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
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EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联...目的探讨EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联免疫吸附法检测EBV mi R-BART17-3p、T细胞免疫球蛋白黏蛋白3(Tim-3)、叉头框蛋白P3(Fox P3)、白细胞介素17A(IL-17A)和转化生长因子-β(TGF-β)的m RNA、蛋白表达水平及含量。采用双荧光素酶报告基因实验考察EBV mi R-BART17-3p对Tim-3表达水平的影响。将15只BALB/C小鼠随机分为空白对照组、模型组、观察组,各5只。腹腔注射抗血小板抗体MWReg30以复制ITP小鼠模型,建模4 d后观察组小鼠予尾静脉注射携带EBV mi R-BART17-3p inhibitor的腺病毒载体。细胞染色并观察形态,检测外周血中TGF-β、IL-17A含量及血小板计数,采用流式细胞仪分别检测CD_(4)^(+)T细胞中Th17和Treg水平,并计算二者百分比。结果与对照组比较,ITP组患儿外周血EBV mi R-BART17-3p表达水平显著升高,Tim-3和TGF-βm RNA表达水平显著降低(P<0.05);Tim-3 m RNA表达水平与EBV mi R-BART17-3p表达水平呈显著负相关(r=-0.732,P<0.001)。Tim-3慢病毒载体p LKO.1-sh-Tim-3(sh-Tim-3)可显著降低Tim-3、Fox P3、TGF-β水平(P<0.05)。mi R-BART17-3p mimic显著升高了CD_(4)^(+)T细胞中mi R-BART17-3p的表达水平,并显著降低了Tim-3、Fox P3、TGF-βm RNA和蛋白表达水平(P<0.05);mi R-BART17-3p mimic可显著降低TGF-β含量,Tim-3+mi R-BART17-3p过表达逆转了mi R-BART17-3p mimic对TGF-β的抑制作用。动物实验结果显示,沉默EBV mi R-BART17-3p可促进Treg分化,减少脾脏和骨髓组织中的巨核细胞计数,并显著增加外周血中血小板计数。结论EBV mi R-BART17-3p可通过Fox P3/Tim-3途径调节ITP患儿Treg/Th17的免疫失衡。展开更多
Background： Fine particulate matter （PM2.5） exacerbates airway inflammation and hyperreactivity in patients with asthma, but the mechanism remains unclear. The aim of this study was to observe the effects of prolon...Background： Fine particulate matter （PM2.5） exacerbates airway inflammation and hyperreactivity in patients with asthma, but the mechanism remains unclear. The aim of this study was to observe the effects of prolonged exposure to high concentrations of PM2.5 on the pathology and airway hyperresponsiveness （AHR） of BALB/c mice undergoing sensitization and challenge with ovalbumin （OVA） and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1 （TIM-1） in this process. Methods： Forty female BALB/c mice were divided into four groups： control group, OVA group, OVA/PM group, and PM group （n - 10 in each group）. Mice in the control group were exposed to filtered clean air. Mice in the OVA group were sensitized and challenged with OVA. Mice in the OVA/PM group were sensitized and challenged as in the OVA group and then exposed to PM2.5 for 4 h per day and 5 days per week for a total of 8 weeks using a nose-only ＂PM2.5 online enrichment system＂ in The Second Hospital of Hebei Medical University. Mice in the PM group were exposed to the PM2.5 online enrichment system only. AHR was detected. Bronchoalveolar lavage fluid （BALF） was collected for cell classification. The levels of interleukin-4 （IL-4）, IL-5, and IL-33 in BALF were measured using enzyme-linked immunosorbent assay. Changes in histological structures were examined by light microscopy, and changes in ultramicrostructures were detected by electron microscopy. