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Objective:To analyze the value of cytotoxic T lymphocyte and natural killer cell levels in prognosis evaluation of patients with ST-elevation myocardial infarction (STEMI).Methods:A total of 158 patients with STEMI who underwent percutaneous coronary intervention in The Second People's Hospital of Liaocheng from September 2020 to August 2021 were included in this study. The ratio of cytotoxic T lymphocytes to natural killer cells was measured immediately after admission and 48 hours after surgery. These patients were followed up for 1 month after treatment. They were divided into the adverse cardiovascular event group (occurrence group) and no adverse cardiovascular event group (non-occurrence group) according to the occurrence of cardiovascular adverse events. The influential factors of the prognosis of STEMI and the correlation between the influential factors and STEMI were analyzed.Results:Among 158 patients with STEMI, 27 patients had adverse cardiovascular events, accounting for 17.09%. There were significant differences in systolic blood pressure, left ventricular ejection fraction, and low-density lipoprotein levels between the occurrence and non-occurrence groups ( t = 2.82, 4.27, 2.32, all P < 0.05). At 48 hours after surgery, the levels of cytotoxic T lymphocytes [(22.75 ± 8.39)%, (29.23 ± 4.61)%] and natural killer cells [(13.73 ± 4.64)%, (20.64 ± 4.52)%] in the peripheral blood in the occurrence and non-occurrence groups were significantly decreased compared with before surgery [ t = -5.05, -83.68, -142.71, -7 084.80, all P < 0.001]. Before and 48 hours after surgery, the levels of cytotoxic T lymphocytes [(27.47 ± 3.35)%, (22.75 ± 8.39)%] and natural killer cells [(21.42 ± 4.36)%, (13.73 ± 4.64)%] in the peripheral blood in the occurrence group were significantly lower than those in the non-occurrence group ( t = 7.68, 13.10, 4.16, 5.76, all P < 0.001). Univariate analysis showed that preoperative cytotoxic T lymphocytes < 27.47%, preoperative natural killer cells < 21.42%, left ventricular ejection fraction, and low-density lipoprotein may be the risk factors that affect the prognosis of patients with STEMI ( P < 0.000, 0.012, 0.019, 0.033). Cox regression analysis showed that preoperative cytotoxic T lymphocytes < 27.47% and preoperative natural killer cells < 21.42% were independent risk factors affecting the prognosis of patients with STEMI (both P < 0.001). Conclusion:Reduced levels of baseline cytotoxic T lymphocytes and natural killer cells in patients with STEMI suggest an increased risk of poor prognosis.
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ObjectiveTo investigate whether cytotoxic T lymphocyte (CTL)-derived exosomes can downregulate HBx expression and inhibit hepatic stellate cell (HSC) activation. MethodsThe supernatants of HepG2, HepGA14, and CTL cells were collected to extract exosomes, which were referred to as NC-exo, HBV-exo, and CTL-exo, respectively). Transmission electron microscopy was used to observe their morphology, and Western Blot was used to measure the expression of the markers of exosomes CD63 and TSG101. NC-exo, HBV-exo, and CTL-exo labeled by BODIPY dye were mixed with HBV-exo at different ratios and were then co-cultured with HSC LX-2 (HSC-LX2). A fluorescence microscope was used to observe whether exosomes could enter LX-2 cells, and an fluorescence microscope was used to observe cell morphological changes; quantitative real-time PCR (qPCR) was used to measure the expression of the activated biomarkers such as transforming growth factor-β1 (TGF-β1), ɑ-smooth muscle actin (ɑ-SMA), and collagen type I (Collagen I) in LX-2 cells. CTL-exo was added to the HepGA14 culture system; then qPCR was used to measure the mRNA expression level of HBV DNA, cccDNA, and HBx in exosomes in HepGA14 cells, and Western Blot was used to measure the protein expression level of HBx in exosomes. The t-test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThe exosomes were all microcysts with a double-layer membrane structure and were circular or elliptical in shape, with the expression of the signature proteins CD63 and TSG101, and the vesicles had a diameter of 50-100 nm. The fluorescence microscope showed that exosomes could enter LX-2 cells, and HSC were enlarged with extended cell processes. The results of qPCR showed that there were significant differences in the expression levels of TGF-β1, ɑ-SMA, and Collagen I genes between the NC-exo, HBV-exo, NC-exo+HBV-exo, and Con groups (F=444.678, 417.144, and 571.508, all P<0.05). After the intervention of HepGA14 cells with CTL-exo, qPCR results showed that compared with the control group, there were significant reductions in the expression levels of HBV DNA and cccDNA in HepGA14 cells (all P<0.05), the relative mRNA expression level of HBx in exosomes (P<0.05), and the protein expression level of HBx (P<0.05). CTL-exo and HBV-exo were mixed at different ratios (2∶1, 5∶1, 10∶1) and were then used for the intervention of LX-2 cells, and qPCR results showed that the expression levels of TGF-β1, ɑ-SMA, and Collagen I genes in LX-2 cells gradually decreased with the increase in the ratio of CTL-exo between groups (P<0.05). ConclusionCTL-exo can downregulate the protein expression of HBx in HBV-exo to inhibit HSC activation, suggesting that CTL-exo has an anti-hepatitis B liver fibrosis effect.
