AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 he...AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T cells.RESULTS： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specificall展开更多
AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay con...AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.展开更多
HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting o...HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A＊0209 and HLA-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study ind展开更多
Cytotoxic T cells （CTLs） play a key role in the control of Hepatitis B virus （HBV） infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have be...Cytotoxic T cells （CTLs） play a key role in the control of Hepatitis B virus （HBV） infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein （HBc） is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool cov- ering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141- 149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti- HBV activity were further determined in HBV and HLA- A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parame- ters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.展开更多
Background:Immunotherapy has been shown to be a promising strategy against human cancers.A better understanding of the immune regulation in hepatocellular carcinoma(HCC)could help the development of immunotherapy agai...Background:Immunotherapy has been shown to be a promising strategy against human cancers.A better understanding of the immune regulation in hepatocellular carcinoma(HCC)could help the development of immunotherapy against HCC.The epidermal growth factor receptor(EGFR)signaling is frequently activated in HCC and plays important roles in tumorigenesis.However,its role in HCC immunity is still largely unknown.This study aimed to investigate the impact of EGFR signaling on programmed death-ligand 1(PD-L1)and human leukocyte antigen class-I(HLA-I)expression in HCC cells and its underlying mechanisms.Methods:The expression of phosphorylated EGFR(p-EGFR),PD-L1,and HLAI(HLA-ABC)in HCC specimens was detected by immunohistochemistry,and their correlations were analyzed.PD-L1 and HLA-ABC expression in EGFRactivated HCC cells were detected by quantitative real-time PCR,Western blotting,and flow cytometry,and T cell-mediated lysis was performed to test the immunosuppressive effects of PD-L1 and HLA-ABC alterations in HCC cells.Furthermore,the underlying mechanisms of EGFR activation-induced PD-L1 up-regulation and HLA-ABC down-regulation were explored by animal experiments,luciferase reporter assay,and gene gain-and loss-of-function studies.Results:p-EGFR was positively correlated with PD-L1 and negatively correlated with HLA-ABC expression in HCCs.EGFR activation by its ligand EGF up-regulated PD-L1 and down-regulated HLA-ABC in HCC cells,which was functionally important and could be abolished by the EGFR inhibitor,gefitinib,both in vitro and in vivo.Mechanistically,enhanced P38 mitogenactivated protein kinase(MAPK)activation down-regulated microRNA-675-5p(miR-675-5p)and up-regulated glycolysis-related enzyme hexokinase 2(HK2);miR-675-5p down-regulation enhanced the stability of PD-L1 mRNA probably via the 3’-untranslated region(3’-UTR)of PD-L1 and thereby caused PD-L1 accumulation,and HK2 up-regulation enhanced aerobic glycolysis and mediated a decrease in HLA-ABC.Conclusions:The EGFR-P38 MAPK axis could up-regulate PD-L展开更多
Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwi...Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are rela- tively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type I profile, which promotes cellular effec- tor mechanisms are thought to be more likely to experi- ence viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells, especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in deter- mining the outcome of acute and chronic HCV infection will be discussed in this review.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30230350 and No. 30371651Major State Basic Research Development Program of China, 973 Program, No. G2000057006
文摘AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T cells.RESULTS： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specificall
基金the National Nature Science Foundation of China,No.39800121
文摘AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.
文摘HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A＊0209 and HLA-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study ind
文摘Cytotoxic T cells （CTLs） play a key role in the control of Hepatitis B virus （HBV） infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein （HBc） is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool cov- ering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141- 149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti- HBV activity were further determined in HBV and HLA- A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parame- ters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.
基金This work was supported by National Natural Science Foundation of China(No.81602493,No.81902926,No.31600746)Natural Science Foundation of Guangdong Frovince(No.2018030310305)Fund from Guangzhou Women and Children’s Medical Center/Internal Medicine Department(No.NKE-PRE-2019-008).