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay in the lung tissues. Western blotting and immunohistochemistry were utilized to analyze the expression of Bcl-2, Bax, and TIM-1 in the lungs. Results： The results showed that AHR in the OVA/PM group was significantly more severe than that in the OVA and PM groups （P 〈 0.05）. AHR in the PM group was also considerably more severe than that in the control group （P 〈 0.05）. The BALF of OVA/PM group （28.00± 6.08 vs. 12.33 ±4.51, t = 4.631, P = 0.002） and PM group （29.00 ± 3.00 vs. 12.33 ± 4.51, t = 4.927, P = 0.001） had more lymphocytes than the BALF of the control group. The number of neutrophils in the BALF of the OVA/PM group （6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020） and PM group （6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020） was much higher than those in the BALF of OVA group （P 〈 0.05）. TUNEL assays showed that the number of apoptotic cells in the OVA/PM group was significantly higher than that in the OVA group （Tunel immunohistochemical scores [IHS%], 1.20 ± 0.18 vs. 0.51 ± 0.03, t = 8.094, P 〈 0.001） and PM group （Tunel IHS%, 1.20 ± 0.18 vs. 0.51 ±0.09, t = 8.094, P〈 0.001）, and that the number of apoptotic cells in the PM group was significantly higher than that in the control group （Tunel IHS%, 0.51 ± 0.09 vs. 0.26 ± 0.03, t = 2.894, P = 0.020）. The concentrations of IL-4 （77.44 ± 11.19 vs. 48.02 ±10.02 pg/ml, t = 4.595, P= 0.002） and IL-5 （15.65 ± 1.19vs. 12.35±0.95pg/ml, t=3.806,P=0.005） and the Bax/Bcl-2 ratio （1.51 ± 0.18 vs. 0.48 ± 0.10, t = 9.654, P 〈 0.001） and TIM-1/β-actin ratio （0.78 ± 0.11 vs. 0.40 ±0.06, t = 6.818, P 〈 0.001） in the OVA/PM group were increased compared to those in the OVA group. The concentrations of IL-4 （77.44 ± 11.19 vs. 41.47 ± 3.40 pg/ml, t = 5.617, P= 0.001） and IL-5 （ 15.65±1. 19 vs. 10.99 ± 1.40 pg/ml, t = 5.374, P = 0.001 ） and the Bax/Bcl-2 ratio （1.51 ±0.18 vs. 0.97 ± 0.16, t = 5.000, P = 0.001） and TIM-1/β-actin ratio （0.78 ± 0.11 vs. 0.31 ± 0.06,t = 8.545, P 〈 0.001 ） in the OVA/PM group were increased compared to those in the PM group. The concentration oflL-4 （41.47 ±3.40 vs. 25.46 ± 2.98 pg/ml, t = 2.501, P = 0.037） and the Bax/Bcl-2 ratio （0.97 ± 0.16 vs. 0.18 ± 0.03, t = 7.439, P 〈 0.001） and TIM-1/β-actin ratio （0.31 ± 0.06 vs. 0.02 ± 0.01, t = 5.109, P = 0.001） in the PM group were also higher than those in the control group. Conclusions： Exacerbated AHR associated with allergic asthma caused by PMz5 is related to increased apoptosis and TIM-1 activation. These data might provide insights into therapeutic targets for the treatment of acute exacerbations of asthma induced by PM2.5.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EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联免疫吸附法检测EBV mi R-BART17-3p、T细胞免疫球蛋白黏蛋白3(Tim-3)、叉头框蛋白P3(Fox P3)、白细胞介素17A(IL-17A)和转化生长因子-β(TGF-β)的m RNA、蛋白表达水平及含量。采用双荧光素酶报告基因实验考察EBV mi R-BART17-3p对Tim-3表达水平的影响。将15只BALB/C小鼠随机分为空白对照组、模型组、观察组,各5只。腹腔注射抗血小板抗体MWReg30以复制ITP小鼠模型,建模4 d后观察组小鼠予尾静脉注射携带EBV mi R-BART17-3p inhibitor的腺病毒载体。细胞染色并观察形态,检测外周血中TGF-β、IL-17A含量及血小板计数,采用流式细胞仪分别检测CD_(4)^(+)T细胞中Th17和Treg水平,并计算二者百分比。结果与对照组比较,ITP组患儿外周血EBV mi R-BART17-3p表达水平显著升高,Tim-3和TGF-βm RNA表达水平显著降低(P<0.05);Tim-3 m RNA表达水平与EBV mi R-BART17-3p表达水平呈显著负相关(r=-0.732,P<0.001)。Tim-3慢病毒载体p LKO.1-sh-Tim-3(sh-Tim-3)可显著降低Tim-3、Fox P3、TGF-β水平(P<0.05)。mi R-BART17-3p mimic显著升高了CD_(4)^(+)T细胞中mi R-BART17-3p的表达水平,并显著降低了Tim-3、Fox P3、TGF-βm RNA和蛋白表达水平(P<0.05);mi R-BART17-3p mimic可显著降低TGF-β含量,Tim-3+mi R-BART17-3p过表达逆转了mi R-BART17-3p mimic对TGF-β的抑制作用。动物实验结果显示,沉默EBV mi R-BART17-3p可促进Treg分化,减少脾脏和骨髓组织中的巨核细胞计数,并显著增加外周血中血小板计数。结论EBV mi R-BART17-3p可通过Fox P3/Tim-3途径调节ITP患儿Treg/Th17的免疫失衡。
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 81770020）.