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With the development of immunotherapy in clinical application, immunotherapy also takes advantage in esophageal squamous cell carcinoma (ESCC). Immune checkpoint inhibitors such as programmed death-1 (PD-1) and its ligand PD-L1 and cytotoxic T lymphocyte antigen-4 show significant antitumor activity and safety in immunotherapy for patients with advanced ESCC.
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Resumen Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb- restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.
Abstract Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb- restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.
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Animales , Femenino , Humanos , Ratones , Salmonelosis Animal/inmunología , Células Dendríticas/inmunología , Linfocitos T Citotóxicos/inmunología , Salmonella typhimurium , Inmunización , Islas de CpG , Antígeno de Histocompatibilidad H-2D/inmunología , Inmunidad Celular , Ratones Endogámicos C57BLRESUMEN
ABSTRACT Background In order to induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)-based immunotherapy for bladder cancer, various tumor antigens can be loaded onto DCs. Objective The aim of this study was to establish a method of immunotherapy for male patients with non-muscle invasive bladder cancer (NMIBC), using bladder cancer-specific CTLs generated in vitro by DCs. Materials and Methods Monocyte-derived DCs from bladder cancer patients were induced to mature in a standard cytokine cocktail (IL-1β, TNF-α, IL-6, and PGE2: standard DCs, sDCs) or anα-type 1-polarized DC (αDC1) cocktail (IL-1β, TNF-α, IFN-α, IFN-γ, and polyinosinic:polycytidylic acid) and loaded with the UVB-irradiated bladder cancer cell line, T24. Antigen-loaded αDC1s were evaluated by morphological and functional assays, and the bladder cancer-specific CTL response was analyzed by cytotoxic assay. Results The αDC1s significantly increased the expression of several molecules pertaining to DC maturation, regardless of whether or not the αDC1s were loaded with tumor antigens, relative to sDCs. The αDC1s demonstrated increased production of interleukin-12 both during maturation and after subsequent stimulation with CD40L that was not significantly affected by loading with tumor antigens as compared to that of sDCs. Bladder cancer-specific CTLs targeting autologous bladder cancer cells were successfully induced by αDC1s loaded with dying T24 cells. Conclusion Autologous αDC1s loaded with an allogeneic bladder cancer cell line resulted in increased bladder cancer-specific CTL responses as compared to that with sDCs, and therefore, may provide a novel source of DC-based vaccines that canbe used in immunotherapy for male patients with NMIBC.
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Humanos , Masculino , Anciano , Neoplasias de la Vejiga Urinaria/terapia , Células Dendríticas/inmunología , Linfocitos T Citotóxicos/inmunología , Citocinas/uso terapéutico , Inmunoterapia/métodos , Neoplasias de la Vejiga Urinaria/inmunología , Diferenciación Celular/inmunología , Resultado del Tratamiento , Línea Celular Tumoral , Inmunoterapia/efectos adversos , Persona de Mediana EdadRESUMEN
Objective:To exptore the potential of autologous dendritic cells (DCs) pulsed with caner/ testis antigen NY-ESO-1 peptides in inducing specific cytotoxic T lymphocyte (CTLs) response and an tineoplastic immune function of specific CTLs.Methods:Fifteen patients with Ⅱ to Ⅲ stage positive HLA-A0201 + and NY-ESO-1 + were enrolled in the Cancer Hospital Chinese Academy of Medical Sciences on the basis of preclinical experiments from November 2014 to October 2015,and their peripheral blood mononuclear cells (PBMCs) and peripheral blood lymphocytes (PBLs) were isolated.The PBMCs were induced into DCs and pulsed with NY-ESO-1 peptide.The phenotypes of DCs were stained with antibodies against HLA-DR+ CD11c +,CD80 +,CD83 + and CD86 +,and subsequently analyzed by multichannel flow cytometry (FCM).The killing effects of CTLs pulsed with HLA-A0201-binding peptide NY-ESO-1 and the potential of autologous DCs pulsed with NY-ESO-1 peptides in inducing specific cytotoxic T lymphocytes (CTLs) responses were determined.The patients were administered two infusions of autologous CTLs for 1 time every two weeks.The total infusion was with 2 times.The immunological responses and clinical responses were examined in 1 week after the final administration.Results:The immunophenotype of DCs pulsed with NY-ESO-1 peptide was analyzed,HLA-DR+ CD11c+ cells (93.6% ± 1.2%),CD80 + cells (87.3% ± 3.6%),CD83 + cells (82.8% ± 2.5%) and CD86 + cells (93.4% ± 6.4%).PBLs isolated from patients primed by DCs pulsed with NY-ESO-1 peptide proliferated continuously and the proliferation index (PI) of the PBLs were analyzed.There was significant difference between the DCs loaded with polypeptides and those unloaded,though it could promote the proliferation of PBLs,but the PI was significantly lower than that of the DCs loaded with NY-ESO-1 peptide (P < 0.05).The average percentage of special CTLs primed by DCs pulsed with NY-ESO-1 peptides was significantly higher than that in the control group (5.2% ± 1.2% vs.0.4% ± 0.1%).CTLs induced by NY-ESO-1 pulsed DCs exerted a stronger killing effect on T2 cell line pulsed with NY-ESO-1 peptide than that in the control group at the ratio of E (effect) to T (target) as 30 ∶ 1,P < 0.05.The cytokine levels in the patients' sera such as IFN-γ,IL-2 and IL-12 were increased after treatments [(132.9 ± 10.2) μg/Lvs.(46.4±3.1) μg/L;(101.3 ±6.4) μg/Lvs.(26.7 ±1.2) μg/L;(51.3 ±2.6) μg/L vs.(26.4 ± 1.1) μg/L;all P < 0.05],and the percentages of antigen-specific CD8 + IFN-γ + increased in these patients (P <0.01).Conclusion:Auto-DCs pulsed with NY-ESO-1 peptides can in duce the proliferation of allogenic CTLs,which elicit specific immune responses ex vivo or in vivo,and boost anticancer immunity markedly.
