BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity...BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies.However,ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma(HCC)patients due to the complex pathological mechanisms of HCC.AIM To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model,aiming to identify more effective immunotherapies and provide more treatment options for HCC patients.METHODS The levels of PD-1 and TIM-3 on CD4+and CD8+T cells from tumor tissues,ascites,and matched adjacent tissues from HCC patients were determined with flow cytometry.An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody(mAb)and/or anti-PD-1 mAb.Tumor growth in each group was measured.Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors.The percentage of CD4+and CD8+T cells in tissue samples from mice was tested with flow cytometry.The percentages of PD-1+CD8+,TIM-3+CD8+,and PD-1+TIM-3+CD8+T cells was accessed by flow cytometry.The levels of the cytokines including tumor necrosis factor alpha(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits.RESULTS We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+and CD8+T cells isolated from tumor tissues and ascites of HCC patients.TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight,while combined blockade had more substantial anti-tumor effects than individual treatment.Then we showed that combined therapy increased T cell infiltration into tumor tissues,and downregulated PD-1 and TIM-3 expression on CD8+T cells in tumor tissues.Moreover,combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ,and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues.Thus,we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model.CONCLUSION Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+T cell-mediated antitumor immune responses.展开更多
To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the p...To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the promoter region of TIM4 was re-sequenced by PCR-sequencing, and linkage disequilibrium was analyzed by SHEsis software. Four single nucleotide polymor- phisms （SNPs） in the promoter region of TIM4 were detected, including two new SNPs （at positions -1609, -153） and two reported SNPs （rs6874202, rs6882076）. The frequency distribution of rs6882076 was different among different races （P〈0.05）. In addition, linkage disequilibrium among the SNPs of the promoter region of TIM4 was found and GGTG was the predominant haplotype. There were four SNPs in the promoter region of TIM4 in asthma patients of Chinese Han population, which were in linkage disequilibrium.展开更多
T cell immunoglobulin mucin (TIM) family plays a key role in regulating immune re-sponses.In this study,the interactions of human TIM family with apoptotic cells were evaluated in order to provide a foundation for fur...T cell immunoglobulin mucin (TIM) family plays a key role in regulating immune re-sponses.In this study,the interactions of human TIM family with apoptotic cells were evaluated in order to provide a foundation for further study on the roles of human TIM genes in apoptosis.Nine kinds of pEGFP-N1 eukaryotic expression vectors containing