title: [Preparation of H-2Db tetramer and its application in enumerating the CD8+ T cells specific for lymphocytic choriomeningitis virus].
abstract: Major histocompatibility complex (MHC) tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i.e. the ectodomain of H-2Db fused to a BirA substrate peptide (BSP), was constructed and overexpressed in E. coli BL21(DE3). Then, the denatured H-2Db-BSP was refolded in the presence of human beta2-microglobulin as well as the GP33-41 peptide (KAVYNFATC, KAV) of LCMV. The biotinylated H-2Db/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2Db KAV tetramer reagent was verified by detecting the CD8+ T cells specific for HCMV in KAV peptide vaccinated C57BL/6 mouse, with a mouse receiving subcutaneous injection of only adjuvant as negative control. The results showed that the tetramer positive rates were 0.27%, 0.11%, and 0.24% within the CD8+ T cell populations in the peripheral blood, draining lymph nodes, and spleen of vaccinated mouse, respectively. There was only very low background staining (< or = 0.01%) of those samples from the control mouse. Beside, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2Db tetramer will facilitate the study of the immune responses of antigen-specific CD8+ T cells in the experimental immunotherapy on the mice with H-2Db allele background.
authors.affiliation: Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Lihui
authors.initials: L
authors.lastname: Xu
authors.forename: Xuesi
authors.initials: X
authors.lastname: Zeng
authors.forename: Jianfang
authors.initials: J
authors.lastname: Sun
authors.forename: Xianhui
authors.initials: X
authors.lastname: He
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D006183:H-2 Antigens / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D007158:Immunologic Techniques; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D037182:T-Cell Antigen Receptor Specificity / Q000276:immunology*
chemical_list: D006183:H-2 Antigens; D015395:Histocompatibility Antigens Class I