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Objective To investigate the ability of inducing antigen-specific cytotoxic T lymphocytes (CTL) stimulated by dendritic cell (DC) fused with MiaPaCa-2 cells in vitro.Methods DC were isolated and cultured from peripheral blood mononuclear cells (PBMCs).50%PEG and 10%DMSO were used to fuse MiaPaCa-2 cells and DC, and DC co-cultured with MiaPaCa-2 cells and DC alone served as control .The fusion efficiency was assessed by flow cytometry ( FCM) and DC-MiaPaCa-2 hybrids were identified as PE-MUC1/FITC-CD86 double positive cells . The survival rate of DC was determined by MTT method . The lymphocyte proliferation stimulated by DC in vitro was evaluated by mixed cell culture with DC in different ratios of 1∶10, 1∶20 and 1∶80.IL-2, IL-10, granzyme B and IFN-γreleased by antigen-specific CTLs were measured by ELISA assay.Results The fusion rate in DC fused with MiaPaCa-2 cells ( fused cells ) was ( 42 .30 ± 7.30)%, which was higher than (7.21 ±1.06)% in DC co-cultured with MiaPaCa-2 cells ( co-cultured cells).The cell viability of DC, co-cultured cells and fused cells was >95.0%, 85.0% and 62.8%, and fused cells had greatly lower cell viability than DC and co-cultured cells (P0.05).At the co-culture ratio of 1∶10, IL-2 secreted by CTL in DC, co-cultured and fused cells was (27.30 ±5.21 ),(897.44 ±93.05),(2 243.80 ±381.46) ng/L; IL-10 was (19.55 ± 2.05), ( 424.60 ±108.25 ), ( 706.53 ±161.29 ) ng/L; Granzyme B was ( 16.23 ±1.23 ), ( 451.07 ± 120.50),(1327.77 ±205.15)ng/L;IFN-γwas (30.11 ±4.32)、(982.00 ±124.68)、(2421.04 ±488.50) ng/L.Cytokines from the antigen-specific CTL induced by DC fused with MiaPaCa-2 cells were significantly higher than those by DC and DC co-cultured with MiaPaCa-2 cells ( all P<0.05).Conclusions The fusion of DC and pancreatic cancer MiaPaCa-2 cells could stimulate tumor antigen-specific CTL in vitro .
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Objective To explore the potential mechanism of severe liver injury shortly after withdrawal of antiviral therapy in chronic hepatitis B (CHB) patients.Methods Forty-nine patients with chronic hepatitis B virus (HBV) infection from the Department of Infectious Diseases of the First Affiliated Hospital of Nanchang University and 8 healthy volunteers from August 2014 to March 2015 were included in this study.All of them were human leukocyte antigen (HLA)-A2-positive.CHB patients were classified into three groups,including 15 cases in immune-tolerance group,20 cases in sustained antiviral treatment group,and 14 cases in recurrence of drug withdrawal group.The frequency of peripheral HLA-A0201-restricted hepatitis B core antigen (HBcAg)18-27 pentamer complex specific CD8+ T cells in CHB patients was analyzed by flow cytometry.Enzyme linked immunospot assay(ELISPOT) was used to detect interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) secretions of HBcAg18-27-specific CD8+ T cells.The experimental data were analyzed using non-parametric U tests.Results In healthy control group,immune-tolerance group,sustained antiviral treatment group and recurrence of drug withdrawal group,the frequencies of HBcAg-specific CD8+T cells were (0.17 ± 0.16) %,(1.46±0.72)%,(3.24± 1.60)% and (4.67±2.43)%,respectively.Compared with healthy control group,the difference were all statistically significant in the three groups (Z=-3.583,-4.018 and-3.823,respectively;all P<0.01).The frequencies of HBcAg-specific CD8+T cells in immune tolerance group or recurrence of drug withdrawal group were both significantly different from that in sustained antiviral therapy group (Z=-3.400 and-2.030,respectively;both P<0.05).The difference between immune-tolerance group and recurrence of drug withdrawal group was also significant (Z =-3.230,P<0.01).The secretion levels of IFN-γ of HBcAg-specific CD8+T cells in healthy control group,immune-tolerance group,sustained antiviral treatment group and recurrence of drug withdrawal group were2 (0-6),16 (2-53),106 (14-254) and 156 (28-395) spot forming cell (SFC)/106 peripheral blood mononuclear cell (PBMC),respectively.The differences between healthy control group and immune-tolerance group,sustained antiviral treatment group or recurrence of drug withdrawal group were all statistically significant (Z=-3.585,-4.069 and-3.824,respectively;all P<0.01).The IFN-γ level of HBcAg-specific CD8+ T cells in recurrence of drug withdrawal group was significantly higher than that in sustained antiviral therapy group (Z=-2.205,P=0.027),and that in sustained antiviral therapy group was significantly higher than that in immune-tolerance group (Z=-4.700,P< 0.01).The TNF-α levels secreted by HBcAg-specific CD8+ T cells in each group were 2 (0-5),16 (2-32),112 (15-283),and 195 (55-537) SFC/106PBMC,respectively.The differences between healthy control group and immune-tolerance group,sustained antiviral treament group or recurrence of drug withdrawal group were all statistically significant (Z=-3.619,-4.069 and-3.824,respectively;all P<0.01).The TNF-α level secreted by HBcAg-specific CD8+T cells in recurrence of drug withdrawal group was significantly higher than that in sustained antiviral therapy group (Z=-2.449,P=0.014),and that in sustained antiviral therapy group was significantly higher than that in immune-tolerance group (Z=-4.350,P<0.01).Conclusions The changes of frequency and immune function of HBcAg-specific CD8+T cells in CHB patients may be one of the reasons causing severe liver damage after irregular withdrawal of nucleoside analogues.
