HBV感染不同病程中病毒载量与肝功能相关指标及免疫功能的变化分析

目的 探讨HBV感染者外周血肝功能主要指标、T细胞亚群以及病毒载量在不同病程中的变化及其临床意义.方法 170例HBV感染患者根据病程分成急性乙肝组(18例)、慢性乙肝组(73例)、肝硬化组(41例)及肝癌组(38例),正常对照组40例.用全自动生化分析仪和流式细胞仪检测各组肝功能相关指标及T细胞亚群含量;采用实时荧光定量PCR法检测HBV-DNA含量.结果 四组实验组与正常对照相比谷丙转氨酶(ALT)、谷草转氨酶(AST)均升高,差异具有统计学意义(P＜0.05),且以急性乙肝升高最为显著,慢性乙肝次之,组间比较可见慢性乙肝患者组比急性乙肝患者组ALT、AST有所下降,且差异具有统计学意义(P＜0.05),而肝癌患者组和肝硬化组之间则变化不显著,差异无统计学意义(P＞0.05);碱性磷酸酶ALP的升高见于肝癌患者组,慢性乙肝患者组ALP水平较低;各组总蛋白(TP)均在正常参考范围内.四组实验组外周血CD3+T细胞均比正常对照组低,其中肝癌组、肝硬化患者组降低最为明显,CD4+T细胞比例上升见于急性乙肝组,而CD8+T细胞的比例在急性乙肝患者组中最低.外周血中HBV病毒核酸载量升高的最为明显的是慢性乙肝患者组,而急性乙肝患者组、肝硬化患者组、肝癌患者组的HBV-DNA水平比较低,以急性乙肝最低,各组与其相比均具有统计学差异(P＜0.05).结论 HBV不同病程中患者的肝功能相应指标、T细胞亚群表达、HBV-DNA含量存在显著差异,联合检测这些项目可为临床正确评估患者的病情及病程提供相应指导,为疾病转归的判断及治疗提供可靠的依据.     

乙型肝炎患者特异性细胞免疫功能检测的初步研究

目的 通过酶联免疫斑点法检测乙型肝炎（乙肝）患者特异性细胞免疫功能，初步研究各型乙肝患者特异性细胞免疫功能的差异。方法 选择急、慢性乙肝、肝炎肝硬化（乙型）、乙肝病毒（HBV）既往感染，接种乙肝疫苗后血清乙肝病毒学标志中仅表面抗体阳性及未接种过乙肝疫苗、未感染过HBV、血清HBV标志全部阴性者共6组研究对象，每组4例，采用酶联免疫斑点法（ELISPOT）检测其外周血单个核细胞中γ-干扰索分泌细胞的数量。结果 急性乙肝患者、慢性乙肝患者及急性乙肝患者、肝炎肝硬化患者外周血单个核细胞γ-干扰素分泌细胞数量明显不同（P=0．0209及P=0．0211）。结论 通过ELISPOT检测乙肝患者外周血单个核细胞γ-干扰索分泌细胞的数量可以了解乙肝患者特异性细胞免疫功能。急性乙肝患者特异性细胞免疫功能明显强于慢性乙肝患者及肝炎肝硬化患者。     

慢性乙型肝炎患者HBV特异性细胞毒性T细胞PD-1的表达研究

目的 检测慢性乙型病毒性肝炎患者外周血中HBV特异性CD8 细胞毒性T淋巴细胞表面细胞程序性死亡受体1(programmed cell death 1,PD-1)的表达情况.方法 采集慢性乙型肝炎患者外周血,直接离体情况下利用MHC-I-肽-五聚体技术标定HBV表位特异性细胞毒性T淋巴细胞以及荧光抗体标记细胞表面PD-1分子,经流式细胞仪检测分析.结果 在慢性乙型肝炎患者,HBV核心抗原18-27特异性CTL表达PD-1上调,达(79.0±12.5)%,显著高于总CD8 T细胞(27.7±14.8)%,以及CMV特异性CTL(20.6±5.9)%.结论 慢性乙肝患者HBV特异性CTL高表达PD-1分子,可能与慢性乙肝患者HBV特异性CTL功能低下密切相关.     

