EBV转化慢性乙型肝炎患者B淋巴母细胞系的建立

目的体外建立慢性乙型肝炎患者永生化的B淋巴母细胞系(LCL)。方法用EB病毒感染自慢性乙型肝炎患者外周血中分离的单个核细胞(PBMC),加入CpGDNA免疫调节基序以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性。光学显微镜下观察LCL的形态特征,利用流式细胞术分析LCL细胞膜表面分子CD19和CD23的表达水平。结果46例患者PBMC经EBV感染4周后,42例转化成永生化B淋巴母细胞系,成功率为91.3%。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。结论转化后的LCL保持了成熟B淋巴细胞的生物学特性,可作为体外研究HBV特异性免疫应答的刺激细胞和靶细胞。     

Epstein—Barr病毒（EBV）及其介导的细胞永生化

Epstein-Barr病毒（EBV）因其与众多肿瘤的相关性以及其在体外能使B淋巴细胞永生化形成淋巴母细胞样细胞系（LCLs)的特性而成为近年来细胞永生化研究的热点之一。研究表明，EBV主要是通过其潜伏蛋白激活细胞因子与其受体相互作用的途径而使细胞永生化的，但是病毒蛋白的具体作用机制尚无定论。本综述了EBV生物学特性方面近年来的研究成果，并以LCLs为切入点初步探讨了EBV介导的细胞永生化。     

094 EBV转化的B淋巴母细胞系的建立及应用

EBV转化B淋巴细胞后,形成永生化的B淋巴母细胞系(B-LCL).B-LCL在体外能无限增殖,广泛应用于医学生物学研究领域.本文就EBV对B淋巴细胞的转化机制、建立B-LCL应注意的问题以及B-LCL在细胞与分子遗传学、免疫学、EBV相关肿瘤的免疫治疗等方面的进展作一简要综述.     

EBV转化的B淋巴母细胞系的建立及应用

EBV转化B淋巴细胞后 ,形成永生化的B淋巴母细胞系 (B LCL)。B LCL在体外能无限增殖 ,广泛应用于医学生物学研究领域。本文就EBV对B淋巴细胞的转化机制、建立B LCL应注意的问题以及B LCL在细胞与分子遗传学、免疫学、EBV相关肿瘤的免疫治疗等方面的进展作一简要综述     

EBV转化的B淋巴母细胞系的建立及应用

EBV转化B淋巴细胞后，形成永生化的B淋巴母细胞系(B-LCL)。B-LCL在体外能无限增殖，广泛应用于医学生物学研究领域。本文就EBV对B淋巴细胞的转化机制、建立B-LCL应注意的问题以及B-LCL在细胞与分子遗传学、免疫学、EBV相关肿瘤的免疫治疗等方面的进展作一简要综述。     

永生化EB病毒转化B细胞对肝癌细胞抗原的提呈作用

目的 建立EB病毒(Epstein—Barr virus，EBV)转化的永生化B细胞(命名为LEBV)并研究其对肝癌细胞抗原的提呈作用。方法 从人外周血中分离出外周血单个核细胞(Perpheral blood mononuclear cell,PBMC)，用等体积的EBV上清混合培养4周以上建立永生化EBV转化B细胞(LEBV)，用流式细胞计数仪(FACS)检测其细胞表面功能相关分子CD86、CN40、HLA-DR和HLA-ABC的表达情况。LEBV与自体淋巴细胞和肝癌细胞抗原共培养3d，结束培养前12h加入37kBq／孔^3H—标记的胸腺嘧啶(^3H—TdR)，收获细胞，用液体闪烁计数仪测定cpm。结果 培养3周以上即培养出EBV转化的永生化B细胞，可连续培养1年以上。经FACS检测，细胞表面分子CD86、CD40、HAL-DR和HLA—ABC的表达率分别为74％、91％、83％和86．7％。该细胞接触肝癌细胞抗原后刺激自体淋巴细胞增殖的能力增强。结论 EBV转化的永生化B细胞对肝癌细胞抗原具有提呈作用。     

