Preparation And Application Of HLA-A*2402 Tetramers Loaded With Pp65_341-349 Antigenic Peptide Of HCMV

Human cytomegalovirus(HCMV) is a ubiquitous human persistent virus infecting most of the world populations and HCMV diseases are frequently seen in immunosuppressed individuals. Furthermore, recent studieshave demonstrated that HCMV seropositivity is one of immunological parameters which predicts incipient mortality in an elderly population. Major histocompatibility complex class I(MHC I) tetramer technology has been widely used for quantitation, phenotypic and functional characteristics of antigen-specific cytotoxic T lymphocytes(CTL), which are believed to play an essential role in the immune defense against cancer and infectious diseases. However, the high polymorphism of MHC I molecules-human leukocyte antigen(HLA) in humans—hampers the universal application of tetramer technology, thus the most frequently observed HLA class I alleles are preferable for tetramer construction. HLA-A*2402 is one of the most common HLA class 1 allele in East Asian populations, especially in the Japanese(allelic frequency 58.1ï¼…) and Chinese populations(allelic frequency 32.9ï¼…).In order to study the CD8⁺ T cell responses in Chinese populations, we described here the generation and application of HLA-A*2402 tetramer loaded with HCMV pp65_341-349 peptide (QYDPVAALF, QYD). The cDNA of HLA-A*2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A*2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide(BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type HLA-A*2402-BSP was not expressed in Escherichia coli(E. coli), while mutant HLA-A*2402-BSP with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A*2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence ofβ₂-microglobulin and OYD peptide. The tetramer was formed by mixing HLA-A*2402-QYD monomers with streptavidin-PE at a molar ratio of 4: 1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24⁺ donors. The titration of HLA-A*2402-QYD tetramer dosage was performed and the optimal dosage for 100μL whole blood was determined to be 0.35μg, under which the mean fluorescence intensity(MFI) of specific staining was higher while unspecific staining of CD8⁺ T cells was quiet low. Furthermore, the HLA-A*2402-QYD tetramer was utilized and the the quantitation, phenotypic and functional characteristics(CD62L,CCR7,CD127,CD28,CD27,PD-1 and Perforin) of HCMV specific CTL from healthy Chinese HLA-A24⁺ donors was investigated. The results showed the frequencies of tetramer⁺ CTL were 0.11ï¼…-1.52ï¼…(mean 0.42ï¼…) within total CD8⁺ T cells, while phenotypic and functional characteristics of tetramer⁺ CD8⁺ T cells indicated that they are highly heterogeneous, suggesting they may be composed of CD8⁺ T cells at various differentiated stages.In conclusion, the cDNA of HLA-A*2402 heavy chain was successfully cloned and HLA-A*2402-BSP was overexpressed in E. coli by codon optimization. Furthermore, HLA-A*2402-QYD tetramer was prepared from this fusion protein and it was biologically functional. This tetramer was utilized and the quantitation, phenotypic and functional characteristics of HCMV-specific CTL from healthy Chinese HLA-A24⁺ donors was investigated, which provides valuable data for further characterization of antigen-specific CD8⁺ T cells from HLA-A24⁺ subjects.