文摘Background:Immunotherapy has been shown to be a promising strategy against human cancers.A better understanding of the immune regulation in hepatocellular carcinoma(HCC)could help the development of immunotherapy against HCC.The epidermal growth factor receptor(EGFR)signaling is frequently activated in HCC and plays important roles in tumorigenesis.However,its role in HCC immunity is still largely unknown.This study aimed to investigate the impact of EGFR signaling on programmed death-ligand 1(PD-L1)and human leukocyte antigen class-I(HLA-I)expression in HCC cells and its underlying mechanisms.Methods:The expression of phosphorylated EGFR(p-EGFR),PD-L1,and HLAI(HLA-ABC)in HCC specimens was detected by immunohistochemistry,and their correlations were analyzed.PD-L1 and HLA-ABC expression in EGFRactivated HCC cells were detected by quantitative real-time PCR,Western blotting,and flow cytometry,and T cell-mediated lysis was performed to test the immunosuppressive effects of PD-L1 and HLA-ABC alterations in HCC cells.Furthermore,the underlying mechanisms of EGFR activation-induced PD-L1 up-regulation and HLA-ABC down-regulation were explored by animal experiments,luciferase reporter assay,and gene gain-and loss-of-function studies.Results:p-EGFR was positively correlated with PD-L1 and negatively correlated with HLA-ABC expression in HCCs.EGFR activation by its ligand EGF up-regulated PD-L1 and down-regulated HLA-ABC in HCC cells,which was functionally important and could be abolished by the EGFR inhibitor,gefitinib,both in vitro and in vivo.Mechanistically,enhanced P38 mitogenactivated protein kinase(MAPK)activation down-regulated microRNA-675-5p(miR-675-5p)and up-regulated glycolysis-related enzyme hexokinase 2(HK2);miR-675-5p down-regulation enhanced the stability of PD-L1 mRNA probably via the 3’-untranslated region(3’-UTR)of PD-L1 and thereby caused PD-L1 accumulation,and HK2 up-regulation enhanced aerobic glycolysis and mediated a decrease in HLA-ABC.Conclusions:The EGFR-P38 MAPK axis could up-regulate PD-L
文摘目的探讨人类白细胞抗原(human leukocyte antigen,HLA)基因多态性与乙型肝炎病毒(HBV)感染的相关性。方法收集云南省昆明市延安医院健康体检者静脉血样本501例,采用酶联免疫吸附试验(ELISA)检测HBV二对半,根据HBV二对半检测结果分为HBV携带组和既往感染组以及健康对照组3组,用序列特异性引物聚合酶链反应(polymerase chain reaction with sequence specific primers,PCR-SSP)基因分型技术检测HLA-A抗原的基因型,将HBV携带组和健康对照组以及HBV既往感染组和健康对照组的HLA-A基因多态性的分布频率进行比较。采用SPSS17.0软件进行数据统计分析。结果健康对照组HLA-A2阳性数占比47.49%,等位基因频率数占比31.29%;健康对照组基因分布频率总体与中华骨髓库发布的中国常见及确认的HLA-A等位基因表一致。HBV携带组HLA-A2阳性数占比63.04%,等位基因频率数占比42.23%,携带者的HLA-A2阳性率和等位基因频率差异有统计学意义(P<0.05);HBV既往感染组HLA-A2阳性数占比56.14%,等位基因频率数占比35.97%,既往感染组的HLA-A2阳性率和等位基因频率差异无统计学意义(P>0.05)。结论HLA-A2基因可能是慢性乙型肝炎HBV携带者的易感基因。
基金the Deutsche Forschungsgemeinschaft and the Wellcome Trust and the James Martin School for the 21st century, Oxford
文摘Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are rela- tively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type I profile, which promotes cellular effec- tor mechanisms are thought to be more likely to experi- ence viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells, especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in deter- mining the outcome of acute and chronic HCV infection will be discussed in this review.
