High level expression of HLA-A*0203-BSP fusion protein in Escherichia coli and construction of soluble HLA-A*0203 monomer and tetramer loaded with Epstein-Barr virus peptide.

Major histocompatibility complex (MHC) tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A*0203 tetramer loaded with Epstein-Barr virus EBNA3(596-604) peptide (SVRDRLARL, SVR). Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A*0203 fused with a BirA substrate peptide (HLA-A*0203-BSP) was constructed and the expression conditions of the fusion protein in Escherichia coli (E. coli) were optimized. The fusion protein was highly expressed in inclusion bodies within E. coli. It was then refolded in the presence of beta2-microglobulin and SVR peptide to form a soluble HLA-A*0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4:1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8+ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8+ T cells from HLA-A*0203 subjects.

Preparation and identification of HLA-A*1101 tetramer loading with human cytomegalovirus pp65 antigen peptide.

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8+ T cells in humans. The tetramer molecule is composed of HLA heavy chain, beta2-microglobulin (beta2m), an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A*1101-restricted CD8+ T cell responses against human cytomegalovirus (HCMV), we established an approach to prepare HLA-A*1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A*1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A*1101 fused with a BirA substrate peptide (HLA-A*1101-BSP) at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichia coli. Moreover, HLA-A*1101-BSP protein was refolded in the presence of beta2m and an HCMV peptide pp65(16-24) (GPISGHVLK, GPI). Soluble HLA-A*1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of > 80%. The HLA-A*1101-GPI tetramers could bind to virus-specific CD8+ T cells, suggesting soluble HLA-A*1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A*1101-restricted CD8+ T cell responses against HCMV infection.

Preparation and characterization of HLA-A*0201 tetramer loaded with IE-1316-324 antigenic peptide of human cytomegalovirus.

Major histocompatibility complex (MHC) class I tetramer technology has become the central technique for analyzing antigen-specific CD8(+) T cell responses and it has been widely used to explore the differentiation and formation of memory CD8(+) T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A*0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp65(495-503) has been successfully applied to investigate HCMV-specific CD8(+) T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A*0201 tetramer loaded with another dominant epitope derived from immediate early (IE)-1(316-324) (VLEETSVML, VLE) of HCMV (A2-VLE) and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 microl whole blood was 0.5 microg with incubation at 4 degrees C for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8(+) T cells of one HLA-A2(+) donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8(+) T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8(+) T cells.

Discovery of novel 1,2,4-thiadiazole derivatives as potent, orally active agonists of sphingosine 1-phosphate receptor subtype 1 (S1P(1)).

A novel series of 1,2,4-thiadiazole compounds was discovered as selective S1P(1) agonists. The extensive structure-activity relationship studies for these analogues were reported. Among them, 17g was identified to show high in vitro potency with reasonable free unbound fraction in plasma (F(u) > 0.5%), good brain penetration (BBR > 0.5), and desirable pharmacokinetic properties in mouse and rat. Oral administration of 1 mg/kg 17g resulted in significant peripheral lymphocytes reduction at 4 h after dose and rapid lymphocytes recovery at 24 h. 17g showed a transient lymphopenia profile in the repeated dose study in mouse. In addition, 17g also demonstrated efficacy comparable to that of FTY720 (1) in the mouse EAE model of MS.

CD8(+) T cells specific for both persistent and non-persistent viruses display distinct differentiation phenotypes but have similar level of PD-1 expression in healthy Chinese individuals.

Anti-viral CD8(+) T cell responses involve an initial expansion and effector phase, followed by contraction phase and formation of CD8(+) memory T cells. During this contraction phase, increased surface expression of the negative regulator PD-1 is associated with functional exhaustion of CD8(+) T cells. Although its role in T cell suppression has been established, the importance of PD-1 in the differentiation of CD8(+) T cells remains unclear. In this study, we examine PD-1 expression in relation to viral specificity of CD8(+) T cells against persistent or non-persistent viruses, and further define differentiation phenotypes of CD8(+) T cells by CD27 and CD28 expression. Surprisingly, the inhibitory receptor PD-1 was expressed by Flu-specific CD8(+) T cells in a level comparable to HCMV-and EBV-specific cells. Moreover, in virus-specific CD8(+) T cells, CD127(+)/CD127(-) and CD62L(+)/CD62L(-) cells expressed similar levels of PD-1 molecules. These results suggest that the PD-1/PD-L1 pathway may play a regulatory role in memory T cell subsets in addition to its association with T-cell exhaustion.

[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.

[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.

[Expression and refolding of a HLA-A*0203-BSP fusion protein and identification of its tetramers].

AIM: To optimize expression condition of HLA-A*0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) (HLA-A*0203-BSP) for E.coli BL21(DE3) transformant and to prepare a functional HLA-A*0203 tetramer loaded with an antigenic peptide derived from EBNA3(596-604) of Epstein-Barr virus (EBV). METHODS: The temperature, IPTG concentration and inductive duration of HLA-A*0203-BSP fusion protein expressed for E.coli BL21(DE3) transformant were optimized. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. The monomer of soluble HLA-A*0203-peptide was generated from the fusion protein by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin (beta2m) and HLA-A*0203 restricted EBV EBNA3(596-604) peptide (SVRDRLARL, SVR). Refolded and purified monomer was then biotinylated with BirA. Following the purification of the obtained biotinylated monomer, the tetramer was formed by incubation with streptavidin-PE at a ratio of 4:1. Flow cytometry (FCM) analysis was performed to determine its binding activity with specific cytotoxic T lymphocytes (CTL). RESULTS: SDS-PAGE and Western blot showed that the optimized expression condition was overnight induction at 37 degrees C with 0.4 mmol/L IPTG. The expressed protein of about 34 kDa in the form of inclusion bodies accumulated up to about 30% of total bacterial protein under the optimized expression condition. The monomer of soluble HLA-A*0203/SVR was successfully generated and purified. Non-reducing SDS-PAGE analysis showed that the biotinylation was above 85%. HLA-A*0203/SVR tetramer was constructed by mixing the monomer with streptavidin-PE at a ratio of 4:1. FCM analysis indicated that this tetramer could bind specific CTL from HLA-A2+ donors. CONCLUSION: HLA-A*0203-BSP fusion protein was overexpressed in E.coli under the optimized condition. The tetramers of HLA-A*0203/SVR were prepared from this fusion protein and it possessed binding activity with specific CTL, which provided a powerful tool for direct visualization and quantification of specific CTL from HLA-A*0203 donors.