Epstein-Barr病毒(EBV)及其介导的细胞永生化

Epstein- Barr病毒 (EBV)因其与众多肿瘤的相关性以及其在体外能使 B淋巴细胞永生化形成淋巴母细胞样细胞系 (L CL s)的特性而成为近年来细胞永生化研究的热点之一。研究表明 ,EBV主要是通过其潜伏蛋白激活细胞因子与其受体相互作用的途径而使细胞永生化的 ,但是病毒蛋白的具体作用机制尚无定论。本文综述了 EBV生物学特性方面近年来的研究成果 ,并以 L CL s为切入点初步探讨了 EBV介导的细胞永生化。     

表达特定免疫球蛋白可变区抗原的人B细胞系建立和其生物学特征

目的建立表达特定免疫球蛋白可变区抗原的人B细胞系。方法在29例EBV转化B淋巴细胞建立永生化淋巴母细胞系中,PCR-SSP方法检测HLA-A*0201表达的细胞系,将表达HLA-A*0201的细胞系通过有限稀释培养方法获得单克隆细胞系ZP-1。分别用PCR方法、流式细胞仪和染色体核型分析检测细胞IgHV家族、细胞表面免疫表型CD分子和染色体核型表达。结果 HLA-A*0201(+)的细胞,经单克隆化培养后获得3个细胞克隆,将其中之一命名为ZP-1。PCR检测显示ZP-1细胞表达IgHV3家族;流式细胞仪检测显示ZP-1细胞表达CD19、lambda链等B细胞免疫表型;核型分析结果显示,ZP-1细胞为正常女性染色体结构。结论成功获得表达特定免疫球蛋白可变区抗原的人B细胞系,为研发B细胞相关疾病的疫苗和分子靶向药物提供良好的实验基础。     

儿童原发性免疫缺陷病患者EBV转化的B淋巴母细胞系的建立及应用

目的: 建立原发性免疫缺陷病人外周血永生化B细胞系,保存原发性免疫缺陷病人特有的基因组资源,为进一步研究原发性免疫缺陷病提供丰富实验材料.方法: 采用密度梯度离心法分离PBMC,加入环孢菌素A、 EB病毒共培养以转化外周血淋巴细胞.结果: 成功建立14例原发性免疫缺陷病人外周血永生化B细胞系,建株成功率100%;对已建立的永生细胞株冻存和复苏,成功率为100%;转化前后制备细胞染色体和G显带核型分析无明显改变.结论: 经EB病毒成功转化外周血B淋巴细胞为永生细胞株,为进一步开展原发性免疫缺陷病的基础研究提供了资料.     

CpG ODN抑制EB病毒复制的体外研究

目的:探讨CpG寡脱氧核苷酸链(CpG ODN)体外对EB病毒(EBV)复制的抑制作用.方法:CpG ODN体外预处理人外周血单个核细胞(PBMC)后感染EBV,用荧光定量PCR和ELISA分别检测培养后PBMC中EBV拷贝数和培养上清液中γ干扰素(IFN-γ)的水平,了解不同剂量、不同时间CpG ODN预处理PBMC中EBV复制和PBMC产生IFN-γ之间的关系.结果:(1)不同浓度CpG ODN预处理组的EBV拷贝数均低于未处理组( P <0.05),其中10 mg/L CpG ODN预处理组EBV拷贝数最低,为(3.85±0.37)×10 8基因拷贝/L;(2)CpG ODN预处理48 h组的EBV拷贝数(11.32±0.83)×10 8基因拷贝/L低于其余各预处理时间组的EBV拷贝数( P<0.05);(3)不同浓度CpG ODN预处理组培养上清液中的IFN-γ均高于PBMC组和PBMC+EBV组( P <0.05),其中 10 mg/L CpG ODN处理组IFN-γ量最高(66.27±6.29)ng/L;(4)CpG ODN预处理48 h的PBMC+CpG ODN+EBV组培养上清液中IFN-γ(51.74±4.09)ng/L高于其余各预处理时间组和PBMC组及PBMC+EBV组( P <0.05).结论:CpG ODN作为一种新型的免疫调节剂,在体外能有效抑制EBV复制,其作用机制可能与诱导PBMC产生IFN-γ有关.     

