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Objective To investigate the serum levels of soluble programmed death-1 (sPD-1) and soluble programmed death-ligand 1 (sPD-L1) in chronic hepatitis B (CHB) patients with clinical cure, the correlation between programmed death-1 (PD-1) and lymphocytes by flow cytometry, and the recovery of hepatitis B virus (HBV)-specific immunity. Methods A total of 26 CHB patients with clinical cure, 26 treatment-naïve CHB patients, and 26 healthy controls who were diagnosed at the outpatient service of Peking University First Hospital from January to May of 2022 were enrolled, and related clinical data and peripheral blood samples were collected. ELISA was used to measure the serum levels of sPD-1 and sPD-L1, and flow cytometry was used to measure the expression of PD-1 in peripheral blood lymphocytes. CHB patients with clinical cure were compared with the treatment-naïve CHB patients and the healthy controls. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between three groups, and the chi-square test was used for comparison of categorical data between groups. The Pearson correlation analysis or the Spearman correlation analysis was used to investigate the correlation between two continuous variables. Results For the 26 CHB patients with clinical cure, the mean time of antiviral therapy was 8.33 years, with entecavir as the antiviral drug. The CHB patients with clinical cure had significantly higher levels of sPD-1 and sPD-L1 than the healthy controls ( P 0.05). Conclusion The serum levels of sPD-1 and sPD-L1 in treatment-naïve CHB patients are mainly associated with exhausted CD8 + T cells in peripheral blood, while there is no significant correlation between serum sPD-1/sPD-L1 and exhausted CD8 + T cells in peripheral blood in CHB patients with clinical cure.
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Objective:To investigate changes in expression of plasma soluble CD100 (sCD100) and membrane-bound CD100 (mCD100) on peripheral T cells in patients with herpes zoster, and to observe the regulatory effect of exogenous CD100 on CD8 + T cells. Methods:A total of 53 patients with herpes zoster attending the Zhumadian Central Hospital from July 2019 to April 2021 were enrolled, so were 25 age- and sex-matched healthy controls. Anticoagulated venous blood samples were collected, plasma and peripheral blood mononuclear cells were isolated, plasma sCD100 levels were detected by enzyme-linked immunosorbent assay, and mCD100 expression on CD4 + and CD8 + T cells was determined by flow cytometry. After the purification of CD8 + T cells, the secretion levels of cytotoxic molecules and cytokines by CD8 + T cells were measured and compared between herpes zoster patients and controls. Some purified CD8 + T cells from herpes zoster patients were stimulated with recombinant human CD100 and recombinant varicella-zoster virus glycoprotein, and the effect of recombinant human CD100 on the secretion of cytotoxic molecules and cytokines by CD8 + T cells was investigated. Comparisons between groups were conducted by t test. Results:Plasma sCD100 levels were significantly lower in the herpes zoster group (1.12 ± 0.23 ng/ml) than in the control group (1.31 ± 0.28 ng/ml, t = 2.97, P = 0.004), the proportion of mCD100 + CD8 + T cells was significantly higher in the herpes zoster group (17.41% ± 4.26%) than in the control group (14.69% ± 3.70%, t = 2.52, P = 0.014), and no significant difference in the proportion of mCD100 + CD4 + T cells was found between the two groups (2.52% ± 0.58% vs. 2.32% ± 0.56%, t = 1.27, P = 0.208). The herpes zoster group showed significantly decreased mRNA expression of perforin and granzyme B in, and lower secretion levels of perforin, granzyme B, interferon-γ and tumor necrosis factor-α by CD8 + T cells compared with the control group (all P < 0.05). After stimulation with recombinant human CD100, levels of perforin, granzyme B, interferon-γ and tumor necrosis factor-α in the culture supernatant of CD8 + T cells (43.68 ± 14.12, 126.8 ± 22.92, 12.79 ± 3.66, 310.0 ± 79.90 pg/ml, respectively ) were significantly higher than those in non-stimulated group (34.55 ± 10.78, 99.04 ± 10.44, 9.53 ± 2.00, 275.6 ± 68.04 pg/ml, respectively, all P < 0.05) . Conclusion:There was an imbalance between sCD100 and mCD100 expression in patients with herpes zoster, and exogenous sCD100 may enhance the cytotoxicity of CD8 + T cells in herpes zoster patients.
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Objective To investigate the change in interleukin-33 (IL-33) in the peripheral blood of hepatocellular carcinoma (HCC) patients and the role and potential mechanism of IL-33 in regulating CD8 + T cell function in HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital from April 2019 to January 2020 and 20 healthy controls were enrolled. Peripheral blood was collected, and plasma and peripheral blood mononucleated cells (PBMCs) were isolated; ELISA was used to measure the plasma levels of IL-33 and its receptor ST2, and quantitative real-time PCR was used to measure the relative mRNA expression levels of IL-33 and ST2 in PBMCs. CD8 + T cells were purified and stimulated with recombinant IL-33; CCK-8 assay was used to assess cell proliferation, enzyme-linked immunospot assay was used to measure the secretion of perforin and granzyme B, and flow cytometry was used to measure the expression of PD-1, LAG-3, and CTLA-4; changes in cell proliferation, secretion of cytotoxic molecules, and immune checkpoint molecules after IL-33 stimulation were compared. CD8 + T cells were co-cultured with HepG2 cells; the expression of lactate dehydrogenase was measured to calculate the proportion of dead HepG2 cells induced by CD8 + T cells, and the change in the killing function of CD8 + T cells after IL-33 stimulation was compared. The t -test or the paired t -test was used for comparison of continuous data between two groups, and a Pearson correlation analysis was performed. Results Compared with the control group, the HCC group had significantly lower plasma level of IL-33 (269.80±63.08 pg/ml vs 339.50±64.43 pg/ml, t =4.072, P 0.05). The proportion of CD8 + T cells was not correlated with the plasma level of IL-33 or ST2 (both P > 0.05). Compared with the control group, the HCC group had significantly lower levels of perforin and granzyme B (both P 0.05), but it promoted the secretion of perforin and granzyme B ( P < 0.05). Compared with the control group, the HCC group had a significant reduction in the killing activity of CD8 + T cells ( P < 0.05), and stimulation with recombinant IL-33 enhanced the killing function of CD8 + T cells, which was mainly reflected in the increases in the proportion of dead HepG2 cells ( P < 0.05) and the secretion of IFNγ and TNFα ( P < 0.05). Conclusion There is a reduction in the plasma level of IL-33 in HCC patients. IL-33 can enhance the killing activity of CD8 + T cells by promoting the secretion of perforin and granzyme B, which provides a new target for the treatment of HCC.