different lengths of the three members of human TIM genes for the expression of TIM-EGFP and the vectors for the expression of TIM-Fc fusion pro-teins were constructed.It was found that human TIM proteins could recognize and bind to apoptotic cells directly,but not to viable cells.The interactions of sTIM-1-EGFP,sTIM-3-EGFP and sTIM-4-EGFP with apoptotic cells were blocked by TIM-1-Ig,TIM-3-Ig and TIM-4-Ig fusion proteins respectively.In addition,human TIM proteins mediated the recognition of apoptotic cells and bound to apoptotic cells directly via the IgV domains.In conclusion,the TIM family may play a key role in the regulation of apoptosis.Our data also suggest that human TIM proteins probably serve as novel proteins for the detection of the early cellular apoptosis.展开更多
目的探讨阻断Kupffer细胞(Kupffer cells,KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)诱导小鼠原位肝移植术后i Treg细胞的产生及其相关机制。方法建立小鼠原位肝移植急性排斥反...目的探讨阻断Kupffer细胞(Kupffer cells,KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)诱导小鼠原位肝移植术后i Treg细胞的产生及其相关机制。方法建立小鼠原位肝移植急性排斥反应模型,分为sham组、control m Ab组、TIM-4 m Ab组。流式细胞仪检测KCs活化,Western blot检测肝脏TIM-4表达,TUNEL检测肝细胞凋亡指数(apoptotic index,AI)。提取KCs,激光共聚焦检测KCs TIM-4表达,将KCs和提取的初始CD4+T细胞进行共培养,激光共聚焦检测Kupffer细胞TIM-4表达,分为control组、control m Ab组以及TIM-4 m Ab组;CFSE检测CD4+T细胞增殖,流式细胞仪检测CD4+CD25+Foxp3+T细胞,ELISA检测IL-4、IL-6、IL-13水平,Western blot检测STAT6表达。建立小鼠移植模型,分为3组:i Treg组移植模型注入i Treg细胞(预先用TIM-4 m Ab处理的TIM-4+KCs与初始CD4+T细胞共培养所诱导),sham组和单纯肝移植组(LT)注入相应的PBS作为对照,检测各组AST、ALT、TBIL水平,HE染色评价肝组织排斥活动指数(rejective activity index,RAI),观察小鼠生存率。结果肝移植术后24、48、72 h KCs的活化分别为15.9%、21.6%、28.3%,术后1、3、7 d TIM-4蛋白表达水平明显高于sham组(P<0.05);control m Ab组AI值(29.23±2.56)明显高于sham组和TIM-4 m Ab组[(0.40±0.49),(11.04±2.28),P<0.05]。KCs与CD4+T细胞共培养后,control、control m Ab以及TIM-4 m Ab组CD4+T细胞的增殖率分别为32.5%、31.6%、17.2%,CD4+CD25+Foxp3+T细胞分化分别为12.4%、13.9%、28.1%,共培养上清液TIM-4 m Ab组IL-4、IL-6、IL-13分泌水平明显低于control m Ab(P<0.05),且加入TIM-4 m Ab明显降低了与TIM-4+KCs共培养的CD4+T细胞p-STAT6蛋白表达(P<0.05),外源性加入IL-4可明显增高CD4+T细胞p-STAT6蛋白的表达(P<0.05)。移植模型注入i Treg细胞,i Tregz组肝功指标明显好转,与LT组比较差异有统计学意义(P<0.05)。肝组织病理学检查,i Treg组RAI平均得分为(3.97±0.67),明显低于LT组[(8.47±0.90),P<0.05],且i Treg组小鼠平均存活时间延长。结论阻断KCs TIM-4能够抑制初始CD4+T细胞IL-4/STAT6信号通路的激活,从而诱导更多i Treg细胞的生成,致使宿主对移植物产生免疫耐受。展开更多
目的:探讨Tim-1基因4个SNP位点及单倍型与宁夏地区回族人群类风湿性关节炎(RA)的关联性,旨在为RA的早期预防提供理论依据。方法:采用序列特异性引物聚合酶链式反应(SSP-PCR)及限制性片段长度多态性(PCR-RFLP)2种方法对108例RA患者及104...目的:探讨Tim-1基因4个SNP位点及单倍型与宁夏地区回族人群类风湿性关节炎(RA)的关联性,旨在为RA的早期预防提供理论依据。方法:采用序列特异性引物聚合酶链式反应(SSP-PCR)及限制性片段长度多态性(PCR-RFLP)2种方法对108例RA患者及104例健康个体Tim-1基因-1637A>G、-1454G>A、-416G>C和-232A>G共4个SNP位点进行检测,对各基因位点基因型频数、等位基因频数及单倍型分布情况进行比较和分析。结果:健康对照组与RA患者组-1637、-232位点基因型频数分布差异有统计学意义,在RA患者中-1637位点等位基因A的频数高于等位基因G(P<0.01)。4个位点共检出15种单倍型,单倍型AGCA(OR值9.611,95% CI 3.13～29.52)、AGCG(OR值4.361,95% CI 2.12～8.96)在宁夏回族RA患者组中的频数高于健康人群(P<0.01);而单倍型GGCA(OR值0.374,95% CI 0.22～0.64)、GGCG(OR值0.199,95% CI 0.08～0.49)及GAGA(OR值0.023,95%CI 0.002～0.26)在宁夏回族人群RA患者组中的频数低于健康对照组(P<0.01)。结论:-1637位点A>G的突变与宁夏回族人群RA发生有关,等位基因A的存在可增加RA发病风险,而等位基因G对RA的发生具有保护作用;AGCA、AGCG这2种单倍型可增加宁夏回族人群RA发生的风险,而单倍型GGCA、GGCG、GAGA对宁夏回族人群RA的发生具有保护作用。展开更多
基金Supported by the First-Class Discipline Construction Founded Project of Ningxia Medical University and the School of Clinical Medicine,No.2020008.