Circulating CD4+ CD25+ regulatory T cells correlate with chronic hepatitis B infection

Circulating CD4+ CD25+ regulatory T cells (Tregs) have been demonstrated to maintain immunotolerance and suppress the antigen-specific or antigen-non-specific T-cell responses, but their role in chronic hepatitis B (CHB) infection in humans has not been well characterized. In this study, we analysed the frequency and phenotypic characteristics of CD4+ CD25+ Tregs in patients of different hepatitis B virus (HBV) infection status, and investigated the effect of Tregs on antiviral immune responses in CHB patients, and the mechanism of this effect. A total of 137 subjects, including 79 CHB patients, 26 asymptomatic HBV carriers (ASCs), 12 acute hepatitis B (AHB) patients and 20 healthy controls, were enrolled in the study. We found that the frequency of CD4+ CD25(high) Tregs in AHB patients was comparable to that in healthy controls, while it was significantly increased in CHB patients. CD4+ CD25+ Tregs produced interleukin (IL)-10 but little or no interferon (IFN)-gamma under anti-CD3 stimulation. In CHB patients, the frequency of CD4+ CD25(high) Tregs positively correlated with serum viral load, and the Tregs were capable of suppressing the proliferation and IFN-gamma production of autologous peripheral blood mononuclear cells (PBMC) mediated by HBV antigen stimulation in vitro. However, combined administration of anti-programmed death-1 (PD-1) and anti-cytotoxic lymphocyte antigen-4 (CTLA-4) monoclonal antibody slightly enhanced the cellular proliferation and significantly increased the IFN-gamma production of PBMC cocultured with Tregs at a ratio of 2:1. Thus, the frequency of circulating CD4+ CD25+ Tregs is increased in patients with CHB, and this may play an important role in viral persistence by modulating virus-specific immune responses.     

Taurocholic acid inhibits the response to interferon-α therapy in patients with HBeAg-positive chronic hepatitis B by impairing CD8+ T and NK cell function

Pegylated interferon-alpha (PegIFNα) therapy has limited effectiveness in hepatitis B e-antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. However, the mechanism underlying this failure is poorly understood. We aimed to investigate the influence of bile acids (BAs), especially taurocholic acid (TCA), on the response to PegIFNα therapy in CHB patients. Here, we used mass spectrometry to determine serum BA profiles in 110 patients with chronic HBV infection and 20 healthy controls (HCs). We found that serum BAs, especially TCA, were significantly elevated in HBeAg-positive CHB patients compared with those in HCs and patients in other phases of chronic HBV infection. Moreover, serum BAs, particularly TCA, inhibited the response to PegIFNα therapy in HBeAg-positive CHB patients. Mechanistically, the expression levels of IFN-γ, TNF-α, granzyme B, and perforin were measured using flow cytometry to assess the effector functions of immune cells in patients with low or high BA levels. We found that BAs reduced the number and proportion and impaired the effector functions of CD3⁺CD8⁺ T cells and natural killer (NK) cells in HBeAg-positive CHB patients. TCA in particular reduced the frequency and impaired the effector functions of CD3⁺CD8⁺ T and NK cells in vitro and in vivo and inhibited the immunoregulatory activity of IFN-α in vitro. Thus, our results show that BAs, especially TCA, inhibit the response to PegIFNα therapy by impairing the effector functions of CD3⁺CD8⁺ T and NK cells in HBeAg-positive CHB patients. Our findings suggest that targeting TCA could be a promising approach for restoring IFN-α responsiveness during CHB treatment.     

Dendritic cells expand Epstein Barr virus specific CD8+ T cell responses more efficiently than EBV transformed B cells

Adoptive transfer of Epstein Barr virus (EBV) specific cytotoxic T lymphocytes (CTLs) has been successfully applied in the treatment of EBV associated post-transplant lymphoproliferative disease (PTLD). In most studies EBV transformed B cells (LCLs) have been used for the induction of EBV specific T cell lines. Application of this approach to other EBV associated tumors is difficult, because LCLs focus T cell expansion toward immunodominant EBV antigens that are not expressed in EBV associated Hodgkin's lymphoma and nasopharyngeal carcinoma. Therefore, we compared dendritic cells (DCs) with LCLs for CD8+ T cell stimulation against dominant and subdominant EBV antigens. DCs expanded tenfold more EBNA3A and LMP2 specific CD8+ T cells than LCL and also stimulated EBV specific CTL from PTLD patients. Both, DCs and LCLs stimulations led to the expansion of high affinity T cells, capable to target EBV transformed B cells. While LCLs and DCs expressed MHC class I and II products at similar levels, DCs showed a higher expression of costimulatory and adhesion molecules. This resulted in more efficient T cell conjugate formation with DCs than with LCLs. We propose the use of DCs for stimulation of EBV specific T cells in active or passive immunotherapy of EBV associated malignancies.     

