Objective; Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta.Apart from its expression in placenta,brain and testis,syncytin has also been foun...Objective; Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta.Apart from its expression in placenta,brain and testis,syncytin has also been found in many cancers.Although syncytin has been proposed to serve as a positive prognostic marker in some cancers,the underlying mechanism is unclear.The aim of this study is to evaluate the effects of syncytin expression on the invasive phenotype of melanoma cells.Methods：The eukaryotic expression plasmid for syncytin-EGFP was constructed and transfected into B16F10 melanoma cells.The effect of syncytin on the invasion potential of rumor cells was evaluated in B16F10 subline cells that stably expressed syncytin-EGFP fusion protein or EGFP alone.Results：The B16F10 sublines that stably expressed syncytin-EGFP or EGFP alone were established respectively and confirmed by immunofluorescent and immtmoblotting assay.Syncytin expression in B16F10 cells was associated with decreased cell proliferation,migration and invasion.Multinucleated giant cells that contained as many as five nuclei were induced in syncytin-expressing cells.In addition,syncytin expression did not alter the sensitivity of B16F10 cells to trichosanthin,a toxin that damages syncytiotrophoblasts more efficiently than other tissues.Conclusions：These results suggest that syncytin expression in some cancers may confine their invasion potential and thus serve as a positive prognostic factor.展开更多
The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate t...The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin （PHA） alone or phorbol dibutyrate （PDB） plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry. The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide （PI） and 7-aminoactinomycin D （7-AAD）, respectively. The results showed that 100 μmol/L and 50 μmol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 μmol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased. Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 μmol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes, suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.展开更多
Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility co...Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility complex （MHC） class I tetramers, however, were derived from Indian-origin macaques due to the dominant use of these animals in history. Although there are significant differences in the immunogenetic background between the two macaque populations, they share a few of common MHC class I alleles. We reported in this study the procedure for preparation of a soluble Mamu-B＊1703 （a MHC class I molecule of Chinese macaques） monomer and tetramer loaded with a dominant simian immunodeficiency virus （SIV） epitope IW9 （IRYPKTFGW） that was identified to be Mamu-B＊1701-restricted in Indian macaques. The DNA fragment encoding the Mamu-B＊1703 extracellular domain fused with a BirA substrate peptide （BSP） was amplified from a previously cloned cDNA and inserted into a prokaratic expression vector. In the presence of the antigenic peptide IW9 and light chain β2-microglobulin, the expressed heavy chain was refolded into a soluble monomer. After biotinylation, four monomers were polymerized as a tetramer by phycoerythrin-conjugated streptavidin. The tetramer, having been confirmed to have the right conformation, was a potential tool for investigation of antigen-specific CD8^＋ T-lymphocytes in SIV vaccine models of Chinese macaques. And our results also suggested that some antigenic peptides reported in Indian-origin macaques could be directly recruited as ligands for construction of Chinese macaque MHC tetramers.展开更多
Programmed death-1 （PD-1）, a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands （PD-L1 and/or PD-L2）. PD-1/PD-L1 pathway plays an impo...Programmed death-1 （PD-1）, a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands （PD-L1 and/or PD-L2）. PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag （designated as sPD-1） was expressed as inclusion bodies in Escherichia coll. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.展开更多
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...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 EBNA3596-604 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. coil It was then refolded in the presence of 132-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.展开更多
The phenotype and function of antigen-specific CD8 T cells are closely associated with the efficacy of a therapeutic vaccination. Here we showed that multiple immunizations with LCMV gp33-41 peptide （KAV） in Freund...The phenotype and function of antigen-specific CD8 T cells are closely associated with the efficacy of a therapeutic vaccination. Here we showed that multiple immunizations with LCMV gp33-41 peptide （KAV） in Freund＇s adjuvant could induce KAV-specific CD8 T cells with low expression of CD127 and CD62L molecules. The inhibitory receptor PD-1 was also expressed on a substantial part of KAV-specific CD8 T cells, and its expression level on KAV-specific CD8 T cells in spleen and lymph nodes was much higher when compared to those in peripheral blood. Furthermore, KAV-specific CD8 T cells could specifically kill KAV-pulsed target cells in vivo but the efficiency was low. These data suggest that prime-boost vaccination schedule with peptide in Freund＇s adjuvant can elicit antigen-specific CD8 T cells of effector-like phenotype with partial functional exhaustion, which may only provide short-term protection against the pathogen. Cellular ＆ Molecular Immunology.展开更多
Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide(LPS)leading to pyroptosis that has critical role in defensing against bacterial infection,whereas its excess activation under pathoge...Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide(LPS)leading to pyroptosis that has critical role in defensing against bacterial infection,whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases.However,there are few known drugs that can control caspase-11 activation.We report here that scutellarin,a flavonoid from Erigeron breviscapus,acted as an inhibitor for caspase-11 activation in macrophages.Scutellarin dosedependently inhibited intracellular LPS-induced release of caspase-11 p26(indicative of caspase-11 activation)and generation of N-terminal fragment of gasdermin D(GSDMD-NT),leading to reduced pyroptosis.It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome as evidenced by reduced apoptosisassociated speck-like protein containing a CARD(ASC)speck formation and decreased interleukin-1 beta(IL-1 b)and caspase-1 p10 secretion,whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1 b and caspase-1 p10 release and ASC speck formation but not pyroptosis.Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression.Moreover,scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A(PKA)-specific sites,and its inhibitory action on caspase-11 activation was largely abrogated by PKAinhibitor H89 or by adenylyl cyclase inhibitor MDL12330 A.Collectively,our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.展开更多
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 a...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 pp6549s.50a 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 μl whole blood was 0.5 μg with incubation at 4℃ 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. Cellular ＆ Molecular Immunology.展开更多
基金supported by grants from the Major State Basic Research Development Program of China(973 Program)(No.2010CB833603)the National Natural Science Foundation of China(No.81173604)
文摘Objective; Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta.Apart from its expression in placenta,brain and testis,syncytin has also been found in many cancers.Although syncytin has been proposed to serve as a positive prognostic marker in some cancers,the underlying mechanism is unclear.The aim of this study is to evaluate the effects of syncytin expression on the invasive phenotype of melanoma cells.Methods：The eukaryotic expression plasmid for syncytin-EGFP was constructed and transfected into B16F10 melanoma cells.The effect of syncytin on the invasion potential of rumor cells was evaluated in B16F10 subline cells that stably expressed syncytin-EGFP fusion protein or EGFP alone.Results：The B16F10 sublines that stably expressed syncytin-EGFP or EGFP alone were established respectively and confirmed by immunofluorescent and immtmoblotting assay.Syncytin expression in B16F10 cells was associated with decreased cell proliferation,migration and invasion.Multinucleated giant cells that contained as many as five nuclei were induced in syncytin-expressing cells.In addition,syncytin expression did not alter the sensitivity of B16F10 cells to trichosanthin,a toxin that damages syncytiotrophoblasts more efficiently than other tissues.Conclusions：These results suggest that syncytin expression in some cancers may confine their invasion potential and thus serve as a positive prognostic factor.
基金supported by the National B asic Research Priorities Program of China(No.G2000057006)the grants from National Natural Science Foundation of China(No.30230350&No.30371651).
文摘The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin （PHA） alone or phorbol dibutyrate （PDB） plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry. The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide （PI） and 7-aminoactinomycin D （7-AAD）, respectively. The results showed that 100 μmol/L and 50 μmol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 μmol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased. Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 μmol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes, suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.
基金supported by Natural Science Fund of Guangdong Province (No.8451063201000340)the Talented Man Initiation Fund of Jinan University (No.51208004, No.51208017) to Dr. DY Ouyang +1 种基金grants from the National Natural Science Foundation of China (No.30572199, No.30230350 and No.30371651) to Prof. XH Hethe Biochemistry and Molecular Biology Key Discipline of Guangdong Province.
