活动性慢性乙型肝炎患者特异性 CD8＋T 细胞转录因子 T-bet 表达及临床意义

目的：分析转录因子 T-bet 在活动性慢性乙型肝炎患者外周血 HBV 特异性 CD8＋ T 细胞中的表达及其临床意义。方法用 HBV 肽五聚体结合流式细胞仪检测 HLA-A2阳性的活动性慢性乙型肝炎患者和急性乙型肝炎患者外周血 HBV 特异性 CD8＋ T 细胞中 T-bet 的表达。比较分析 HBV 特异性 CD8＋ T 细胞中 T-bet 表达在急性、慢性乙型肝炎之间的差异,并进一步分析慢性乙型肝炎患者 HBV 特异性 CD8＋ T 细胞中 T-bet 表达与 HBV DNA 水平、ALT 水平以及临床预后之间的相关性。结果急性乙型肝炎患者外周血 HBV 特异性 CD8＋ T 细胞中 T-bet 表达水平显著高于活动性慢性乙型肝炎患者。活动性慢性乙型肝炎患者及 CHB 患者外周血 HBV 特异性 CD8＋ T 细胞中 T-bet 表达水平与 HBV DNA 水平及 ALT 水平无明显相关性,HBV 特异性 CD8＋ T 细胞中 T-bet 表达水平高的活动性慢性乙型肝炎患者发生HBeAg 血清转换率显著升高。结论活动性慢性乙型肝炎患者特异性 CD8＋ T 细胞转录因子 T-bet 表达与 HBV 感染的免疫控制密切相关。     

不同临床类型HBV感染者外周血T淋巴细胞亚群的变化

目的研究HBV感染者外周血T淋巴细胞亚群的变化及其临床意义.方法在33例慢性乙型肝炎轻度、50例中度、32例重度、64例肝硬化、51例慢性乙型肝炎肝衰竭和30例健康人,采用特异性荧光抗体标记法和流式细胞仪检测外周血T淋巴细胞亚群.结果慢性乙型肝炎重度、乙型肝炎肝硬化和慢性乙型肝炎肝衰竭患者与正常对照组比,CD3^＋、CD4^＋、CD8^＋细胞数和CD4^＋/CD8^＋比值显著下降（P〈0.05）,以慢性乙型肝炎肝衰竭下降最显著;HBeAg阴性患者外周血CD3^＋、CD4^＋和CD8^＋细胞数较HBeAg阳性患者显著减少（P〈0.05）;HBV DNA载量与患者外周血CD3^＋、CD4^＋、CD8^＋细胞数和CD4^＋/CD8^＋比值均无明显相关性.结论严重的HBV感染者存在不同程度的细胞免疫功能低下及免疫调节紊乱,细胞免疫功能下降程度与病毒复制水平无明显相关.     

慢性乙型肝炎患者外周血 CD8＋T 淋巴细胞功能的变化

目的：探讨慢性乙型肝炎患者 CD8＋ T 淋巴细胞功能的变化。方法利用主要组织相容性复合物（MHC）-抗原肽四聚体标记技术检测 HBV 的特异性 CD8＋ T 淋巴细胞,并检测其细胞因子的分泌能力,分析其与病毒学指标及临床生化指标的关系。结果人类白细胞相关抗原（HLA）-A 基因型为 A02或 A24的42例患者中,有11例患者至少检出一种被四聚体识别的 HBV 特异性 CD8＋ T 淋巴细胞,白细胞介素2（IL-2）及γ（IFN-γ）干扰素的分泌能力相对于其自身的外周血总 CD8＋ T 淋巴细胞显著降低（t＝14．231、10．450,P ＜0．01）。检出抗原特异性 CD8＋ T 淋巴细胞的患者,其血清ALT 的水平较阴性患者高（t＝2．306,P ＜0．05）,而 HBV DNA 水平却相对低（t＝－4．447,P ＜0．001）。结论慢性乙型肝炎患者外周血的抗原特异性 CD8＋ T 淋巴细胞的功能存在明显的缺陷,可能与病毒抗原的持续刺激导致功能耗竭有关。     

