Xianhui He

High Frequencies Cytomegalovirus pp65495–503-Specific CD8+ T Cells in Healthy Young and Elderly Chinese Donors: Characterization of Their Phenotypes and TCR Vβ Usage

Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus which persists lifelong after primary infection and can lead to a significant disease in the immunocompromised individuals. CD8+ T cells are believed to play a crucial role in both the elimination of active infection and maintenance of HCMV latency. Large expansions of CD8+ T cells specific for a single epitope of HCMV have been well documented in Caucasoid population. To date, no similar study has been performed in Chinese populations. Here we report the characteristics of HCMV-specific CD8+ T cells in healthy young and elderly Chinese donors using pp65495–503-loaded HLA-A*0201 tetramers. Cells were stained with a combination of the tetramers and antibodies for CD28 and CD57 or a panel of TCR Vβ and analyzed by three-color flow cytometry. The frequencies of pp65495–503-specific T cells within total CD8+ T cell population were between 0.14 and 6.84% (mean 2.45%) in the young donors and were from 0.33 to 6.89% (mean 1.95%) in the elderly donors, respectively. There was no significant difference between the two groups. The expression of CD28 was decreased whereas CD57 expression was increased in tetramer-negative CD8+ T cells in the elderly when compared with the young group. However, neither of these changes was found within tetramer-positive cell populations. Moreover, TCR Vβ usage within tetramer-positive population was predominated by certain TCR Vβ subsets. These results demonstrate that large expansions of HCMV-specific CD8+ T cells with certain subsets TCR Vβ exist both in the healthy young and in the elderly Chinese individuals, which may play a role in the maintenance of virus latency but have potential detrimental influence on the immune responses to other pathogens or vaccinations.

Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif.

AbstractAim:To investigate the effect of incorporation of an isoleucine zipper (IZ) motif into CD40 on binding activity of CD40 for the CD40 ligand (CD40L).Methods:Prokaryotic expression vectors for 2 soluble CD40 derivatives, shCD40His and shCD40IZ containing an IZ dowain, were constructed and expressed in Escherichia coli. The recombinant proteins were purified to homogeneity after refolding from inclusion bodies. Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography, and their binding activity for CD40L on Jurkat T cells was determined by flow cytometry.Results:shCD40His and shCD40IZ were generated. Both of them possessed significant binding activity for the cognate ligand CD40L expressed on the cell surface. shCD40IZ had much higher binding activity to its ligand (CD40L) than did shCD40His. Furthermore, size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution.Conclusion:Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.

[Preparation and identification of human soluble sPD-L1 and its antibodies].

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.

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, 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.

[Improved expression of HLA-A* 2402-BSP in Escherichia coli and its tetramer preparation].

HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test 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 gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene 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 of beta2-microglobulin and QYD peptide. The tetramer was subsequently 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 and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.