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Objective:To investigate the modulatory function of interleukin-7 (IL-7)/CD 127 signaling pathway on CD 8+T cells in patients with myasthenia gravis (MG). Methods:Fifty-seven treatment-naive MG patients who were hospitalized in Department of Neurology, Nanyang Central Hospital between 2017 and 2020 as well as 35 healthy controls were enrolled. Peripheral blood was collected, while plasma and peripheral blood mononuclear cells were isolated. Plasma IL-7 and soluble CD 127 (sCD 127) were measured by enzyme linked immunosorbent assay (ELISA). Membrane-bound CD 127 (mCD 127) percentage in CD 8+T cells was measured by flow cytometry. The differences of above indices between different gender, onset age, afflicting with thymoma, or different Osserman type and their correlation with Quantitative Myasthenia Gravis (QMG) score were analyzed. Purified CD 8+T cells from MG patients were stimulated with recombinant human IL-7 (5 μg/L). Changes of sCD 127 and mCD 127 level were analyzed. Levels of perforin, granzyme B, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the cultured supernatants were measured by ELISA. Immune checkpoint molecules mRNA in CD 8+T cells was semi-quantified by real-time fluorescence quantitative polymerase chain reaction. Results:Plasma IL-7 level was up-regulated in MG patients compared with controls [(293.4±74.7) pg/ml vs (233.8±70.8) pg/ml, t=3.78, P<0.001], while sCD 127 level was down-regulated in MG patients compared with controls [(102.7±13.7) pg/ml vs (131.2±20.9) pg/ml, t=7.91, P<0.001]. Peripheral CD 8+T cells percentage was up-regulated in MG patients compared with controls (35.4%±7.1% vs 30.2%±7.5%, t=3.31, P=0.001), and mCD 127+CD 8+T cell percentage was also elevated (45.5%±7.7% vs 34.7%±11.5%, t=5.44, P<0.001). There were no significant differences of above indices between different gender, onset age, afflicting with thymoma, or different Osserman type. There was no significant correlation between above indices and QMG score. There were no significant differences of sCD 127 in cultured supernatants, mCD 127+CD 8+T cell percentage, or immune checkpoint molecules mRNA expression between CD 8+T cells from MG patients with and without IL-7 stimulation. IL-7 stimulation promoted the secretion of perforin [(208.1±67.2) pg/ml vs (168.8±46.2) pg/ml, t=2.16, P=0.038], granzyme B [(941.8±273.9) pg/ml vs (782.4±137.2) pg/ml, t=2.33, P=0.025], and IFN-γ [(19.1±5.2) pg/ml vs (15.3±4.5) pg/ml, t=2.47, P=0.018] by CD 8+T cells. However, there was no remarkable difference of TNF-α production between CD 8+T cells with and without IL-7 stimulation. Conclusion:Elevated IL-7-mediated signaling pathway enhanced the secretion of cytotoxic molecules and cytokines by CD 8+T cells, leading to increased activity of CD 8+T cells in MG patients.
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Objective To investigate the effect of liver CD8 + T lymphocytes on co-cultured hepatic stellate cells (HSCs) after the application of Fuzheng Huayu prescription in a moues model of acute liver injury, as well as the mechanism of action of Fuzheng Huayu prescription in preventing liver fibrosis. Methods A total of 18 specific pathogen-free male C57BL/6NCrl Vr mice were randomly divided into normal group, model group, and Fuzheng Huayu prescription group, with 6 mice in each group. The mice in the Fuzheng Huayu prescription group were given Fuzheng Huayu prescription for 5 days in advance. At 12 hours before the experiment, 10% CCl 4 was injected intraperitoneally at a dose of 2 mL/kg body weight. Blood was collected from the main abdominal vein, and the serum was separated to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Part of the liver was used for pathological observation. After the mice were pretreated with medication in vivo, flow cytometry was used for the sorting of mouse liver CD8 + T lymphocytes, which were then co-cultured with the mouse HSC cell line (JS 1) in a 96-well plate at a ratio of 2∶ 1, and after co-culture for 24 and 48 hours, qPCR was used to measure the changes in the mRNA expression of Col.I and α-SMA. An analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the SNK- q test or the least significant difference t -test was used for further comparison between two groups. Results The model group had significantly higher activities of ALT and AST than the normal group (both P < 0.000 1), and compared with the model group, the Fuzheng Huayu prescription group had a significantly lower degree of increase in ALT activity ( P < 0.001). HE staining showed that the Fuzheng Huayu prescription group had a significantly lower degree of hepatocyte degeneration and necrosis compared with the model group. Compared with the normal group, the total lymphocytes, CD45, CD4 - CD8 + T and CD8 + CD28 - T in the model group increased significantly, while the proportion of the above lymphocytes in the Fuzheng Huayu formula group decreased significantly compared with the model group ( P < 0.001). CD8 + T lymphocytes isolated from the liver of mice in each group were co-cultured with JS 1 for 48 hours, and compared with the control group (JS 1 cultured alone) and the normal group, the model group had a significant increase in the mRNA expression of α-SMA (both P < 0.01) and significantly higher mRNA expression of Col.I than the control group and the normal group (normal mouse liver CD8 + T lymphocytes co-cultured with JS 1) (both P < 0.001). The Fuzheng Huayu prescription group had significantly lower mRNA expression levels of α-SMA and Col.I than the model group (both P < 0.01). Conclusion Fuzheng Huayu prescription can indirectly inhibit activated HSCs by altering the functional phenotype of CD8 + T lymphocytes in mouse liver.