慢性HBV感染者CD8^＋ T细胞免疫反应的研究

目的观察不同慢性HBV感染者外周血CD8^＋ T细胞的细胞内毒性颗粒和细胞因子的表达水平。方法用rHBcAg作为刺激剂,采用免疫荧光染色结合流式细胞术分析慢性乙型肝炎中度、重度患者以及无症状HBV携带者外周血CD8^＋ T细胞的IFN-γ/IL-4和穿孔素/颗粒酶B表达水平。结果慢性乙型肝炎重度患者CD8^＋T细胞的穿孔素及颗粒酶B百分率显著高于正常对照（P〈0.01）和HBV携带者（P〈0.05,P〈0.01）,中度患者显著高于正常对照（P〈0.05）。2组慢性乙型肝炎IFN-γ＋/CD8^＋ T细胞（Tc1）百分率均高于正常对照和HBV携带者（P〈0.05）。HBV携带者CD8^＋T细胞的穿孔素和颗粒酶B表达以及Tc1百分率与健康对照差异无统计学意义。IL-4^＋/CD8^＋ T细（Tc2）表达各组间差异无统计学意义（P〉0.05）。结论 CD8^＋ T细胞中穿孔素/颗粒酶B表达率的增加以及Tc1细胞增加与慢性HBV感染的肝损伤有关。     

结核杆菌热休克蛋白质70作为乙型肝炎核心抗原细胞毒性T淋巴细胞表位肽载体的研究

目的探讨结核杆菌热休克蛋白质70（TB．HSP70）作为乙型肝炎病毒（HBV）核心抗原细胞毒性T淋巴细胞（CTL）表位肽载体，诱导HBV特异性免疫应答的可能性。方法体外观察重组TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物诱导慢性乙型肝炎患者外周血淋巴细胞增殖以及HBV特异性细胞毒活性；以Balb／c小鼠为体内研究对象，行流式细胞术分析免疫后小鼠外周血和脾细胞中CD4^＋与CD8^＋T淋巴细胞及自然杀伤（NK）细胞的比率，并观察能否诱导HBV特异性免疫保护作用。结果体内外研究表明TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物能有效地诱导HBV特异性细胞毒活性，体内能激活CD4^＋与CD8^＋T淋巴细胞及NK细胞增殖。在体内，TB．HSP70-CTL融合蛋白质较复合物能更有效地激活免疫应答，其杀伤率为28．9％。CD8^＋T淋巴细胞在脾细胞中比率为43．9％，NK细胞为13．6％。而单纯的TB．HSP70和CTL表位肽并不能有效地引起机体的免疫应答。结论TB．HSP70能够作为乙型肝炎核心抗原CTL表位肽的载体，提高小分子表位肽的免疫原性。     

急性乙型肝炎HBcAg特异性CTL克隆建立及初步分析

目的建立急性乙型肝炎（乙肝）患者的HBcAg特异性CD8^＋T细胞克隆。方法一例急性乙肝患者经用序列特异引物聚合酶链反应分型技术,测得其人类白细胞抗原（human leukocyte antigen,HLA）-A基因型为1101,其外周血单个核细胞采用重组HHBcAg和合成的HLA—A＊1101限制性表位肽（HBV croe88—96）刺激,并以有限稀释法对活化的T细胞进行克隆化,建立HBcAg特异性的CD8^＋T细胞克隆。用免疫荧光染色、流式细胞术以及乳酸脱氢酶释放实验鉴定所获克隆。结果获得13株HBcAg＋特异性CD8^＋咖胞克隆（6侏为Tc1,3株为Tc2,4株为Tc0）。其中12株具有明显的特异性细胞毒沂陛（37．5％-85．5％,效／靶比为2．5：1）,1株（A10株,Tc0）细胞毒活性仅为10．2％。结论应用HLA—A＊1101限制性表位肽（HBV croe88—96）刺激急性乙肝患者的外周血单个核细胞,建立了13株肽表位特异性的CD8^＋T细胞克隆,为进一步研究打下基础。     

