BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a h...BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.展开更多
Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^...Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.展开更多
Objective:To investigate the changes of Mg^(2+) levels in serum and peripheral blood mononuclear cells(PBMCs)of patients with COVID-19 and its effects on the functions of CD8^(+)T lymphocytes and NK cells.Methods:A to...Objective:To investigate the changes of Mg^(2+) levels in serum and peripheral blood mononuclear cells(PBMCs)of patients with COVID-19 and its effects on the functions of CD8^(+)T lymphocytes and NK cells.Methods:A total of 165 COVID-19 patients hospitalized in Ezhou Central Hospital from January 20 to February 20,2020 were divided into mild/common group(98 cases)and severe/critical group(67 cases).At the same time,34 healthy persons were selected as the control group.Peripheral blood was collected and PBMCs were isolated,the level of Mg^(2+) in serum and PBMCs was detected.The subsets of CD8^(+)T lymphocytes and NK cell and the expression levels of their surface inhibitory molecular PD-1 and activator molecular NKG2D were detected by flow cytometry.The correlation between Mg^(2+) concentration and the expression levels of PD-1 and NKG2D was also analyzed.Results:Compared with the control group,the concentration of Mg^(2+) in serum and PBMCs,the counts of CD8^(+)T lymphocytes and NK cell in patients with mild/common and severe/critical groups were significantly reduced(P<0.05),while the expression level of surface inhibitory molecular PD-1 were significantly increased(P<0.05),while the expression level of the activation molecule NKG2D were significantly decreased(P<0.05).However,the changes of the above indicators in patients with severe/critical group were greater than those in the mild/common group(P<0.05).In addition,the Mg^(2+) concentration in COVID-19 patients was negatively correlated with the expression level of PD-1 on CD8^(+)T lymphocytes and NK cells(P<0.05),and positively correlated with the expression levels of NKG2D(P<0.05).Conclusion:The concentration of Mg^(2+) in the serum and PBMCs of COVID-19 patients is significantly reduced,which may cause the function of CD8^(+)T lymphocytes and NK cells to be inhibited.展开更多
AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC ...AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box(Fox)p3, cytotoxic T-lymphocyte-associated protein(CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP^-CD4^+ and LAP^+CD4^+ T cells were isolated using a magnetic cellsorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor(TGF)-β.RESULTS The proportion of LAP^+CD4^+ T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P < 0.001). Among patients, the proportion of LAP^+CD4^+ T cells was significantly higher in tumor tissues(11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P < 0.001). We also observed positive correlations between the proportion of LAP^+CD4^+ T cells and TNM stage(P < 0.001), distant metastasis(P < 0.001) and serum level of carcinoembryonic antigen(P < 0.05). Magnetic-activated cell sorting gave an overall enrichment of LAP^+CD4^+ T cells (95.02% ± 2.87%), which was similar for LAP^-CD4^+ T cells(94.75% ± 2.76%). In contrast to LAP^-CD4^+ T cells, LAP^+CD4^+ T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5(P < 0.01). LAP^+CD4^+ T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAPCD4+ T cells.CONCLUSION LAP^+CD4^+ T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.展开更多
Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to ex...Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.展开更多
基金Supported by High-End Talent Funding Project in Hebei Province,No.A202003005Hebei Provincial Health Commission Office,No.G2019074Natural Science Foundation of Hebei Province,No.H2019209355。
文摘BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.
基金supported by the Natural Science Foundation of Sichuan Province (Grant No.2022NSFSC1415)the Special Project of Sichuan Province Traditional Chinese Medicine Administration (Grant No. 2020JC0124)+1 种基金the Management Project of General Hospital of Western Theater Command (Grants No. 2021-XZYG-C22 and 2021-XZYG-C21)the Spark Young Innovative Talent Project of General Hospital of Western Theater Command。
文摘Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.