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Objective:To prepare 99Tc m-hydrazinonicotinamide(HYNIC)-αCD8/Fab ( 99Tc m-αCD8/Fab), and explore the predictive value of 99Tc m-αCD8/Fab SPECT/CT imaging for the efficacy of anti-programmed death-1 (PD-1) immunotherapy. Methods:The αCD8/Fab was modified with HYNIC- N-hydroxysuccinimide (NHS) and IRDye800-NHS to form HYNIC-αCD8/Fab and IRDye800-αCD8/Fab (Dye-αCD8/Fab), respectively. 99Tc m-αCD8/Fab was prepared in sodium bicarbonate buffer (pH=8.5), with SnCl 2 being used as the reducing agent. The labeling yield and radiochemical purity of 99Tc m-αCD8/Fab and its stability in PBS and fetal bovine serum (FBS) were tested in vitro. The mouse spleen and human peripheral blood lymphocytes were isolated for cell-specific binding and blocking experiments of 99Tc m-αCD8/Fab in vitro. SPECT/CT imaging was used to analyze the specific binding ability of the 99Tc m-αCD8/Fab probe in CT26 colon cancer mouse models (BALB/c). The near infrared fluorescence imaging and SPECT/CT imaging were performed to detect the intra-tumoral CD8 + T cell infiltration after anti-PD-1 therapy in tumor bearing mice, and the results were further verified by HE and immunofluorescence staining. CD8 + T cell depletion study was performed to determine the role of CD8 + T cells in the tumor responses to anti-PD-1 therapy. Two-way analysis of variance was used to compare the data difference. Results:The labeling yield of 99Tc m-αCD8/Fab was 90% with a high radiochemical purity (95%) and good stability in vitro (radiochemical purity still more than 80% after 720 min in PBS and FBS). 99Tc m-αCD8/Fab could specifically bind to mouse CD8 + T cells ((10.30±0.81) percent added radioactive dose (%AD)/10 6 cells), compared with the binding ability in human peripheral blood lymphocytes group and CD8 antibody blocking group ((1.78±0.61) and (1.59±0.25) %AD/10 6 cells; F=10.07, P<0.001). SPECT/CT imaging showed that 99Tc m-αCD8/Fab had markedly higher tumor uptake in the CT26 colon cancer mouse models. Near-infrared fluorescence imaging showed that the tumor uptake of 99Tc m-αCD8/Fab in the responsive group was significantly higher than in the nonresponsive group after anti-PD-1 treatment ((8.9±1.1)% vs (7.1±0.8)%; F=4.69, P=0.024), and SPECT/CT imaging found the similar result. HE and immunofluorescence staining of tumor and lymph nodes showed that the proportion of lymphocyte infiltration was higher in the responsive group. Furthermore, CD8 + T cell depletion significantly reversed the therapeutic effect of anti-PD-1 immunotherapy in tumor-bearing mice. Conclusions:In this study, 99Tc m-αCD8/Fab was successfully obtained. CD8-specific SPECT imaging could sensitively visualize the tumor-infiltrating CD8 + T cells, suggesting the potential application value to predict and evaluate the efficacy of immunotherapy in the clinical settings.
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Objective:This study aims to reveal the special immune infiltrating environment and possible immune escape mechanism of giant cell tumor of bone through single-cell sequencing data.Methods:The fresh samples obtained from 4 patients with primary giant cell tumor of bone from January 2018 to December 2021 were collected, and single-cell transcriptome sequencing was performed on the 10X platform to explore the characteristics and immune environment of giant cell tumor of bone by using t-distributed stochastic neighbor embedding ( t-SNE). The main cell types and signal pathways of immune cell regulation and function in giant cell tumor of bone were observed by cell communication analysis. Results:Cell clustering, the definition of basic cell types, the classification of immune cells, and the mutual regulatory relationship between cell types were analyzed for 35 643 single-cell data from 4 giant cell tumor samples of bone. It was found that giant cell tumor of bone was composed of 9 basic cell types, in which the immune cells were mainly CD8 + T cells (51%) and the non-immune cells were mainly fibroblast like spindle stromal cells (74%). The immune infiltration of giant cell tumor of bone is dominated by cytotoxic CD8 + T cells and lacks exhausted CD8 + T cells. CD4 + T cells are characterized by high expression of immune checkpoint genes CTLA4 and TIGIT. In giant cell tumor of bone, immune cells mainly act on multinucleated osteoclast like giant cells through PARs and CCL signaling pathways, but not stromal cells. Conclusion:This study defined the main cell types of giant cell tumor of bone through single cell sequencing data, and further revealed the composition characteristics of its immune infiltration, and found that the target of its immune cells was mainly multinuclear osteoclast like giant cells, which provided effective information for further understanding the occurrence and development of giant cell tumor of bone.