乙型肝炎相关肝癌患者乙型肝炎病毒不同抗原肽特异性CD3+CD8+人白细胞抗原-A2+细胞表达

目的 探讨乙型肝炎相关肝细胞癌(HCC)患者外周血单个核细胞(PBMC)中HBV不同抗原肽特异性CD3+ CD8+人白细胞抗原( HLA)-A2+细胞的表达.方法 从4例HLA-A2阳性乙型肝炎相关HCC患者外周血中分离PBMC,分别与HBV抗原肽sAg( FLLTRILTI、GLSPTVWLSV、WLSLLVPFV),HBV eore(FLPSDFFPSV)和HBV pol(FLLSLGIHL)及抗-CD3-pacific blue、抗-CD8-异硫氰酸荧光素共育,流式细胞仪分析HBV/HLA-A2-CD3-CD8阳性细胞,克隆培养,筛选出克隆培养的抗HBV T淋巴细胞;再与含有HBV的肝癌细胞株HepG2( HLA-A2+)共育,ELISA法检测其分泌IFN-γ水平.结果 4例患者体内受GLSPTVWLSV肽诱导的特异性抗HBV T淋巴细胞占所有CD8+细胞的1.44％±0.04％,高于FLLTRILTI肽的0.68％±0.08％、FLPSDFFPSV肽的1.06％±0.09％、FLLSLGIHL肽的0.56％±0.04％和WLSLLVPFV肽的0.46％±0.08％(t=0.001,P＜0.05).将GLSPTVWLSV/HLA-A2获得的HBV/HLA-A2 PentamerCD3-CD8阳性细胞克隆,获得2株抗HBV CD8 T淋巴细胞,与负荷GLSPTVWLSV肽段的HepG2 (HLA-A2+)细胞共育,CD8 T淋巴细胞分泌较高水平的IFN-γ.结论 乙型肝炎相关HCC患者外周血中存在针对不同HBV抗原肽的特异性抗HBV CD8 T淋巴细胞,其表达量与抗原肽段有关.     

慢性乙型肝炎患者PB MC中H BV特异性IL-21表达及其对CD8^＋T淋巴细胞功能的影响

目的：探讨慢性乙型肝炎患者外周血单个核细胞（PBMC）中 HBV特异性IL-21的表达及其对CD8＋T淋巴细胞功能的调节作用。方法以 HBVc18-27（10μg/mL）多肽刺激46例不同临床类型慢性乙型肝炎患者PBMC。ELISA检测上清液IL-21水平。在 HBVc18-27（10μg/mL）多肽和IL-21（50 U/mL）或IL-2（100 ng/mL）培养条件下,应用 MHC-肽五聚体结合流式细胞术检测PBMC中 HBV特异性CTL细胞频数变化;免疫磁珠分离纯化免疫耐受期和免疫激活期慢性乙型肝炎患者PBMC中CD8＋T淋巴细胞,以 HBcAg（10μg/mL）预刺激CD8-PBMC 5 h后,采用Transwell 小室共培养技术,在阻断IL-21通路条件下,应用实时PCR检测CD8^＋T淋巴细胞IFN-γmRNA的表达。结果 HBV特异性IL-21在免疫控制期和免疫激活期慢性乙型肝炎患者P B MC中表达水平显著高于免疫耐受期患者,而在免疫控制期和免疫激活期两组之间无显著差异。与 PBS 对照组相比,IL-21组 HBV 特异性 CTL 细胞频数明显增高,差异有统计学意义（P＜0．01）,而与IL-2组相比差异无统计学意义。以IL-21抗体阻断IL-21通路后,CD8^＋T 淋巴细胞IFN-γmRNA 表达水平显著下降,差异有统计学意义。结论免疫控制期和免疫激活期慢性乙型肝炎患者PBMC 均可生成一定水平的 HBV 特异性IL-21,并能增强外周CD8＋T淋巴细胞的增殖和IFN-γ表达水平。     

慢性H BV感染者外周血中Fo xp3的表达及IFN-γ分泌的变化

目的：观察不同类型 HBV 感染者和健康者 PBMC 中 Foxp3的表达和 IFN-γ含量的变化情况,探讨在HBV感染慢性化中CD4^＋CD25^＋调节性T细胞发挥的作用。方法采用RT-PCR 检测不同类型 HBV 感染者和健康人外周血中Foxp3的表达情况。应用 ELISA 的方法检测外周血血清中 IFN-γ的含量。结果急性乙型肝炎患者Foxp3的表达与健康对照组比较差异无统计学意义,而血清中 IFN-γ的含量有所增加（P＞0．05）;慢性乙型肝炎患者Foxp3的表达均明显高于健康对照组（P＜0．01）,血清中IFN-γ的含量则有所减少。结论慢性乙型肝炎患者 CD4^＋CD25^＋调节性T细胞活性较健康对照组增强,且Th1细胞活性和体液免疫均受到抑制,CD4＋CD25＋调节性T 细胞在HBV感染慢性化中起着一定的作用。     