文摘Objective:To investigate the changes of Mg^(2+) levels in serum and peripheral blood mononuclear cells(PBMCs)of patients with COVID-19 and its effects on the functions of CD8^(+)T lymphocytes and NK cells.Methods:A total of 165 COVID-19 patients hospitalized in Ezhou Central Hospital from January 20 to February 20,2020 were divided into mild/common group(98 cases)and severe/critical group(67 cases).At the same time,34 healthy persons were selected as the control group.Peripheral blood was collected and PBMCs were isolated,the level of Mg^(2+) in serum and PBMCs was detected.The subsets of CD8^(+)T lymphocytes and NK cell and the expression levels of their surface inhibitory molecular PD-1 and activator molecular NKG2D were detected by flow cytometry.The correlation between Mg^(2+) concentration and the expression levels of PD-1 and NKG2D was also analyzed.Results:Compared with the control group,the concentration of Mg^(2+) in serum and PBMCs,the counts of CD8^(+)T lymphocytes and NK cell in patients with mild/common and severe/critical groups were significantly reduced(P<0.05),while the expression level of surface inhibitory molecular PD-1 were significantly increased(P<0.05),while the expression level of the activation molecule NKG2D were significantly decreased(P<0.05).However,the changes of the above indicators in patients with severe/critical group were greater than those in the mild/common group(P<0.05).In addition,the Mg^(2+) concentration in COVID-19 patients was negatively correlated with the expression level of PD-1 on CD8^(+)T lymphocytes and NK cells(P<0.05),and positively correlated with the expression levels of NKG2D(P<0.05).Conclusion:The concentration of Mg^(2+) in the serum and PBMCs of COVID-19 patients is significantly reduced,which may cause the function of CD8^(+)T lymphocytes and NK cells to be inhibited.
基金Supported by the National Natural Science Foundation of China,No.81260316
文摘AIM To investigate the abundance and potential functions of LAP^+CD4^+ T cells in colorectal cancer(CRC). METHODS Proportions of LAP^+CD4^+ T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box(Fox)p3, cytotoxic T-lymphocyte-associated protein(CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP^-CD4^+ and LAP^+CD4^+ T cells were isolated using a magnetic cellsorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor(TGF)-β.RESULTS The proportion of LAP^+CD4^+ T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P < 0.001). Among patients, the proportion of LAP^+CD4^+ T cells was significantly higher in tumor tissues(11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P < 0.001). We also observed positive correlations between the proportion of LAP^+CD4^+ T cells and TNM stage(P < 0.001), distant metastasis(P < 0.001) and serum level of carcinoembryonic antigen(P < 0.05). Magnetic-activated cell sorting gave an overall enrichment of LAP^+CD4^+ T cells (95.02% ± 2.87%), which was similar for LAP^-CD4^+ T cells(94.75% ± 2.76%). In contrast to LAP^-CD4^+ T cells, LAP^+CD4^+ T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5(P < 0.01). LAP^+CD4^+ T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAPCD4+ T cells.CONCLUSION LAP^+CD4^+ T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.
基金Fund supported by the Healthcare Technology Plan of Zhejiang Provincial Health Bureau(No.2016KYB292)the Technology Plan of Science and Technology Bureau of Jiaxing,Zhejiang province(No.2016AY23054)~~
文摘Objective To investigate the expression and regulation of programmed cell death protein 1(PD1),B lymphocyte and T lymphocyte attenuator(BTLA)in peripheral blood of patients with non-small cell lung cancer(NSCLC);to examine the correlation of the mRNA levels between PD and BTLA in NSCLC.Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8^+T cells andγδ+T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals.We compared the expression of PD1 and BTLA on the surfaces ofγδ+T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid.The correlations of PD1 and BTLA,as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform.Results The frequency of PD1 on the surfaces of CD8^+T cells was significantly higher than that of theγδT cells in both healthy controls(t=2.324,P=0.024)and NSCLC patients(t=2.498,P=0.015).The frequency of PD1 on CD8^+T cells,rather than onγδ+T cells,was significantly upregulated in advanced NSCLC patients compared with that in healthy controls(t=4.829,P<0.001).The PD1+BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients(t=2.422,P=0.0185).No differences in percentage of PD1+γδ+and BTLA+γδ+T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment.PD1 was positively correlated with BTLA in both lung adenocarcinoma(r=0.54;P<0.05)and lung squamous cell carcinoma(r=0.78;P<0.05).Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8^+T cells andγδT cells in advanced NSCLC,suggesting that these molecules were involved in regulating the inactivation of CD8^+T cells andγδ+T cells,immune escape and tumor invasion.