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Objective:To investigate the effects of intravenous thrombolysis with alteplase on immune function and quality of life in patients with cerebral infarction.Methods:Sixty-nine patients with cerebral infarction who received treatment in Rizhao Central Hospital, China between January 2014 and January 2019 were included in this study. They were randomly assigned to receive either intravenous thrombolysis with urokinase (control group, n = 34) or intravenous thrombolysis with alteplase (observation group, n = 35). Therapeutic efficacy and cerebral blood perfusion, immune function and quality of life before and after treatment were evaluated. Results:Effective rate in the observation group was significantly higher than that in the control group [82.86% (29/35) vs. 58.82% (20/34), χ2 = 4.840, P < 0.05]. After treatment, the transit time and peak time in the ischemic area in the observation group were (131.25 ± 25.41) seconds and (99.52 ± 17.50) seconds respectively, which were significantly shorter than those in the control group [(165.33 ± 31.05) seconds, (108.45 ± 12.52) seconds, t = 6.580, 3.215, both P < 0.05). The cerebral blood flow and cerebral blood volume in the observation group were (72.51 ± 21.35) mL/100 mg and (95.36 ± 31.25) mL/100 mg, respectively, which were significantly higher than those in the control group [(62.42 ± 19.35) mL/100 mg, (84.20 ± 28.05) mL/100 mg, t = 2.712, 2.243, both P < 0.05). After treatment, the proportion of CD 8+ cells in the observation group was significantly lower than that in the control group [(25.37 ± 3.73)% vs. (27.42 ± 3.25)%, t = 4.261, P < 0.05]. The proportions of CD 3+, CD 4+, CD 3-CD 16+CD 56+ cells in the observation group were (56.32 ± 6.57)%, (34.69 ± 3.44)%, (13.34 ± 3.75)%, respectively, which were significantly higher than those in the control group [(53.32 ± 4.05)%, (31.69 ± 3.72)%, (11.28 ± 3.06)%, t = 5.395, 3.694, 4.179, P < 0.05]. After treatment, the scores of all dimensions of Short Form 36 Health Status Questionnaire in the observation group were significantly higher than those in the control group (all P < 0.05). Conclusion:Intravenous thrombolysis with alteplase is superior to intravenous thrombolysis with urokinase in the treatment of cerebral infarction because it can better improve immune function and quality of life.
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ObjectiveTo investigate the mechanism of gamma-chain (γC) cytokines in regulating the expression of T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) in CD8+ T cells of chronic hepatitis B (CHB) patients. MethodsA total of 23 CHB patients who attended Tangdu Hospital, Fourth Military Medical University, from January to May, 2017, were enrolled. Peripheral blood was collected from all patients, and Ficoll density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were stimulated with interleukin-7 (IL-7), interleukin-15 (IL-15), and interleukin-21, respectively, and then anti-γC antibody and/or anti-IL-7Rα, anti-IL-2Rβ, and anti-IL-21R were added to the culture solution. After 96 hours of culture, flow cytometry was used to measure the expression of TIM-3, interleukin-2 (IL-2), interleukin-10 (IL-10), and interferon-γ (IFNγ) and the phosphorylation level of signal transducer and activator of transcription (STAT) in CD8+ T cells. A one-way analysis of variance and the least significant difference t-test were used for comparison of continuous data. ResultsThe CD8+ T cells stimulated by IL-7 and IL-15 had a significantly higher percentage of TIM-3-positive CD8+ T cells than those without stimulation (t=9.966 and 9074, P＜0.05), as well as significantly higher expression levels of IL-2, IL-10, and IFN-γ and phosphorylation levels of STAT-5 and STAT-1 (all P＜0.05). Stimulation with anti-IL-7Rα and anti-γC antibody significantly reduced the elevated expression levels of TIM-3, IL-2, and IL-10 in the IL-7 stimulation group (t=5.537, 6.224, and 4.500, P＜0.05). Stimulation with anti-IL-2Rβ alone or in combination with anti-γC antibody significantly reduced the expression levels of TIM-3 and IL-2 and the phosphorylation level of STAT-1 in the IL-15 stimulation group (P ＜0.05). ConclusionIL-7 and IL-15 can upregulate the expression of TIM-3 in CD8+ T cells of CHB patients, possibly via the γC receptor-mediated STAT-cytokine signaling pathway.
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Objective To investigate the influence of interleukin-6(IL-6) on the expression and function of programmed death-1(PD-1) in patients with hepatocellular carcinoma (HCC) by measuring the plasma level of IL-6 and the expression of PD-1 in peripheral blood CD8 + T cells from HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital or The Second Affiliated Hospital of Air Force Medical University & Tangdu Hospital of Fourth Military Medical University from January to September 2019 were enrolled as HCC group, and 19 healthy controls, matched for age and sex, were enrolled as HC group. Peripheral blood was collected, and plasma and peripheral blood mononuclear cells were isolated to separate CD8 + T cells. ELISA was used to measure the plasma level of IL-6, and flow cytometry was used to measure the expression level of PD-1 in CD8 + T cells. The separated CD8 + T cells were stimulated with anti-IL-6 neutralizing antibody for 24 hours; CCK-8 assay was used to measure cell proliferation, ELISA was used to measure the levels of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in supernatant, real-time PCR was used to measure the relative mRNA expression levels of perforin, granzyme B, and granulysin, and Western blot was used to measure the phosphorylation levels of STAT3 and Src. The t -test or the paired t -test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between groups. Results Compared with the HC group, the HCC group had a significant increase in the plasma level of IL-6 (99.67±20.92 pg/mL vs 81.05±16.76 pg/mL, t =3.427, P =0.001 1). There was no significant difference in the percentage of CD3 + CD8 + T cells between the HCC group and the HC group, while there was a significant increase in the percentage of PD-1 + CD8 + cells in HCC patients (3.79%±1.36% vs 2.20%±0.47%, t =5.335, P < 0.000 1). In the patients with HCC, although anti-IL-6 neutralizing antibody for inhibiting IL-6 in CD8 + T cells did not affect cell proliferation, it downregulated the expression of PD-1 (2.67%±0.91% vs 3.33%±1.12%, t =2.177, P =0.035) and increased the secretion of IFNγ (13.50±3.82 pg/mL vs 10.82±1.37 pg/mL, t =3.170, P =0.002 8), and there were also significant increases in the relative mRNA expression levels of perforin and granzyme B ( t =6.161 and 14.140, both P < 0.000 1) and a significant reduction in the level of phosphorylated STAT3 ( P < 0.000 1). Conclusion Anti-IL-6 neutralizing antibody can enhance the function of CD8 + T cells in HCC patients possibly by increasing the levels of perforin and granzyme B, improving the secretion of cytokines, and inhibiting the expression of PD-1.