文摘BACKGROUND Immune checkpoint inhibitors(ICIs)targeting programmed cell death protein 1(PD-1)and T cell immunoglobulin and mucin domain-containing protein 3(TIM-3)are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies.However,ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma(HCC)patients due to the complex pathological mechanisms of HCC.AIM To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model,aiming to identify more effective immunotherapies and provide more treatment options for HCC patients.METHODS The levels of PD-1 and TIM-3 on CD4+and CD8+T cells from tumor tissues,ascites,and matched adjacent tissues from HCC patients were determined with flow cytometry.An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody(mAb)and/or anti-PD-1 mAb.Tumor growth in each group was measured.Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors.The percentage of CD4+and CD8+T cells in tissue samples from mice was tested with flow cytometry.The percentages of PD-1+CD8+,TIM-3+CD8+,and PD-1+TIM-3+CD8+T cells was accessed by flow cytometry.The levels of the cytokines including tumor necrosis factor alpha(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits.RESULTS We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+and CD8+T cells isolated from tumor tissues and ascites of HCC patients.TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight,while combined blockade had more substantial anti-tumor effects than individual treatment.Then we showed that combined therapy increased T cell infiltration into tumor tissues,and downregulated PD-1 and TIM-3 expression on CD8+T cells in tumor tissues.Moreover,combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ,and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues.Thus,we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model.CONCLUSION Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+T cell-mediated antitumor immune responses.
基金the National Natural Sciences Foundation of China (No. 30672008)
文摘To investigate the distribution characteristics and linkage disequilibrium of T cell immunoglobulin domain and mucin domain protein 4 （TIM4） promoter polymorphisms in asthma patients of Chinese Han population, the promoter region of TIM4 was re-sequenced by PCR-sequencing, and linkage disequilibrium was analyzed by SHEsis software. Four single nucleotide polymor- phisms （SNPs） in the promoter region of TIM4 were detected, including two new SNPs （at positions -1609, -153） and two reported SNPs （rs6874202, rs6882076）. The frequency distribution of rs6882076 was different among different races （P〈0.05）. In addition, linkage disequilibrium among the SNPs of the promoter region of TIM4 was found and GGTG was the predominant haplotype. There were four SNPs in the promoter region of TIM4 in asthma patients of Chinese Han population, which were in linkage disequilibrium.
基金supported by a grant from the National Natural Science Foundation of China (No. 30672008)
文摘T cell immunoglobulin mucin (TIM) family plays a key role in regulating immune re-sponses.In this study,the interactions of human TIM family with apoptotic cells were evaluated in order to provide a foundation for further study on the roles of human TIM genes in apoptosis.Nine kinds of pEGFP-N1 eukaryotic expression vectors containing different lengths of the three members of human TIM genes for the expression of TIM-EGFP and the vectors for the expression of TIM-Fc fusion pro-teins were constructed.It was found that human TIM proteins could recognize and bind to apoptotic cells directly,but not to viable cells.The interactions of sTIM-1-EGFP,sTIM-3-EGFP and sTIM-4-EGFP with apoptotic cells were blocked by TIM-1-Ig,TIM-3-Ig and TIM-4-Ig fusion proteins respectively.In addition,human TIM proteins mediated the recognition of apoptotic cells and bound to apoptotic cells directly via the IgV domains.In conclusion,the TIM family may play a key role in the regulation of apoptosis.Our data also suggest that human TIM proteins probably serve as novel proteins for the detection of the early cellular apoptosis.