Characteristics of Epstein-Barr virus transformed B cell lines from patients with Alzheimer's disease and age-matched controls.

The characteristics of B cell lines isolated from patients with Alzheimer's disease (AD) and age-matched controls were investigated after having been transformed by Epstein-Barr virus (EBV). After isolation of mononuclear blood cells and in vivo or in vitro EBV infection, 35 and 21 lymphoblastoid cell lines (LCLs) were generated from 19 patients with AD (mean age 79.4 years) and 21 age-matched controls (mean age 80.0 years), respectively. B lymphocytes from AD patients were immortalised more easily than those from controls; the percentage of in vitro EBV infected LCLs (B95-LCLs) obtained in the AD group was significantly higher (76.2% versus 33.3% in the control group) and the mean time required for establishment was significantly lower (20.2 and 21.9 days versus 26.7 and 60.9 days in the control group). The EBV receptor and surface immunoglobulin (Ig) analyses showed no difference between the two groups. The expression of Epstein-Barr early antigens (EA) and viral capsid antigens (VCAs) revealed a tendency to higher viral replication in LCLs from AD patients; however, VCA expression remained limited to a small number of cells and did not affect overall cell growth. Finally, qualitative and quantitative differences were observed in the pattern of Ig production. Whereas spontaneously established LCLs from AD patients were generally monoclonal (80% of LCLs versus 33% in the control group), B95-LCLs were all polyclonal and secreted more IgM and IgA than those from controls; the mean IgM level was significantly higher in B95-LCLs from the AD group. These results suggest that B cells derived from AD patients seemed to be less differentiated than cells from age-matched controls.     

Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones.

We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells (PBMCs) were cultured with EBV, and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+, CD4+, and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-superinfected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+, CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-superinfected and non-superinfected LCLs. Proliferative and cytotoxic responses to allogenic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections.     

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.     

HBcAg-induced upregulated 4-1BB ligand on B cells contributes to B-cell hyperactivation during chronic hepatitis B infection

During the natural history of chronic hepatitis B infection (CHB), the function of B cells is still obscure. Several limited studies have suggested that B cells are highly active in patients with CHB. In the present study, we reported that the 4-1BB ligand (4-1BBL) expression on B cells was significantly higher in patients with CHB than that in healthy subjects, meanwhile, the patients with CHB had higher serological IgG levels. Further, after being stimulated with sCD40L or hepatitis B core antigen (HBcAg), B cells had higher levels of 4-1BBL. After being cocultured with 4-1BBL+ B cells, the expressions of CD69 and 4-1BB on CD4+ T cells were significantly higher than that cocultured with 4-1BB− B cells. Cytokines including interleukin (IL)-2, IL-4, and IL-6 were significantly higher in the supernatant from 4-1BBL+ B cells coculture group than those from coculture group of 4-1BBL− B cell group, respectively; while IFN-γ and TNF-α in cocultured supernatant of 4-1BBL+ B cell group were significantly lower. Consistently, the total IgG levels in culture supernatant were significantly higher in 4-1BBL+ B cell group. Thus, hyperactive status of B cells in patients with CHB could be partially derived from the higher 4-1BBL expression on B cells triggered by HBcAg. 4-1BBL signaling pathway is involved in B cells activation, and further regulates B cell-T cell interaction by modulating the cytokines secretion, which might be critical in B cells dysfunction during CHB infection.     

慢性乙型肝炎病人HBcAg特异性CTL的体外诱导及活性

目的 从慢性乙型肝炎病人外周血单个核细胞（PBMC）中诱导和扩增乙型肝炎病毒核心抗原（HBcAg）特异性细胞毒性T淋巴细胞（CTL）。方法 含乙型肝炎病毒重组逆转录病毒载体转染病人自身永生化B细胞作为抗原递呈细胞，联合重组HBcAg和IL-2，从病人PBMC中诱导和扩增HBcAg特异性CTL，^51Cr释放法检测其细胞毒活性。结果 此方法诱导的TCL可以有针对性的杀伤靶细胞，而不能够杀伤空载体转染及未经转染的B细胞。结论 所建立的方法可以在慢性乙型肝炎病人的PBMC中诱导出HBcAg特异性CTL活性。