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Objective To investigate the influence of interleukin-6(IL-6) on the expression and function of programmed death-1(PD-1) in patients with hepatocellular carcinoma (HCC) by measuring the plasma level of IL-6 and the expression of PD-1 in peripheral blood CD8 + T cells from HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital or The Second Affiliated Hospital of Air Force Medical University & Tangdu Hospital of Fourth Military Medical University from January to September 2019 were enrolled as HCC group, and 19 healthy controls, matched for age and sex, were enrolled as HC group. Peripheral blood was collected, and plasma and peripheral blood mononuclear cells were isolated to separate CD8 + T cells. ELISA was used to measure the plasma level of IL-6, and flow cytometry was used to measure the expression level of PD-1 in CD8 + T cells. The separated CD8 + T cells were stimulated with anti-IL-6 neutralizing antibody for 24 hours; CCK-8 assay was used to measure cell proliferation, ELISA was used to measure the levels of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in supernatant, real-time PCR was used to measure the relative mRNA expression levels of perforin, granzyme B, and granulysin, and Western blot was used to measure the phosphorylation levels of STAT3 and Src. The t -test or the paired t -test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between groups. Results Compared with the HC group, the HCC group had a significant increase in the plasma level of IL-6 (99.67±20.92 pg/mL vs 81.05±16.76 pg/mL, t =3.427, P =0.001 1). There was no significant difference in the percentage of CD3 + CD8 + T cells between the HCC group and the HC group, while there was a significant increase in the percentage of PD-1 + CD8 + cells in HCC patients (3.79%±1.36% vs 2.20%±0.47%, t =5.335, P < 0.000 1). In the patients with HCC, although anti-IL-6 neutralizing antibody for inhibiting IL-6 in CD8 + T cells did not affect cell proliferation, it downregulated the expression of PD-1 (2.67%±0.91% vs 3.33%±1.12%, t =2.177, P =0.035) and increased the secretion of IFNγ (13.50±3.82 pg/mL vs 10.82±1.37 pg/mL, t =3.170, P =0.002 8), and there were also significant increases in the relative mRNA expression levels of perforin and granzyme B ( t =6.161 and 14.140, both P < 0.000 1) and a significant reduction in the level of phosphorylated STAT3 ( P < 0.000 1). Conclusion Anti-IL-6 neutralizing antibody can enhance the function of CD8 + T cells in HCC patients possibly by increasing the levels of perforin and granzyme B, improving the secretion of cytokines, and inhibiting the expression of PD-1.
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Objective@#To explore the effect of acupoint application combined with conventional anti-tuberculosis treatment on pulmonary tuberculosis and its effect on the immune function of patients.@*Methods@#A total of 64 patients with pulmonary tuberculosis who visited Hangzhou Red Cross Hospital from March 2016 to September 2017 were selected in the study.The patients were randomly divided into control group (n=30) and observation group (n=34) according to the digital table.The clinical curative effect (obvious absorption, absorption, no change and deterioration), lung function (FVC, FEV1 and FVC/FEV1), immune function(CD3+, CD4+, CD8+, CD4+/CD8+, IgC, IgA, IgM) were compared between the two groups.@*Results@#The absorptive rate of the control group was 46.67%, which was significantly lower than that of the observation group(76.47%, χ2=6.04, P<0.05). After treatment, the levels of FVC, FEV1 and FVC/FEV1 of the two groups were significantly increased compared with before treatment..After treatment, the levels of FVC, FEV1 and FVC/FEV1 in the observation group were (2.20±0.48)L, (2.50±0.34)L, (87.44±13.60)%, respectively, which were significantly higher than those in the control group [(1.63±0.32)L, (2.02±0.44)L, (80.28±12.66)%] (t=6.607, 5.687, 2.558, all P<0.05). After treatment, the CD3+, CD4+, CD8+, CD4+/CD8+, IgC, IgA, IgM levles in the observation group were significantly increased compared with before treatment (all P<0.05). After treatment, the CD3+, CD4+, CD8+, CD4+/CD8+, IgC, IgA, IgM levles in the observation group were (73.25±6.17)%, (38.65±5.75)%, (36.58±3.17)%, (1.52±0.65), (15.49±1.49)g/L, (3.07±1.30)g/L, (1.94±0.50)g/L, respectively, which were significantly higher than those in the control group[(68.43±6.4)%, (34.72±5.68)%, (35.02±3.08)%, (1.16±0.78), (12.61±1.64)g/L, (2.23±0.90)g/L, (1.68±0.35)g/L] (t=3.590, 3.223, 2.340, 2.340, 8.594, 3.550, 2.846, all P<0.05).@*Conclusion@#Conventional anti-tuberculosis chemotherapy drugs combined with acupuncture has better effect than conventional anti-tuberculosis chemotherapy drugs.It can significantly improve the immune function of patients, improve the success rate of treatment, and speed up the improvement of the patients' condition.