慢性乙型肝炎树突状细胞表型和功能的变化与免疫耐受

目的 观察慢性乙型肝炎病毒（HBV）感染者树突状细胞（DC）形态和功能的改变。方法 从13例慢性乙型肝炎患者和11例健康人外周血中分离和培养DC,观察DC的形态,用流式细胞仪检测DC表面标记HLA－DR、CD1a、CD80和CD86的表达,用^3H-TdR掺入法测定DC诱导混合性淋巴细胞反应（MLR）的能力。结果 正常DC较慢性乙型肝炎患者在形态上更为典型,不规则,HLA－DR、CD80和CD86分子的表达水平较高（P＜0.05）,诱导MLR的能力较强（P＜0.05）。结论 慢性乙型肝炎患者外周血DC处于不完全成熟状态,其免疫刺激能力较低。     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

重型肝炎患者乙型肝炎病毒特异性细胞毒性T淋巴细胞应答功能研究

目的 用主要组织相容性复合物（MHC）抗原肽四聚体（Tetramer）流式细胞技术，分析乙型重型肝炎患者外周血中特异性细胞毒性T淋巴细胞（CTL）应答状况，并探讨其临床意义。方法 采用Tetramer流式细胞技术检测乙型重型肝炎患者外周血中受人类白细胞抗原Ⅰ类分子限制的三类特异性CD8^＋CTL细胞数量；采用酶联免疫吸附斑点试验技术，测定经特异性乙型肝炎病毒（HBV）肽段诱导培养的特异性CTL表达膜内细胞因子IFNγ、TNFα、IL-4和IL—10等的水平；采用Promega CytoTox96非放射性细胞毒试验技术，测定经特异性HBV肽段诱导培养的特异性CTL杀伤靶细胞能力。结果 急性乙型重型肝炎组外周血中针对HBVcore18-27表位的特异性CTL数量高于慢性乙型重型肝炎组（P〈0．05），而低于急性乙型肝炎组（P〈0．05）；急性乙型重型肝炎组表达干扰素γ和肿瘤坏死因子α较慢性乙型重型肝炎组高（P值均〈0．05）；急性乙型重型肝炎组HBVcore18—27特异性CTL裂解靶细胞能力高于慢性乙型重型肝炎组（P〈0．05）。结论 急性乙型重型肝炎患者特异性CTL应答作用增强，而慢性乙型重型肝炎患者特异性CTI。应答缺乏。急性乙型重型肝炎患者外周血特异性CTL持续存在，可能与促进病毒清除等相关。     

HLA–B27–restricted CD8+ T cell response to cartilage‐derived self peptides in ankylosing spondylitis

Objective

Several hypotheses have been proposed to explain the strong association between HLA–B27 and ankylosing spondylitis (AS). Among these, the arthritogenic peptide theory proposes that certain B27 subtype alleles bind specific arthritogenic peptide(s) due to their unique amino acid anchor residues. Cartilage antigens have been discussed as candidate targets for the immune response in AS. The recognition of HLA–B27–peptide complexes by self‐reactive CD8+ T cells might contribute to joint‐specific tissue damage. Therefore, we investigated the presence of autoreactive CD8+ T cells specific for cartilage‐derived peptides in patients with AS.

Methods

An HLA–B27–binding prediction program and a proteasome‐cutting prediction program for the human 20S proteasome were used to screen 18 human cartilage proteins for potentially immunogenic nonamer peptides. The peptides identified were used to stimulate peripheral blood mononuclear cells from 20 HLA–B27–positive patients with AS and synovial fluid (SF) mononuclear cells from 7 HLA–B27–positive patients with AS. Activation of T cells was measured by antigen‐specific intracellular cytokine staining and quantified by flow cytometry.

Results

From the screening analysis, we identified 121 nonamer peptides. Of these, 1 peptide derived from type II collagen and 1 from type VI collagen were stimulatory for peripheral blood CD8+ T cells in only 1 of 20 patients. However, in 4 of 7 SF samples the same type VI collagen–derived nonamer peptide stimulated SF CD8+ T cells, but none of the other peptides was stimulatory. This CD8+ T cell response could be blocked by an anti–HLA–B27 antibody, confirming an HLA–B27–restricted immune response.