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Objective To evaluate the effect ofT-cell immunoglobulin and mucin domain-3 (TIM-3) on TRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells.Methods A recombinant plasmid pFUSE-TIM-3-mIgG2Aae1-Fc2 encoding TIM-3 was constructed.Then,the recombinant plasmid and an empty plasmid pFUSE-mIgG2Aae1-Fc2 were transfected into human 293T epithelial cells followed by 48-hour culture for the preparation of supernatants containing TIM-3 and Ig-tail respectively.C57BL/6 mice were immunized with the TRP-2180-188 peptide vaccine for 4 sessions.One week after the last vaccination,C57BL/6 mice were sacrificed,and spleen lymphocytes were collected and then cultured with the TRP-21180-188 peptide and interleukin-2 (IL-2) for 5 days,with lymphocytes untreated with the TRP-2180-188 peptide or IL-2 serving as the control group.Mitomycin-treated B16F10 murine melanoma cells and TRP-2180-188 peptide-stimulated lymphocytes were co-cultured with the presence of supernatants of 293T cells that had been cultured for 48 hours (blank control group),TIM-3-containing supernatants (TIM-3 group) and Ig-tail-containing supernatants (negative control group) separately.After 24 and 48 hours of co-culture,cell counting kit-8 (CCK-8) assay was performed to estimate the proliferative activity of lymphocytes,enzyme-linked immunosorbent assay (ELISA) to determine the supernatant levels of interferon (INF)-γ and tumor necrosis factor (TNF)-α,flow cytometry to determine the percentage of CD8 + T cells in the co-culture system.Results Enzyme digestion and sequence analysis showed that the TIM-3 gene was successfully inserted into the eukaryotic expression plasmid.After 48-hour culture,TIM-3 and Ig-tail expressions were detected in the supernatants of 293T cells transfected with the recombinant plasmid and empty plasmid respectively.As CCK-8 assay showed,the proliferative activity of lymphocytes was significantly lower in the TIM-3 group than in the blank control group and negative control group after 24-and 48-hour culture (78.06% ± 6.37% vs.100.00% ± 10.42% and 108.70% ± 9.90% at 24 hours,42.93% ± 5.93% vs.100.00% ± 6.24% and 168.00% ± 2.98%at 48 hours,all P < 0.05),so was the ratio of cellular proliferative activity at 48 hours to that at 24 hours (all P < 0.05).Compared with the blank control group and negative control group,the TIM-3 group showed significantly decreased supernatant levels of IFN-γ and TNF-α after 24-hour (IFN-γ:192.96 γ 5.05 ng/L vs.216.44 ± 7.85 ng/L and 223.67 ±7.79 ng/L,both P< 0.05;TNF-α:58.43 ± 0.26 ng/L vs.26.43 ± 0.01 ng/L and 86.85 ± 1.12 ng/L,both P< 0.05) and 48-hour culture (IFN-γ:54.95 ± 0.57 ng/L vs.230.06 ± 4.23 ng/L and 167.24 ± 3.33 ng/L,both P < 0.05;TNF-α:30.23 ±0.26 ng/L vs.26.84 ± 0.20 ng/L and 45.34 ± 0.22 ng/L,both P < 0.05).In addition,the median percentage of CD8+ T cells was significantly increased in the TIM-3 group compared with the blank control group and negative control group after 24-and 48-hour culture (3.30% vs.0.421% and 2.22% at 24 hours,4.06% vs.0.577% and 0.691% at 48 hours,all P< 0.05).Conclusion TIM-3 in vitro can suppress the proliferative activity of and secretion of IFN-γand TNF-α by lymphocytes,but increase the percentage of CD8 + T cells in the co-culture system of TRP-2180-188 peptide-stimulated lymphocytes and B16F10 cells.