文摘Background： Fine particulate matter （PM2.5） exacerbates airway inflammation and hyperreactivity in patients with asthma, but the mechanism remains unclear. The aim of this study was to observe the effects of prolonged exposure to high concentrations of PM2.5 on the pathology and airway hyperresponsiveness （AHR） of BALB/c mice undergoing sensitization and challenge with ovalbumin （OVA） and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1 （TIM-1） in this process. Methods： Forty female BALB/c mice were divided into four groups： control group, OVA group, OVA/PM group, and PM group （n - 10 in each group）. Mice in the control group were exposed to filtered clean air. Mice in the OVA group were sensitized and challenged with OVA. Mice in the OVA/PM group were sensitized and challenged as in the OVA group and then exposed to PM2.5 for 4 h per day and 5 days per week for a total of 8 weeks using a nose-only ＂PM2.5 online enrichment system＂ in The Second Hospital of Hebei Medical University. Mice in the PM group were exposed to the PM2.5 online enrichment system only. AHR was detected. Bronchoalveolar lavage fluid （BALF） was collected for cell classification. The levels of interleukin-4 （IL-4）, IL-5, and IL-33 in BALF were measured using enzyme-linked immunosorbent assay. Changes in histological structures were examined by light microscopy, and changes in ultramicrostructures were detected by electron microscopy. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay in the lung tissues. Western blotting and immunohistochemistry were utilized to analyze the expression of Bcl-2, Bax, and TIM-1 in the lungs. Results： The results showed that AHR in the OVA/PM group was significantly more severe than that in the OVA and PM groups （P 〈 0.05）. AHR in the PM group was also considerably more severe than that in the control group （P 〈 0.05）. The BALF of OVA/PM group （28.00± 6.08 vs. 12.33 ±4.51, t = 4.631, P = 0.002） and PM group （29.00 ± 3.00 vs. 12.33 ± 4.51, t = 4.927, P = 0.001） had more lymphocytes than the BALF of the control group. The number of neutrophils in the BALF of the OVA/PM group （6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020） and PM group （6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020） was much higher than those in the BALF of OVA group （P 〈 0.05）. TUNEL assays showed that the number of apoptotic cells in the OVA/PM group was significantly higher than that in the OVA group （Tunel immunohistochemical scores [IHS%], 1.20 ± 0.18 vs. 0.51 ± 0.03, t = 8.094, P 〈 0.001） and PM group （Tunel IHS%, 1.20 ± 0.18 vs. 0.51 ±0.09, t = 8.094, P〈 0.001）, and that the number of apoptotic cells in the PM group was significantly higher than that in the control group （Tunel IHS%, 0.51 ± 0.09 vs. 0.26 ± 0.03, t = 2.894, P = 0.020）. The concentrations of IL-4 （77.44 ± 11.19 vs. 48.02 ±10.02 pg/ml, t = 4.595, P= 0.002） and IL-5 （15.65 ± 1.19vs. 12.35±0.95pg/ml, t=3.806,P=0.005） and the Bax/Bcl-2 ratio （1.51 ± 0.18 vs. 0.48 ± 0.10, t = 9.654, P 〈 0.001） and TIM-1/β-actin ratio （0.78 ± 0.11 vs. 0.40 ±0.06, t = 6.818, P 〈 0.001） in the OVA/PM group were increased compared to those in the OVA group. The concentrations of IL-4 （77.44 ± 11.19 vs. 41.47 ± 3.40 pg/ml, t = 5.617, P= 0.001） and IL-5 （ 15.65±1. 19 vs. 10.99 ± 1.40 pg/ml, t = 5.374, P = 0.001 ） and the Bax/Bcl-2 ratio （1.51 ±0.18 vs. 0.97 ± 0.16, t = 5.000, P = 0.001） and TIM-1/β-actin ratio （0.78 ± 0.11 vs. 0.31 ± 0.06,t = 8.545, P 〈 0.001 ） in the OVA/PM group were increased compared to those in the PM group. The concentration oflL-4 （41.47 ±3.40 vs. 25.46 ± 2.98 pg/ml, t = 2.501, P = 0.037） and the Bax/Bcl-2 ratio （0.97 ± 0.16 vs. 0.18 ± 0.03, t = 7.439, P 〈 0.001） and TIM-1/β-actin ratio （0.31 ± 0.06 vs. 0.02 ± 0.01, t = 5.109, P = 0.001） in the PM group were also higher than those in the control group. Conclusions： Exacerbated AHR associated with allergic asthma caused by PMz5 is related to increased apoptosis and TIM-1 activation. These data might provide insights into therapeutic targets for the treatment of acute exacerbations of asthma induced by PM2.5.