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Objective@#To investigate the factors associated with CD4+ /CD8+ T lymphocyte ratio normalization in acquired immunodeficiency syndrome (AIDS) patients after antiretroviral therapy (ART).@*Methods@#The data of 1 188 human immunodeficiency virus (HIV)/AIDS patients from the national ART reporting system in Yuxi City, Yunnan Province between January 1, 2006 and December 31, 2016 were retrospectively collected and analyzed. The rate of CD4+ /CD8+ T lymphocyte ratio normalization after ART was calculated by lifetable. Cox proportional hazard models were used to analyze the factors associated with CD4+ /CD8+ T lymphocyte normalization in AIDS patients after ART. The Wilcoxon rank sum test was used for comparison between groups.@*Results@#The follow-up time was 3.8 (1.0-10.8) years. CD4+ /CD8+ T lymphocyte ratio normalization was documented in 95 patients with the rate of 1.89 per 100 person-years (95% confidence interval(CI) 1.52-2.27) after ART. The average time from ART to CD4+ /CD8+ T lymphocyte ratio normalized was 9.4 years. The cumulative normalization rate was 0.02 for the first year, 0.06 for the third year, 0.11 for the fifth year, 0.19 for the seventh year and 0.37 for the ninth year. By Cox proportional hazard models, the probability of CD4+ /CD8+ T lymphocyte ratio normalization in patients infected HIV by heterosexual contacts was 3.709 (95%CI 1.781-7.726) times higher than those by intravenous injection. The probability of CD4+ /CD8+ T lymphocyte ratio normalization in patients with baseline CD4+ T lymphocytes of 350-499 and more than 500 cell/μL groups were 2.792 (95%CI 1.196-6.519) and 3.832 (95%CI 1.648-8.913) times higher than those with baseline CD4+ T lymphocytes less than 200 cell/μL, respectively. The probability of normalization after ART in patients with higher baseline CD4+ /CD8+ T lymphocyte ratio was higher than those with baseline CD4+ /CD8+ T lymphocyte ratio≤ 0.20 (hazard ratio>1, all P<0.01).@*Conclusion@#The CD4+ /CD8+ T lymphocyte ratio normalization in AIDS patients after ART is associated with baseline CD4+ T lymphocyte counts, baseline CD4+ /CD8+ T lymphocyte ratio and HIV transmission mode.
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Objective To investigate the factors associated with CD 4 +／CD8 +T lymphocyte ratio normalization in acquired immunodeficiency syndrome ( AIDS) patients after antiretroviral therapy ( ART). Methods The data of 1 188 human immunodeficiency virus ( HIV)／AIDS patients from the national ART reporting system in Yuxi City , Yunnan Province between January 1, 2006 and December 31, 2016 were retrospectively collected and analyzed.The rate of CD4 +／CD8 +T lymphocyte ratio normalization after ART was calculated by lifetable.Cox proportional hazard models were used to analyze the factors associated with CD 4+／CD8+T lymphocyte normalization in AIDS patients after ART.The Wilcoxon rank sum test was used for comparison between groups.Results The follow-up time was 3.8 (1.0 -10.8 ) years.CD4+／CD8+T lymphocyte ratio normalization was documented in 95 patients with the rate of 1.89 per 100 person-years (95%confidence interval (CI) 1.52-2.27) after ART.The average time from ART to CD4 +／CD8+T lymphocyte ratio normalized was 9.4 years.The cumulative normalization rate was 0.02 for the first year, 0.06 for the third year, 0.11 for the fifth year, 0.19 for the seventh year and 0.37 for the ninth year.By Cox proportional hazard models, the probability of CD4+／CD8 +T lymphocyte ratio normalization in patients infected HIV by heterosexual contacts was 3.709 (95%CI 1.781-7.726) times higher than those by intravenous injection.The probability of CD4 +／CD8 +T lymphocyte ratio normalization in patients with baseline CD 4+T lymphocytes of 350-499 and more than 500 cell／μL groups were 2.792 (95%CI 1.196-6.519) and 3.832 (95%CI 1.648-8.913) times higher than those with baseline CD 4 +T lymphocytes less than 200 cell／μL, respectively.The probability of normalization after ART in patients with higher baseline CD 4+／CD8+T lymphocyte ratio was higher than those with baseline CD 4 +／CD8+T lymphocyte ratio≤0.20 ( hazard ratio ＞1, all P＜0.01). Conclusion The CD4 +／CD8 +T lymphocyte ratio normalization in AIDS patients after ART is associated with baseline CD4+T lymphocyte counts, baseline CD4 +／CD8 +T lymphocyte ratio and HIV transmission mode.
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Objective To explore the effect of acupoint application combined with conventional anti-tuber-culosis treatment on pulmonary tuberculosis and its effect on the immune function of patients.Methods A total of 64 patients with pulmonary tuberculosis who visited Hangzhou Red Cross Hospital from March 2016 to September 2017 were selected in the study.The patients were randomly divided into control group ( n =30) and observation group (n=34) according to the digital table.The clinical curative effect (obvious absorption,absorption,no change and deterioration),lung function ( FVC,FEV1 and FVC/FEV1 ),immune function ( CD+3 ,CD+4 ,CD+8 ,CD+4 /CD+8 , IgC,IgA, IgM) were compared between the two groups. Results The absorptive rate of the control group was 46.67% ,which was significantly lower than that of the observation group(76.47% ,χ2 =6.04,P<0.05).After treat-ment,the levels of FVC,FEV1 and FVC/FEV1 of the two groups were significantly increased compared with before treatment..After treatment,the levels of FVC,FEV1 and FVC/FEV1 in the observation group were (2.20 ± 0.48)L, (2.50 ± 0.34)L,(87.44 ± 13.60)% ,respectively,which were significantly higher than those in the control group [(1.63 ± 0.32)L,(2.02 ± 0.44) L,(80.28 ± 12.66)% ] (t=6.607,5.687,2.558,all P<0.05).After treat-ment,the CD+3 ,CD+4 ,CD+8 ,CD+4 /CD+8 ,IgC,IgA,IgM levles in the observation group were significantly increased compared with before treatment ( all P <0.05). After treatment, the CD+3 , CD+4 , CD+8 , CD+4 /CD+8 , IgC, IgA, IgM levles in the observation group were (73.25 ± 6.17)% ,(38.65 ± 5.75)% ,(36.58 ± 3.17)% ,(1.52 ± 0.65), (15.49 ± 1.49)g/L,(3.07 ± 1.30)g/L,(1.94 ± 0.50)g/L,respectively,which were significantly higher than those in the control group [( 68.43 ± 6.4 )% , ( 34.72 ± 5.68 )% , ( 35.02 ± 3.08 )% , ( 1.16 ± 0.78 ), ( 12.61 ± 1.64)g/L,(2.23 ± 0.90)g/L,(1.68 ± 0.35)g/L] (t=3.590,3.223,2.340,2.340,8.594,3.550,2.846,all P<0.05 ). Conclusion Conventional anti - tuberculosis chemotherapy drugs combined with acupuncture has better effect than conventional anti-tuberculosis chemotherapy drugs.It can significantly improve the immune function of patients,improve the success rate of treatment,and speed up the improvement of the patients＇condition.