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Objective To compare the killing effect of cytotoxic T lymphocytes(CTLs) induced by dendritic cells transduced recombinant adenovirus associated virus (rAAV ) with different hepatitis B virus gene fragment (rAAV‐HBV‐S ,C ,E ,X ) . Methods Peripheral blood mononuclear cells(PBMCs) from chronic hepatitis B patients were isolated and transduced recombinant adeno‐associated virus with different hepatitis B virus (HBV) antigen gene fragment (rAAV‐HBV‐S ,C ,E ,X) ,then GM‐CSF ,IL‐4 and TNFαwere added to cultivate for 7 days to generate mature dendritic cells (DCs) .The state of DCs were observed and differen‐tiation antigen molecules (CD)were detected by flow cytometry(FACS)to evaluate their maturation and function .Cytotoxic T lym‐phocytes (CTLs) were induced by the mixed cuture of DCs with prepared T lymphocytes .HepaG2 .2 .15 and HepaG2 were targed cells and the killing effect of CTLs wered compared using MTS assay .Results The expression of phenotype CD14 ,CD80 ,CD83 , CD86 from DCs transduced with rAAV‐HBV‐S ,C ,E ,X were compared ,respectively .Of which ,CD80 ,CD86 were significantly dif‐ferent(P0 .05) .Conclusion HBV‐S ,C ,E ,X genes all induce M HC‐I dependently cytotoxic T‐lym‐phocytes specific response based adeno‐associated virus (AAV) vector delivery into dendritic cells .
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Objective To investigate the association between the difference of specific cytotoxic lymphocyte (CTL) and liver function of patients with hepatitis B virus (HBV) genotype B and C infections and interleukin (IL)‐7 induced follicular helper T lymphocytes (Tfh) .Methods Sixty‐seven patients with chronic hepatitis B (CHB) hospitalized at Wuxi No .5 People′s Hospital from August 2013 to January 2015 were collected and 30 healthy blood donors were set as healthy control group .The peripheral blood IL‐7 , Tfh ,IL‐21 ,HBV specific‐CTL ,nonspecific CTL ,levels of HBV DNA ,alanine aminotransferase (ALT) and total bilirubin (TBil) were compared between patients with genotype B and C infection ,hepatitis B e antigen (HBeAg)‐positive and HBeAg‐negative CHB ,high ALT level and low ALT level .Multivariate regression analysis was performed to determine the factors associated with IL‐7 .The t test was used for quantitative data and chi‐square test was used for categorical data .Results Of the 67 CHB patients with average age of (35 .1 ± 11 .4) ,48 were male and 19 were female;32 were infected with genotype C and 35 were infected with genotype B ;40 were HBeAg‐positive CHB and 27 were HBeAg‐negative CHB ;17 were with high ALT levels and 50 were with low ALT levels .IL‐7 ,Tfh ,IL‐21 and HBV specific‐CTL levels in the peripheral blood of genotype C‐infected patients were (20 .79 ± 4 .82 ) ng/L , (3 .93 ± 0 .82)% ,(24 .77 ± 7 .52) ng/L and (0 .20 ± 0 .04)% ,respectively ,while in genotype B‐infected patients , those were (29 .13 ± 8 .17) ng/L ,(5 .92 ± 1 .92)% ,(39 .94 ± 24 .00) ng/L and (0 .40 ± 0 .06)% , respectively .Levels of IL‐7 , Tfh ,IL‐21 and HBV specific‐CTL in genotype C‐infected patients were significantly lower than those in genotype B‐infected patients (t= 5 .027 ,5 .595 ,3 .553 and 15 .133 , respectively ;all P<0 .01) .Nonspecific CTL ,HBV DNA ,ALT and TBil levels in the peripheral blood of genotype C‐infected patients were all significantly higher than those in genotype B infected‐patients (t=4 .899 ,6 .815 ,2 .763 and 4 .899 ,respectively ;all P< 0 .01) .IL‐7 ,Tfh ,IL‐21 ,HBV specific‐CTL levels in the peripheral blood of HBeAg‐positive patients were significantly lower than those in HBeAg‐negative patients (all P<0 .01) .Nonspecific CTL ,HBV DNA ,ALT and TBil levels in the peripheral blood of HBeAg‐positive patients were all significantly higher than those in HBeAg‐negative patients (all P<0 .05) .IL‐7 ,Tfh ,IL‐21 ,HBV specific‐CTL levels in the peripheral blood of patients with high ALT levels were all significantly lower than those in patients with low ALT levels (all P<0 .01) .Nonspecific CTL and HBV DNA levels in the peripheral blood of patients with high ALT levels were both significantly higher than those in patients with low ALT levels (both P<0 .01) .HBV DNA ,IL‐21 and nonspecific CTL were all correlated with IL‐7 (all P<0 .01) .Conclusion The differences of HBV specific‐CTL and liver function in CHB patients infected with genotype B and C may be correlated with interleukin‐7 induced Tfhcells.