慢性乙肝患者外周血HBcAg18-27V／I变异体特异性淋巴细胞分析

目的分析不同HBcAg18-27V／I变异体特异性淋巴细胞的频数在慢性乙肝患者外周血中的差异。方法收集44例ALT≥2×ULN的慢性乙肝患者外周血淋巴细胞，通过PCR方法分析感染者病毒学背景（HBV基因型、HBcAg18-27V／I、HLA-A2基因型），应用流式细胞仪分别检测HBcAg18-27V-Tetramer（＋）CD8（＋）和HBcAg18-27I-Tetramer（＋）CD8（＋）的淋巴细胞频数。结果华南地区慢性乙肝患者主要为HLA-A2基因型（22／44）；感染的HBV主要为B基因型（35／44）。其中HBcAg18-27为I变异体的占优势（43／44），在A2阳性组中，HBcAg18-27I-Tetramer（＋）CD8（＋）的淋巴细胞频数与HBcAg18-27V-Tetramer（＋）CD8（＋）的淋巴细胞频数（0．41±0．52 VS 0．28＋0．43）比较差异有统计学意义（P〈0．001）。结论在HBV基因型B／C为主的地区．应用HBcAg18-27V表位进行细胞免疫学研究而不考虑HBV病毒学背景的结果可能导致一定的偏差。     

不同亚群CD8＋T细胞细胞毒作用对慢性乙型肝炎的影响

目的观察慢性乙型肝炎不同分期及干扰素-a（interferon-a,IFN-a）治疗前后外周血不同亚群CD8＋T细胞细胞毒作用的变化,探讨其在慢性乙型肝炎发病机理及抗病毒治疗中的作用。方法采集20例慢乙肝免疫耐受期患者和20例免疫清除期患者IFN—a治疗前后的外周全血,采用流式细胞仪检测CD8^high和CD8^lowT细胞的颗粒酶B（Granzyme B,GrB）和溶酶体相关膜蛋白-1（Lysosome—associated membrane protein．1,LAMP-1,CD107a）表达变化,并进行分析。结果（1）慢乙肝患者免疫清除期不同亚群CD8＋T细胞GrB和CD107a的表达水平均高于免疫耐受期;（2）不同分期慢乙肝患者CD8^lowT细胞GrB和CD107a的表达水平均高于CD8^highT细胞;（3）IFN—a治疗后CD8^highT细胞的GrB和CD107a表达水平和治疗前相比具有升高趋势,而CD8^lowT细胞反而具有下降趋势;（4）不同亚群CD8＋T细胞的GrB和CD107a表达水平与HBV—DNA载量在免疫耐受期呈正相关,而在免疫清除期呈负相关。结论（1）不同亚群CD8＋T细胞的GrB和CD107a分子在慢乙肝疾病进程及抗病毒治疗中均起重要作用,CD8^lowT细胞的细胞毒作用强于CD8^highT细胞,而CD8^highT细胞的细胞毒效应在IFN—a治疗过程中逐渐增强。（2）不同亚群CD8＋T细胞的GrB和CD107a分子水平与HBV病毒载量的相关性在一定程度上可以提示机体免疫应答与病毒之间的关系。     

乙型肝炎病毒感染者外周血CD4＋T细胞CD25-CD127-表达的检测及临床意义

目的 检测乙型肝炎病毒（ HBV）感染者外周血CD4＋T细胞表面CD25 - CD127 -的表达情况及临床意义.方法 用流式细胞术比较分析53例慢性乙肝患者、53例HBV携带者和26例正常对照人群CD4＋T细胞表面CD25 - CD127 -的表达情况,并对20例用干扰素治疗的HBV-DNA阳性慢性乙肝患者随访.结果 ①与正常对照组比较,慢性乙肝患者、HBV携带者CD4＋ CD25 -CD127 -T细胞均显著升高,两者比较有统计学差异（Q =4.559,P＜0.05;Q=6.230,P＜0.05）;②HBV- DNA阳性患者（n=77） CD4＋ CD25 - CD127 -T细胞显著低于HBV- DNA阴性患者（n=29）,两者比较有统计学意义（t =2.290,P=0.024）;③与治疗前比,慢性乙肝患者干扰素治疗12周后CD4＋CD25- CD127 -T细胞显著降低,两者比较差异有统计学意义（t =2.469,P=0.024）.结论 乙型肝炎病毒感染者外周血CD4＋ CD25 - CD127 -T细胞与病毒的感染和清除相关,外源性干扰素可降低CD4＋ CD25 - CD127 -T细胞.     