Conclusion

Our findings suggest that cartilage‐directed cellular autoimmunity might play an important role in joint‐specific tissue damage in patients with AS. Future research is necessary to determine whether the identified peptide is of pathogenetic relevance.

     

Programmed death-1 expression is associated with the disease status in hepatitis B virus infection

AIM： TO define the potential role of programmed death-i/programmed death-ligand （PD-1/PD-L） pathway in different hepatitis B virus （HBV） infection disease status; we examined the expression of PD-1 on antigen specific CD8＋T cells in peripheral blood of patients with chronic hepatitis B （CriB） and acute exacerbation of hepatitis B （AEHB） infection.
METHODS： The PD-1 level on CD8＋ T lymphocytes and the number of HBV specific CD8＋ T lymphocytes in patients and healthy controls （HCs） were analyzed by staining with pentameric peptide-human leukocyte antigen2 （HLA2） complexes combined with flow cytometry. Real-time quantitative polymerase chain reaction （PCR） was used to measure the serum HBV- DNA levels.
RESULTS： The level of PD-1 expression on total CD8＋ T cells in CHB patients （13.86% ± 3.38%） was significantly higher than that in AEHB patients （6.80%± 2.19%, P 〈 0.01） and healthy individuals （4.63% ± 1.23%, P 〈 0.01）. Compared to AEHB patients （0.81% ± 0.73%）, lower frequency of HBV-specific CD8＋ T cells was detected in chronic hepatitis B patients （0.37% ± 0.43%, P 〈 0.05）. There was an inverse correlation between the strength of HBV-specific CD8＋ T-cell response and the level of PD-1 expression. Besides, there was a significant positive correlation between HBV viral load and the percentage of PD-1 expression on CD8＋ T cells in CriB and AEHB subjects （R = 0.541, P 〈 0.01）. However, PD-1 expression was not associated with disease flare-ups as indicated by alanine aminotransferase （ALT） levels （R = 0.066, P 〉 0.05）.
CONCLUSION： Our results confirm previous reports that HBV specific CD8＋T-cell response in the peripheral blood is more intense in patients with AEHB than in chronic hepatitis B wlth persistent viral infection. Moreover, there is a negative correlation between the level of PD-1 and the intensity of virus specific CD8＋ T cell response.     

Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen

BACKGROUND & AIMS: Impaired T-cell reactivity is believed to be the dominant cause of chronic hepatitis B virus (HBV) infection. We characterized HBV-specific T-cell responses in chronic hepatitis B surface antigen carriers who received bone marrow from HLA-identical donors with natural immunity to HBV and seroconverted to antibody to hepatitis B surface antigen. METHODS: T-cell reactivity to HBV antigens and peptides was assessed in a proliferation assay, the frequency of HBV core- and surface-specific T cells was quantified directly by ELISPOT assays, and T-cell subsets were analyzed by flow cytometry. RESULTS: CD4+ T-cell reactivity to HBV core was common in bone marrow donors and the corresponding recipients after hepatitis B surface antigen clearance, whereas none reacted to surface, pre-S1, or pre-S2 antigens. Furthermore, CD4+ T cells from donor/recipient pairs recognized similar epitopes on hepatitis B core antigen; using polymerase chain reaction for the Y chromosome, the recipients' CD4+ T lymphocytes were confirmed to be of donor origin. The frequency of core-specific CD4+ and CD8+ T cells was several-fold higher than those specific for surface antigen. CONCLUSIONS: This study provides the first evidence in humans that transfer of hepatitis B core antigen-reactive T cells is associated with resolution of chronic HBV infection. Therapeutic immunization with HBV core gene or protein deserves further investigation in patients with chronic hepatitis B.     

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.     