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Objective To investigate the expression of interferon inducible protein-10 (IP-10) in peripheral blood mononuclear cells (PBMC) of patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF), and its correlation with disease severity.Methods Eighty patients with HBV-ACLF, 60 patients with chronic hepatitis B (CHB), and 25 healthy controls were enrolled from the Affiliated Hospital of Hubei University of Arts and Science during October 2013 and February 2015.IP-10 mRNA in PBMC was measured by real time quantitative PCR.Independent sample t test was used to analyze the difference in IP-10 mRNA expression between HBV-ACLF patients with model for end-stage liver disease (MELD) < 30 and ≥30, and Pearson correlation test was performed to analyze the correlations of IP-10 mRNA expression with alanine aminotransferase (ALT), total bilirubin (TBil) and international normalized ratio (INR).Results The expressions of IP-10 mRNA in HBV-ACLF patients was 1.00 ± 0.19, which was higher than those in CHB patients and healthy controls (0.64 ± 0.08 and 0.41 ± 0.06, t =3.841 and 16.661, all P < 0.01).The expression of IP-10 mRNA in HBV-ACLF patients with MELD < 30 was 0.96 ±0.19, which was lower than that in patients with MELD ≥ 30 (1.14 ± 0.21, t =-2.283, P <0.05).Pearson correlation analysis showed that IP-10 mRNA level in HBV-ACLF patients was positively correlated with ALT, TBil and INR (r =0.697, 0.738 and 0.775, all P < 0.01).Conclusion IP-10 mRNA is over-expressed in PBMC of patients with HBV-ACLF, and it is correlated with disease severity, which suggests that IP-10 may play an important role in the progression of liver failure.
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Objective To explore the changes of programmed death receptor-1 (PD-1) in chronic hepatitis B (CHB) patients with different baseline of hepatitis B virus (HBV) DNA levels after treatment with adefovir dipivoxil (ADV).Methods One hundred CHB patients with positive hepatitis B e antigen (HBeAg),1 × 104 copy/mL≤HBV DNA≤1 × 107 copy/mL,and positive human leukocyte antigen-A2 were divided into two groups according to the baseline HBV DNA level:47 cases in low virus load group whose HBV DNA level was ≤1 × 105 copy/mL; 53 cases in high virus load group whose HBV DNA level was>1 × 105 copy/mL.Both groups were treated with ADV 10 mg/d.Serum HBV DNA,HBeAg seroconversion rate,alanine aminotransferase (ALT) and total bilirubin (TBil) levels of both groups before treatment and 12 months after treatment were compared.Flow cytometry was used to test peripheral blood HBV-specific cytotoxic T lymphocyte (CTL) surface PD-1 and peripheral blood HBV-specific CTL level.Categorical data were tested by x2 test; quantitative data was compared with t-test.Results Peripheral blood HBV-specific CTL surface PD-1 of CHB patients in low virus load group was 20.17 %±1.69%,which was lower than that in high virus load group (41.38%±2.30%,t =53.02,P<0.01) ; peripheral blood HBV specific CTL levels in two groups were 0.37%±0.02% and 0.17%± 0.02%,respectively (t=50.47,P<0.01) ; ALT and TBil levels in low virus load group were both lower than those of high virus load group (t=13.07,P<0.01; t=5.06,P<0.01).Twelve months after treatment,HBV DNA of 25 cases (53.2%) in low virus load group and 10 cases (18.9%) in high virus load group were lower than the detectable level (HBV DNA<500 copy/mL,x2 =12.89,P<0.01);HBeAg seroconversion was achieved in 15 cases(31.9%) and 1 case (1.9%),respectively (x2 =16.72,P<0.01) ; peripheral blood HBV-specific CTL surface PD-1 expression levels were 9.00 % ±1.38 % and 29.40 % ± 3.76 %,respectively (t =36.80,P< 0.01) ; peripheral blood HBV-specific CTL levels were 0.65%±0.10% and0.48%±0.07%,respectively (t=9.61,P<0.01).Conclusions After treatment with ADV,along with the decrease of HBV DNA load,HBV-specific CTL surface PD-1 expression decreases,while HBV-specific CTL level increases.The changes in low virus load group are much more remarkable.
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Objective To investigate the specific anti-tumor immune response induced by dendritic cells (DCs) transfected with total RNA of human pancreatic cancer Capan-2 cells.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) derived from six patients with pancreatic cancer.Total RNA of Capan-2 cells and MUC4 mRNA were transfected into DCs by electroporation.The survival rate of transfected DCs was determined by MTT method and the expression of MUC4 mRNA in DCs was detected by Western blotting.The activity of cytotoxic T lymphocyte cells (CTLs) induced by DCs transfected with total RNA of Capan-2 cells were evaluated by IFN-γ ELISA and the induction of specific CTL response to the killing effect on pancreatic cancer cell in vitro were measured by 51 Cr standard cytotoxicity test.Results The survival rate of DCs transfected with total RNA of Capan-2 cells (DC-Capan-2-total RNA) showed a decrease in a time dependent manner and the survival rate was reduced to 60.81% after transfection for 96 h.The survival rate of MUC4 mRNA transfection DCs (DC-MUC4 mRNA) was stable at around 80%.The difference of DCs surviral rate between the two groups was statistically significant (P < 0.05).The amount of MUC4 protein expression of DC-Capan-2-total RNA was significantly lower than that of DC-MUC4 mRNA (P <0.05).The quantity of CTL IFN-γ release induced by DC-Capan-2-total RNA was (89.34 ± 3.85)U/mL and the quantity of DC-MUC4 mRNA induced CTL IFN-γ release was (21.77 ± 21.77)U/ml There was statistically significant between the two groups (P <0.05).In addition,the specific CTLs induced by DC-Capan-2-total RNA could effectively identify and kill the HLA-A2+/MUC4+ Capan-2 and the HLA-A2+/MUC4-PANC 1 cells,and could not effectively identify and kill the HLA-A2 /MUC4-MiaPaCa-2 cells and the HLA-A2-/MUC4 + AsPC-1 cells.Conclusions A more pronounced CTL anti-tumor immune response can be induced by DCs transfected with total RNA of Capan-2 cells compared with a single tumor associated antigen,but it is limited by MHC class Ⅰ antigen presented.
