Preparation of H-2Db tetramer and its application in enumerating the CD8+ T cells specific for lymphocytic choriomeningitis virus.
- Author:
Yi LIU
1
;
Lihui XU
;
Xuesi ZENG
;
Jianfang SUN
;
Xianhui HE
Author Information
1. Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibody Specificity;
CD8-Positive T-Lymphocytes;
immunology;
H-2 Antigens;
genetics;
Histocompatibility Antigens Class I;
genetics;
Immunologic Techniques;
Lymphocytic choriomeningitis virus;
immunology;
Male;
Mice;
Mice, Inbred C57BL;
T-Cell Antigen Receptor Specificity;
immunology
- From:
Chinese Journal of Biotechnology
2008;24(2):278-284
- CountryChina
- Language:Chinese
-
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.