Toll样受体2介导的Th17细胞活化在HBV感染中的作用

目的探讨乙型肝炎患者外周血单个核细胞(PBMC)中Toll样受体2(TLR2)与Th17细胞的相关性,为阐述HBV感染诱导炎症应答机制提供理论和实验依据。方法选取2012年7月-2013年7月唐都医院感染科门诊和住院的34例乙型肝炎初治患者,其中24例慢性乙型肝炎和10例急性乙型肝炎;另外选取健康对照者10例,分离PBMC,利用HBV C基因型Envelope区肽段(特异性)或佛波酯联合伊屋诺霉素(非特异性)刺激,流式细胞术检测TLR2表达及Th17细胞百分比。进一步用TLR2的激动剂刺激PBMC,检测Th17细胞变化情况。组间比较采用Kruskal-Wallis H检验。结果在非特异性刺激条件下,Th17细胞在慢性乙型肝炎患者体内的百分比(4.08±1.78)%显著高于急性乙型肝炎患者(1.85±1.28)%及健康对照者(2.09±0.53)%(P=0.000 9、0.000 4),而TLR2+及IL-17A+TLR2+的表达在急、慢性乙型肝炎患者与健康人外周血中差异均无统计学意义(P均0.05)。在特异性刺激条件下Th17及TLR2的表达在慢性乙型肝炎患者体内的表达显著高于急性乙型肝炎组[(5.45±1.61)%vs(3.20±1.13)%;(5.19±3.18)%vs(1.88±1.30)%],差异具有统计学意义(P=0.000 6、0.000 6)。加入TLR2激动剂后急、慢性乙型肝炎患者体内Th17细胞的比例均显著升高,但在急性乙型肝炎患者中,刺激前后差异无统计学意义(P0.05)。结论 TLR2可以直接影响Th17细胞的应答,从而促进乙型肝炎中炎症应答反应。     

The impact of HBV-DNA fluctuations on virus-specific CD8+ T cells in HBeAg+ chronic hepatitis B patients treated with a steroid and lamivudine

Restoration of anti-viral immune response may be a requisite for sustained virological response to treatment in chronic hepatitis B patients. Over a 13-month period, we examined the dynamics of hepatitis B virus (HBV)-specific CD8+ cells in six human leucocyte antigen (HLA)-A2+ hepatitis B e antigen (HBeAg)+ 'immunotolerant' chronic hepatitis B patients treated sequentially with corticosteroid and lamivudine. Our results show that the combination treatment did not result in a sustained restoration of anti-viral specific CD8+ cells in five of the six patients studied. However, HBV-specific CD8+ cells, despite being severely compromised, were not totally deleted. Paradoxically, steroid treatment was not associated with inhibition but with a minimal increase of the HBV-specific CD8 response, and we observed that nucleocapsid-specific CD8 responses were not rescued by stable and prolonged inhibition but became detectable after rapid rebounds of HBV replication. In most patients, the transient and minimal restoration of HBV-specific immunity was not associated with clinical benefits. Our results describe a dynamic relationship between HBV-specific CD8+ cells and HBV-DNA values, that could potentially be used for a better design of HBV treatment in HBeAg+ 'immunotolerant' chronic hepatitis B patients.     

CD4+CD25+Foxp3+调节性T细胞在慢性乙型肝炎患者中的作用

目的 探讨CD4+CD25+Foxp3+调节性T细胞与乙型肝炎慢性化和病毒清除之间的关系.方法 收集慢性活动性乙型肝炎(CAH)患者19例、HBV携带者(AsC)21例、HBV感染恢复者12例和健康对照者15例.通过流式细胞术分析外周血CD4+CD25+Foxp3+T细胞的表型和频率,磁珠分选(MACS)CD4+CD25+T细胞,实时荧光定量PCR方法 分析Foxp3 mRNA基因在CD4+CD25+T细胞的表达水平.统计学处理采用单因素方差分析或非参数检验.结果CAH或AsC组外周血CD4+CD25+Foxp3+T细胞频率以及CD4+CD25+T细胞中Foxp3 mRNA的表达水平显著高于健康对照组或HBV感染恢复者(F=6.8,F=3.72,均P<0.05).免疫组织化学染色发现,CAH患者肝组织Foxp3'T细胞浸润累积较对照组明显增高,但AsC较CAH减少.HBeAg阳性患者(包括CAH和AsC)CD4'CD25'T细胞频率显著高于HBeAg阴性患者(t=2.3,P<0.05),抗-HBe阴性患者显著高于抗-HBe阳性患者(t=2.4,P<0.05).CD4+CD25+Foxp3+T细胞频率与慢性乙型肝炎患者血清中HBV病毒载量存在正相关(r=0.56,P<0.01).结论 慢性乙型肝炎患者CD4+CD25+Foxp3+调节性T细胞异常与乙型肝炎慢性化和病毒清除有关.     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

BACKGROUND/AIMS: The aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia. METHODS: HLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24+ patients with acute hepatitis B. RESULTS: An HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117-125 and P756-764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24+ patients with acute hepatitis B, respectively. CONCLUSIONS: We identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.