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Viral infections remain a major cause of morbidity in patients with immunodeficiency,such as recipients of hematopoietic stem cell transplantation (HSCT).Adoptive transfer of donor-derived virusspecific cytotoxic T lymphocytes is a strategy to restore virus-specific immunity to prevent or treat viral diseases and has been tested in the clinical setting for more than 20 years.The 55th ASH annual meeting reported lots of basic and clinical experience about CTL and summarized lots of trails to evaluate it,which will give us some new thought about the antivirus therapy after HSCT.
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Objective To investigate the ability of induction of specific cytotoxic T lymphocytes (CTL) stimulated by dendritic cells (DCs) co-transfected with MUC1 and survivin mRNA of human pancreatic cancer,and to provide the experimental basis for the treatment of human pancreatic cancer with multi-epitope DC vaccine.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) of 6 patients with pancreatic cancer.Human pancreatic cancer cell line MiaPaCa-2 was routinely cultured,after being transcripted and amplified by RT-PCR,MUC1 and survivin mRNA were co-transfected or individually transfected into DCs by electroporation,and they were named as DC-MUC1,DC-survivin,DC-MUC1 + survivin.The expression of MUC1 and survivin mRNA in DCs were detected by real-time PCR.The survival rate of transfected DCs were determined by MTT method.The lymphocyte proliferation ability was evaluated by mixed cell culture method.The Th1 cytokine releasing of antigen-specific CTLs were measured by ELISA assay.Results Mature DCs were obtained,the positive expression rates of surface markers CD40,HLA-DR,CD83 and CD86 were 34.31%,50.21%,89.17% and 73.62%,respectively.The expression amount of MUC1 mRNA of DC-MUC1 was 36.24 ± 5.17,and the expression amount of survivin mRNA of DC-survivin was 34.53 ± 4.02,while the expression amounts of MUC1,survivin mRNA of DC-MUC1 + surviving were 31.79 ±4.26 and 14.67 ± 2.96,which were significantly lower than that in individual transfection group (P < 0.05).The survival rate of DC-MUC1 + surviving was decreased in a time dependent manner,which was significantly lower than that in individual transfection group (about 50.21% vs 80% at 24 h,P <0.05).When DC/T cells ratio was 1∶ 10,1∶ 20,the autologous T cell proliferation index of MUC1 and survivin mRNA in co-transfection DC group was significantly higher than that in individual transfection group (P < 0.05) ;when DC/T cells ratio was 1∶ 40,1∶ 80,the difference of proliferation index was not statistically significant.When DC/T cells ratio was 1∶ 10,after 14 d culture,the expressions of IL-2 in DC-MUC1,DC-survivin,DC-MUC1 + surviving were (892.73 ± 32.9),(713.62 ± 56.37),(1884.37 ± 95.21) pg/ml,and the expressions of granzyme B were (501.62 ± 12.30),(203.84 ± 12.55),(1193.15 ± 86.04) pg/ml ; and the expressions of IFN-γ were (981.50 ± 47.82),(696.05 ± 41.66),(2237.94 ± 189.55) pg/mL.The corresponding values in DC-MUC1 + surviving group were significantly higher than those in individual transfection group (P < 0.05) ; while the difference of IL-10 was not statistically significant.Conclusions DCs co-transfected with MUC1 and survivin mRNA have a stronger ability to stimulate specific CTL in vitro than individual antigen loaded DCs.
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Viral infections remain the common cause of morbidity and mortality in immunocompromised patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT).Virus antigens can be recognized by cytotoxic T-lymphocytes (CTL) in the context of HLA class Ⅰ molecules.T-cell-based immunotherapies are now being included in the clinical practice of transplant recipients to prevent and treat viral infections and complications associated with CMV,AdV and EBV.Here,the current status and future feasibility of CTL immunotherapy for virus infections after allo-HSCT are discussed.
