Preparation of H-2Db Tetramer and Its Application in Enumerating the CD8+ T Cells Specific for Lymphocytic Choriomeningitis Virus

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, the H-2Db tetramer was prepared and its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV) was verified. First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of C57BL/6 mice. The expression vector for H-2Db-BSP, that is 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 β2-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 mice, with mice receiving subcutaneous injection of only adjuvant as negative control. The results indicated 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 mice, respectively. There was only very low background staining (≤0.01%) of those samples from the control mice. Besides, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2Db tetramers 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.