title: Preparation and characterization of HLA-A*0201 tetramer loaded with IE-1316-324 antigenic peptide of human cytomegalovirus.
abstract: 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 pp65(495-503) 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 microl whole blood was 0.5 microg with incubation at 4 degrees C 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.
authors.affiliation: Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Lihui
authors.initials: L
authors.lastname: Xu
authors.forename: Qingbing
authors.initials: Q
authors.lastname: Zha
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.forename: Qiantao
authors.initials: Q
authors.lastname: Jia
authors.forename: Fengyao
authors.initials: F
authors.lastname: Li
authors.forename: Xianhui
authors.initials: X
authors.lastname: He
mesh_terms: D001710:Biotin / Q000737:chemistry; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003587:Cytomegalovirus / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005805:Genes, MHC Class I / Q000276:immunology; D015234:HLA-A Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D015789:HLA-A2 Antigen; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D018984:Epitopes, T-Lymphocyte; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; C088645:IE1 protein, cytomegalovirus; C464970:IE1-pp65 recombinant protein, Human cytomegalovirus; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D001710:Biotin