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Objective To evaluate the effect of calcipotriol on the proliferation of and cytokine secretion by melanocytes and perilesional CD8+ cytotoxic T lymphocytes (CTLs) from patients with vitiligo.Methods Melanocytes isolated from abdominal skin and CD8+ CTLs from perilesional skin of patients with vitiligo were subjected to successive culture in vitro.After several passages,the melanocytes and CD8+ CTLs were cultured alone or in combination with or without the presence of various concentrations of calcipotriol for 24 to 48 hours.MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt) method was used to evaluate the proliferative activity of cells,enzyme-linked immunosorbent assay to determine the levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the culture supematant of cells,flow cytometry to detect cell apoptosis.Some co-cultured melanocytes and CTLs were treated with calcipotriol of 10-8 mol/L and anti-IL-6 antibody of various concentrations (0,1,2,2.5,5,10 mg/L) for two days followed by enumeration of cells.The concentrations of 108 and 10-9 mol/L (calcipotriol) were chosen for relevant tests.Results There was a marked apoptosis in MCs after coculture with CD8+ CTLs.The 24-hour treatment with calcipotriol of 104 and 10-9 mol/L had no obvious effect on the proliferation of melanocytes cultured alone (both P > 0.05),but accelerated the proliferation of melanocytes cocultured with CTLs (both P <0.05) as well as that of CD8+ CTLs cultured alone or in combination with melanocytes (all P <0.05).A statistical decrease was observed in IL-6,TNF-α and IFN-γlevels in the supernatant of cocultured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone,and calcipotriol of 10-9 mol/L intensified the decrease in supernatant IL-6 level (t =2.89,P <0.05),but no statistical changes were noted for the level of TNF-α or IFN-γin the supernatant of the coculture system after treatment with calcipotriol of 104 or 104 mol/L compared with that before treatment (both P > 0.05).In the coculture system pretreated with calcipotriol of 10-8 mol/L,the number of CD8+ CTLs significantly decreased (t =3.15,P <0.05),whereas that of melanocytes significantly increased (t =3.53,P <0.05) after the treatment with anti-IL-6 antibody of 5 mg/L.Conclusions Perilesional CD8+ CTLs have a specific cytotoxic effect on melanocytes,and calcipotriol may inhibit the cytotoxic effect of CD8+ CTLs by suppressing the secretion of IL-6,which may partly explain the therapeutic mechanism of calcipotriol for vitiligo.
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Objective To investigate the anti-tumor immune response induced by human pancreatic cancer mucin 4 mRNA and human telomerase reverse transcriptase (hTERT) mRNA cotransfected dendritic cells (DC),and to provide the experimental evidences for the treatment of pancreatic cancer with multi-epitope loaded DC vaccine.Methods DC were isolated from peripheral blood mononuclear cells (PBMC) of six patients with HLA-A2+ pancreatic cancer and cultured.Mucin 4 mRNA and hTERT mRNA were transcripted and amplified in vitro,which were transfected into DC separately or in order by eleetroporation.DC were cultured for 48 hours.The expressions of mucin 4 and hTERT in DC were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot.The survival rates of transfected DC were determined by methylthiazolyl tetrazolium (MTT) method.The cytotoxic T lymphocyte (CTL) activation induced by mucin 4 mRNA and hTERT mRNA transfected DC were evaluated by interferon (IFN)-γ release assays (enzyme linked immunosorbent assay) method.The cytotoxicity of CTL induced by mucin 4 mRNA and hTERT mRNA transfected DC in pancreatic cancer cell lines MiaPaCa-2,Capan-2,AsPC-1 and Pane-1 was measured by 51Cr standard cytotoxicity test.Student t test was performed for statistical analysis.Results After in order transfection of mucin 4 mRNA and hTERT mRNA for 48 h,the relative quantity of the expression of mucin 4 and hTERT in DC were 30.09±5.24 and 12.87±3.36,which were lower than the relative quantity of the expression in DC transfected separately (38.54±6.21 and 36.35±5.03,t=3.469,6.721,both P<0.05).After transfected in order for 96 hours,the survival rate of DC decreased to 52.17%,which was lower than that of DC transfected separately (around 80%).The quantity of IFN-γ releasing of specific CTL induced by mucin 4 mRNA and hTERT mRNA cotransfected DC was (32.57±2.01) U/mL in 24 hours,which was higher than that of CTL induced by DC transfected with mucin 4 mRNA ((23.06±4.74) U/mL) or hTERT mRNA ((16.82±3.67) U/mL) separately (1=5.092,7.141,both P<0.05).After co-transfected with mucin4 mRNA and hTERT mRNA,DC could effectivly induce HLA-A2+/mucin 4+/hTERT+ specific CTL immune responses,however there was no significant cytotoxicity in HLA-A2+ pancreatic cancer cells.Conclusion The induction of CTL anti-tumor immune response by DC co-transfected with mucin4 mRNA and hTERT mRNA is more significant compared with that by single antigen loaded DC.
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Objective To evaluate the protective effects of tea polyphenols against the destruction of melanocytes by CD8+ T cells from vitiligo patients.Methods Skin tissue was resected from the margin of vitiligo lesions followed by the isolation and culture of CD8+ T lymphocytes,and from the normal skin of vitiligo patients followed by the isolation and culture of melanocytes.Flow cytometry was carried out to evaluate the purity of CD8+ T cells.The melanocytes were cocultured with the CD8 + T cells at different ratios followed by the evaluation of killing effect of CD8+ T cells.Various concentrations (200 and 400 μg/ml) of tea polyphenols were added into the co-culture system of CD8+ T cells and melanocytes at a ratio of 5 ∶ 1 followed by an additional culture of 48 hours.Then,flow cytometry was performed to detect the apoptosis in melanocytes in the coculture system.Results CD8+ T lymphocytes were successfully obtained from the marginal area of vitiligo lesions with a purity of more than 90%,which highly expressed the antigens CD137 and CD69.The coculture with CD8+ T cells markedly accelerated the apoptosis in melanocytes,while the accelerative effect was inhibited by tea polyphenols of 200 and 400 μg/ml.Conclusions The CD8+ T cells infiltrating the edge of vitiligo lesions display a potential destructive effect on autologous melanocytes from vitiligo patients,and tea polyphenols have a protective effect against the destruction of melanocytes by CD8+ T cells.