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Objective: To investigate the inhibitory effect of lycium barbarum polysaccharides (LBPs) on growth of glioma in rats and its possible mechanism. Methods: The rats were divided into LBP A group, LBP B group, LBP C group, LBP D group, LBP E group and F group (control group) to be fed with 400, 200, 100, 50 and 25 mg · kg-1 · d-1 LBP and drinking water, respectively. The model of rat C6 glioma in situ was established through surgery. The survival of the glioma-bearing rats was observed, and the tumor volume was examined. The percentage of CD3+CD8+T cells in rat blood was detected by FCM method. The expressions of CD3 and CD8 in glioma tissues in rats were detected by immunohistochemistry. After injection of Evans blue solution through femoral vein, the effect of LBP on the permeability of blood-brain barrier in rats was observed, and the change of ultrastructure of brain tissues was observed under a scanning electron microscope. The expressions of CD8 and annexin A1 (ANXA1) mRNAs and ANXA1 protein in glioma tissues in rats were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. Results: The survival of glioma-bearing rats in LBP A, LBP B and LBP C groups was superior to that in F group (all P < 0.05). The tumor volume of glioma-bearing rats in LBP A, LBP B and LBP C groups was smaller than that in F group (all P < 0.05). The percentage of CD3+CD8+T cells in blood of glioma-bearing rats in LBP C group was higher than that in F group [(18.9± 1.4)% vs (11.5±0.7)%, P < 0.01]. The number of CD3+ and CD8+ T cells in glioma tissues in rats in LBP C group was higher than that in F group (both P < 0.01). The amount of Evans blue solution into the brain tissues in LBP C group was increased, the capillary endothelial cells were shrinkable, and the basement membrane was uneven with partial fracture. The expression level of CD8 mRNA in glioma tissues in rats was up-regulated (P < 0.01), whereas the expression levels of ANXA1 mRNA and protein were down-regulated (both P < 0.01). Conclusion: LBPs can inhibit the growth of glioma and prolong the survival of glioma-bearing rats. The mechanism may be related to the inhibitory effect of LBPs on the growth of tumor by regulating the blood-brain barrier and promote the invasion of CD8+T cells into the brain.
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Objective@#To explore the changes of the peripheral invariant natural killer T (iNKT) cells in patients with human immunodeficiency virus (HIV) infection. @*Methods@#A total of 101 patients with HIV infection including 52 asymptomatic patients and 49 acquired immunodeficiency syndrome (AIDS) patients were enrolled in the study from June 2016 to July 2017. Flow cytometry was used to detect iNKT cells, CD4+ T cells and CD8+ T cells, and the relationship among them and HIV RNA was studied. At same time, 12 healthy persons were enrolled as control group. T test or variance analysis, rank sum test, Chi-square test and Fisher exact test were used for statistical analysis. @*Results@#In HIV infected asymptomatic patients, AIDS patients and healthy controls, iNKT cells were 0.135% (0.066%, 0.228%), 0.058% (0.034%, 0.100%) and 0.385% (0.205%, 0.600%), respectively, and the difference was statistical significant (Z=40.113, P<0.01). CD4+ T cell counts in the three groups were (340.82±119.26) cells/μL, (72.73±61.84) cells/μL and (555.17±229.43) cells/μL, respectively, and the difference was statistical significant (t=113.79, P<0.01); CD8+ T cell counts in the three groups were (842.29±423.68) cells/μL, (540.43±257.85) cells/μL and (875.92±516.45) cells/μL, respectively, and the difference was statistical significant (t=9.423, P<0.01). Ratios of CD4+ /CD8+ T cells in the three groups were 0.490 (0.240, 0.695), 0.120 (0.030, 0.210) and 0.600 (0.475, 0.895), respectively, and the difference was statistical significant (Z=53.603, P<0.01). iNKT cell counts in patients with or without hepatitis B virus infection, pneumocystis pneumonia, oral mold infection, treponema pallidum, latent tuberculosis or EB virus infection were not significantly different (Z=0.244, 2.325, 2.393, 0.168, 1.183 and 0.454, respectively, all P>0.05). There were correlations between iNKT cells and CD4+ T cells, CD4+ /CD8+ T cells (r=0.513 and 0.261, respectively, both P<0.05), and no relationship was found between iNKT cells and CD8+ T cells (r=0.155, P=0.126). In HIV infected asymptomatic patients and AIDS patients, iNKT cells was not associated with HIV RNA (r=-0.113 and -0.111, respectively, both P>0.05). @*Conclusions@#The level of peripheral iNKT cells in HIV infected patients decreases with the disease progression. To certain extent, iNKT cells can reflect the severity of immune damaging in HIV infected patients.
