then refolded in the presence of 132-microglobulin and SVR peptide to form a soluble HLA-A＊0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4：1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8^＋ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8^＋ T cells from HLA-A＊0203 subjects.

Preparation and Identification of HLA-A*1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8^＋ T cells in humans. The tetramer molecule is composed of HLA heavy chain, β2-microglobulin （β2m）, an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A＊1101-restricted CD8^＋ T cell responses against human cytomegalovirus （HCMV）, we established an approach to prepare HLA-A＊1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A＊1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A＊1101 fused with a BirA substrate peptide （HLA-A＊1101-BSP） at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （GPISGHVLK, GPI）. Soluble HLA-A＊1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of 〉 80%. The HLA-A＊1101-GPI tetramers could bind to virus-specific CD8^＋ T cells, suggesting soluble HLA-A＊1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A＊1101-restricted CD8^＋ T cell responses against HCMV infection.

Preparation and Characterization of HLA-A*0201 Tetramer Loaded with IE-1_(316-324) Antigenic Peptide of Human Cytomegalovirus

Major histocompatibility complex （MHC） class I tetramer technology has become the central technique for analyzing antigen-specific CD8^＋ T cell responses and it has been widely used to explore the differentiation and formation of memory CD8^＋ T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A＊0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp6549s.50a has been successfully applied to investigate HCMV-specific CD8^＋ T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A＊0201 tetramer loaded with another dominant epitope derived from immediate early （IE）-1 316.324 （VLEETSVML, VLE） of HCMV （A2-VLE） and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 μl whole blood was 0.5 μg with incubation at 4℃ for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8^＋ T cells of one HLA-A2^＋ donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8^＋ T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8^＋ T cells. Cellular ＆ Molecular Immunology.