文摘目的探讨阻断Kupffer细胞(Kupffer cells,KCs)T细胞免疫球蛋白黏蛋白4(T-cell immunoglobulin and mucin-domain-containing molecule 4,TIM-4)诱导小鼠原位肝移植术后i Treg细胞的产生及其相关机制。方法建立小鼠原位肝移植急性排斥反应模型,分为sham组、control m Ab组、TIM-4 m Ab组。流式细胞仪检测KCs活化,Western blot检测肝脏TIM-4表达,TUNEL检测肝细胞凋亡指数(apoptotic index,AI)。提取KCs,激光共聚焦检测KCs TIM-4表达,将KCs和提取的初始CD4+T细胞进行共培养,激光共聚焦检测Kupffer细胞TIM-4表达,分为control组、control m Ab组以及TIM-4 m Ab组;CFSE检测CD4+T细胞增殖,流式细胞仪检测CD4+CD25+Foxp3+T细胞,ELISA检测IL-4、IL-6、IL-13水平,Western blot检测STAT6表达。建立小鼠移植模型,分为3组:i Treg组移植模型注入i Treg细胞(预先用TIM-4 m Ab处理的TIM-4+KCs与初始CD4+T细胞共培养所诱导),sham组和单纯肝移植组(LT)注入相应的PBS作为对照,检测各组AST、ALT、TBIL水平,HE染色评价肝组织排斥活动指数(rejective activity index,RAI),观察小鼠生存率。结果肝移植术后24、48、72 h KCs的活化分别为15.9%、21.6%、28.3%,术后1、3、7 d TIM-4蛋白表达水平明显高于sham组(P<0.05);control m Ab组AI值(29.23±2.56)明显高于sham组和TIM-4 m Ab组[(0.40±0.49),(11.04±2.28),P<0.05]。KCs与CD4+T细胞共培养后,control、control m Ab以及TIM-4 m Ab组CD4+T细胞的增殖率分别为32.5%、31.6%、17.2%,CD4+CD25+Foxp3+T细胞分化分别为12.4%、13.9%、28.1%,共培养上清液TIM-4 m Ab组IL-4、IL-6、IL-13分泌水平明显低于control m Ab(P<0.05),且加入TIM-4 m Ab明显降低了与TIM-4+KCs共培养的CD4+T细胞p-STAT6蛋白表达(P<0.05),外源性加入IL-4可明显增高CD4+T细胞p-STAT6蛋白的表达(P<0.05)。移植模型注入i Treg细胞,i Tregz组肝功指标明显好转,与LT组比较差异有统计学意义(P<0.05)。肝组织病理学检查,i Treg组RAI平均得分为(3.97±0.67),明显低于LT组[(8.47±0.90),P<0.05],且i Treg组小鼠平均存活时间延长。结论阻断KCs TIM-4能够抑制初始CD4+T细胞IL-4/STAT6信号通路的激活,从而诱导更多i Treg细胞的生成,致使宿主对移植物产生免疫耐受。
文摘目的:探讨Tim-1基因4个SNP位点及单倍型与宁夏地区回族人群类风湿性关节炎(RA)的关联性,旨在为RA的早期预防提供理论依据。方法:采用序列特异性引物聚合酶链式反应(SSP-PCR)及限制性片段长度多态性(PCR-RFLP)2种方法对108例RA患者及104例健康个体Tim-1基因-1637A>G、-1454G>A、-416G>C和-232A>G共4个SNP位点进行检测,对各基因位点基因型频数、等位基因频数及单倍型分布情况进行比较和分析。结果:健康对照组与RA患者组-1637、-232位点基因型频数分布差异有统计学意义,在RA患者中-1637位点等位基因A的频数高于等位基因G(P<0.01)。4个位点共检出15种单倍型,单倍型AGCA(OR值9.611,95% CI 3.13～29.52)、AGCG(OR值4.361,95% CI 2.12～8.96)在宁夏回族RA患者组中的频数高于健康人群(P<0.01);而单倍型GGCA(OR值0.374,95% CI 0.22～0.64)、GGCG(OR值0.199,95% CI 0.08～0.49)及GAGA(OR值0.023,95%CI 0.002～0.26)在宁夏回族人群RA患者组中的频数低于健康对照组(P<0.01)。结论:-1637位点A>G的突变与宁夏回族人群RA发生有关,等位基因A的存在可增加RA发病风险,而等位基因G对RA的发生具有保护作用;AGCA、AGCG这2种单倍型可增加宁夏回族人群RA发生的风险,而单倍型GGCA、GGCG、GAGA对宁夏回族人群RA的发生具有保护作用。