文摘Chinese-descent rhesus macaques have become more prevalent for HIV infection and vaccine investigation than Indian-origin macaques. Most of the currently available data and reagents such as major histocompatibility complex （MHC） class I tetramers, however, were derived from Indian-origin macaques due to the dominant use of these animals in history. Although there are significant differences in the immunogenetic background between the two macaque populations, they share a few of common MHC class I alleles. We reported in this study the procedure for preparation of a soluble Mamu-B＊1703 （a MHC class I molecule of Chinese macaques） monomer and tetramer loaded with a dominant simian immunodeficiency virus （SIV） epitope IW9 （IRYPKTFGW） that was identified to be Mamu-B＊1701-restricted in Indian macaques. The DNA fragment encoding the Mamu-B＊1703 extracellular domain fused with a BirA substrate peptide （BSP） was amplified from a previously cloned cDNA and inserted into a prokaratic expression vector. In the presence of the antigenic peptide IW9 and light chain β2-microglobulin, the expressed heavy chain was refolded into a soluble monomer. After biotinylation, four monomers were polymerized as a tetramer by phycoerythrin-conjugated streptavidin. The tetramer, having been confirmed to have the right conformation, was a potential tool for investigation of antigen-specific CD8^＋ T-lymphocytes in SIV vaccine models of Chinese macaques. And our results also suggested that some antigenic peptides reported in Indian-origin macaques could be directly recruited as ligands for construction of Chinese macaque MHC tetramers.
文摘Programmed death-1 （PD-1）, a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands （PD-L1 and/or PD-L2）. PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag （designated as sPD-1） was expressed as inclusion bodies in Escherichia coll. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.
文摘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 EBNA3596-604 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. coil It was then refolded in the presence of 132-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.
基金supported by the grants from the National Natural Science Foundation of China(No.30672474,No.30572199,No.30230350,and No.30371651)grants from Institute of Dermatology,Chinese Academy of Medical Sciences&Peking Union Medical College.
文摘The phenotype and function of antigen-specific CD8 T cells are closely associated with the efficacy of a therapeutic vaccination. Here we showed that multiple immunizations with LCMV gp33-41 peptide （KAV） in Freund＇s adjuvant could induce KAV-specific CD8 T cells with low expression of CD127 and CD62L molecules. The inhibitory receptor PD-1 was also expressed on a substantial part of KAV-specific CD8 T cells, and its expression level on KAV-specific CD8 T cells in spleen and lymph nodes was much higher when compared to those in peripheral blood. Furthermore, KAV-specific CD8 T cells could specifically kill KAV-pulsed target cells in vivo but the efficiency was low. These data suggest that prime-boost vaccination schedule with peptide in Freund＇s adjuvant can elicit antigen-specific CD8 T cells of effector-like phenotype with partial functional exhaustion, which may only provide short-term protection against the pathogen. Cellular ＆ Molecular Immunology.
基金supported by the National Natural Science Foundation of China(Nos.81773965,81873064,and 81673664)
文摘Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide(LPS)leading to pyroptosis that has critical role in defensing against bacterial infection,whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases.However,there are few known drugs that can control caspase-11 activation.We report here that scutellarin,a flavonoid from Erigeron breviscapus,acted as an inhibitor for caspase-11 activation in macrophages.Scutellarin dosedependently inhibited intracellular LPS-induced release of caspase-11 p26(indicative of caspase-11 activation)and generation of N-terminal fragment of gasdermin D(GSDMD-NT),leading to reduced pyroptosis.It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome as evidenced by reduced apoptosisassociated speck-like protein containing a CARD(ASC)speck formation and decreased interleukin-1 beta(IL-1 b)and caspase-1 p10 secretion,whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1 b and caspase-1 p10 release and ASC speck formation but not pyroptosis.Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression.Moreover,scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A(PKA)-specific sites,and its inhibitory action on caspase-11 activation was largely abrogated by PKAinhibitor H89 or by adenylyl cyclase inhibitor MDL12330 A.Collectively,our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.
基金This work was supported by Key Project of the National Natural Science Foundation of China （No.30230350）.
文摘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 pp6549s.50a 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 μl whole blood was 0.5 μg with incubation at 4℃ 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. Cellular ＆ Molecular Immunology.