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Objective To identify the phenotype of CD8αα+ T cells locally infiltrating psoriatic skin lesions,and to investigate their role in the occurrence of psoriasis.Methods Skin lesions were obtained from 8 patients with confirmed plaque psoriasis in the progressive stage,who visited the Department of Dermatology in Xijing Hospital affiliated to the Fourth Military Medical University from January to December in 2017.Of the 8 patients,4 were male and 4 were female,with ages ranging from 24 to 50 years.Normal skin tissues were obtained from discarded skin tissues of 8 healthy controls in plastic surgery.Of the 8 healthy controls,4 were male and 4 were female,with ages ranged from 23 to 46 years.Immunofluorescence technique was used to investigate the distribution of CD8αα+ T cells,determine the proportion of CD8αα+ T cell subsets,identify the immunological phenotype of CD8αα+ T cells and measure the expression of interleukin-17A (IL-17A).Results Infiltration of CD8 + T cells was observed in the dermis and epidermis of the 8 psoriatic skin lesions,and the proportion of CD8αα+ T cells was 88.48％ ± 7.39％.However,only a few CD8+ T cells locally infiltrated the control skin tissues,and the proportion of CD8αα+ T cells was 14.43％ ± 13.14％.There was a significant difference in the proportion of CD8αα+ T cells between the psoriatic skin lesions and control skin tissues (t =11.5,P ＜ 0.01).CD8αα+ T cells in the psoriatic epidermis expressed CD103 (a marker of tissue-resident cells),while CD8αα+ T cells in the psoriatic dermis did not express CD103.CD8αα+ T cells in the psoriatic lesions were identified as CD45RA CCR7 effector T cells,and did not express Foxp3,CD25 and CD122 (markers of CD8+ regulatory T cells).The proportion of CD8αα+ T cells producing IL-17A in the psoriatic lesions was 24.85％ ± 4.25％,while CD8αα+ T cells in the control skin tissues did not produce IL-17A.There was a significant difference in the proportion of CDSαα+ T cells producing IL-17A between the psoriatic skin lesions and control skin tissues (t =5.853,P ＜ 0.01).Conclusion CD8αα+ T cells infiltrating psoriatic lesions are effector memory T cells,and may contribute to the occurrence and development of psoriasis by producing IL-17A.
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Objective To study the correlation between CD8 + T cells' balance and prognosis for patients with diffuse axonal injury(DAI).Methods To collect the 41 patients with DAI as observation group,with 33 cases light craniocerebral injury (LCI) patients and 35 healthy volunteers as control group.We detect the peripheral blood CD8 + CD28 + T cells and CD8 + CD28-T cell percentage,and calculate the ratio of both.The subjects working curve method (ROC) of.the above CD8 + T cells and its ratio were used to predict the prognosis of patients with DAI efficiency evaluation.Results (1) Compared with control group,CD8 + CD28 + T cells in patients with DAI and LCI increased significantly,and DAI was significantly higher than that of LCI group (P ＜ 0.05).Compared with control group,CD8 + CD28-T cells in patients with DAI were decreased,but no statistical difference was found between LCI and the control group,but DAI group was significantly lower than the LCI group (P ＜ 0.05).(2) CD8 + CD28-T cells and CD8 + CD28 +/CD8 + CD28-ratio were correlated with DAI diagnosis (P =0.003,0.000).When the percentage of CD8 + CD28-T cells =2.95％,the sensitivity of the diagnosis of DAI was 87.49％,86.21％;When the ratio of CD8 + CD28 +/CD8 + CD28-=7.39,the sensitivity was 92.63％,90.71％.(3) a total of 7 cases of patients died within 1 week after injury,CD8 + CD28 + T cells and CD8 + CD28 +/CD8 + CD28-ratio were significantly negative correlated with survival time (r =-0.739,-0.834,P =0.021,0.002),and CD8 + CD28-T cells were significantly positive correlated with survival time (r =0.782,P =0.006).Conclu sions Decreasing CD8 + CD28-T cells and higher CD8 + CD28 +/CD8 + CD28-ratio in patients with DAI immunological characteristics,especially the ratio (balance) is closely corrected with the diagnosis and prognosis of DAI.
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Objective To explore the risk factors of pulmonary artery hypertension (PAH) and the its relationship with T cell subsets to provide a foundation for the prevention and treatment of PAH.Methods 154 maintained hemodialysis (MHD) patients in our dialysis center were recruited according to the criterion and divided into two groups subsequently:PAH group (pulmonary artery systolic pressure,PASP ＞ 35 mmHg) and non-PAH group (PASP≤35 mmHg).The related clinical,biochemical and ultrasonic cardiogram data were collected and peripheral blood was acquired to detect the expressions of the surface antigen CD3,CD4,CD8 and CD69 with flow cytometry.Logistic regression analysis was used to find out the relationship between PAH and T cell subsets.Results There was no significant difference between 56 cases of PAH and 98 cases of non-PAH as regards gender,age,mean systolic and diastolic pressure,dialysis durations,morbidities of hypertension and diabetes,smoking rate,and left ventricular diameter.Compared with the non-PAH group,the PAH group demonstrated a lower percent of CD8 T cells and CD8 CD69 T cells,but a much higher left atrial diameter (LAD),Interventricular septum thickness,left ventricular posterior wall thickness,and NT-proBNP.The percentage of T cells,CD4 T cells and CD4 CD69 T cells showed no difference between the two groups.Multivariate analysis confirmed that PAH was negatively independently associated with the percentage of CD8 T cells and CD8CD69 T cells.Conclusions The decreased percentage of CD8 T cells and CD8CD69 T cells in the peripheral blood is a risk factor of PAH in maintained hemodialysis patients,and CD8 T cells may play an important role in the genesis of PAH.