HBV抗原肽-HLA-A*2402复合物四聚体的构建及初步检测应用

目的：构建2种HBV抗原肽-HLA-A＋2402复合物四聚体，并初步用该2种四聚体检测针对不同抗原肽特异性的细胞毒性T细胞（CTL）。方法：原核高效表达HLA-A＋2402-BSP和β2m蛋白后，分别与乙型肝炎病毒抗原肽P01756-764和Corell7-125在体外复性折叠成可溶性的HLA-A＋2402抗原肽复合物单体，经BirA酶作用并通过凝胶过滤层析法纯化复合物单体，分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成2种HBV抗原肽四聚体。最后进行流式细胞术检测。结果：Dot-ELISA和ELISA检测显示获得了2种具有天然构象的生物素化的HBV抗原肽-HLA-A＋2402复合物单体。结论：构建的2种四聚体可以检测乙型肝炎感染者体内特异性的CTL。     

HBV转基因小鼠CD4＋CD25＋调节性T细胞免疫抑制功能的研究

目的通过对HBV转基因小鼠CD4＋、CD4＋CD25＋调节性T细胞数量和免疫抑制功能的研究,探讨CD4＋CD25＋调节性T细胞在乙肝免疫耐受中的作用。方法用流式细胞术对12只HBV转基因小鼠和12只正常小鼠外周血CD4＋、CD4＋CD25＋调节性T细胞的频率进行检测;磁珠分选小鼠脾CD4＋CD25-T细胞和CD4＋CD25＋T细胞,分为HBsAg刺激组和ConA刺激组体外单独和共培养,ELISA方法检测诱生的细胞因子IL-2。结果与正常小鼠比较,HBV转基因小鼠外周血CD4＋、CD4＋CD25＋调节性T细胞数量差异无统计学意义（P〉0.05）。HBsAg刺激组,CD4＋CD25-T细胞单独或共培养,HBV转基因小鼠CD4＋CD25-T细胞诱生IL-2水平显著低于正常小鼠（P〈0.01）;ConA刺激组,HBV转基因小鼠CD4＋CD25-T细胞诱生IL-2水平与正常小鼠相比则差异无统计学意义（P〉0.05）;两组小鼠CD4＋CD25-T细胞单独培养时诱生IL-2水平均显著高于共培养（P〈0.01）。结论与正常小鼠比较,HBV转基因小鼠CD4＋、CD4＋CD25＋调节性T细胞数量和免疫抑制功能差异无统计学意义,但T细胞水平对乙肝病毒存在特异性免疫耐受。CD4＋CD25＋调节性T细胞可抑制CD4＋、CD8＋T细胞。     

转基因表达HBV抗原特异性细胞毒性T淋巴细胞受体

目的 通过逆转录病毒介导HBV抗原特异性细胞毒性T细胞(CTL)的T细胞受体(TCR)转基因表达,初步观察其结合活性.方法 从HLA-A2阳性急性乙肝患者外周血中诱导、分选、克隆和扩增HBV抗原特异性CTL;提取细胞RNA,用RT-PCR、5'-RACE和OVER-LAP PCR等方法获取TCR的α和β链编码基因;构建TCR重组逆转录病毒,介导特异性TCR分别在人Jurkat T细胞和HLA-A2阳性健康人CD8 T淋巴细胞上表达.结果 从1例HLA-A2阳性急性乙肝患者样本中分别获得了2组TCR Vα、Vβ配对,分别命名为α21β13、α15β13,包装的重组逆转录病毒滴度为(1.5 ～5.0)×105 IU/mL,用针对目标TCR的特异性Vβ链抗体(抗Vβ13 TCR-PE)和HLA-A2限制性表位特异性五聚体(pentamer)进行免疫荧光染色,重组TCR在T细胞表面获得表达:其中在Jurkat细胞上转入的Vβ13链表达细胞占1.06％ ～2.25％,在HLA-A2阳性健康人T细胞上Vβ13阳性细胞和pentamer阳性细胞分别占到1.03％ ～2.06％和1.05％ ～1.12％,在HLA-A2阴性健康人T细胞上Vβ13阳性细胞和pentamer 阳性细胞均低于0.05％.结论 通过逆转录病毒介导可以使HBV特异性CTL TCR获得转基因表达,具有结合HLA-A2限制性表位的活性.     

基于HCA587的HLA-A2限制性表位肽的体内免疫原性研究

目的检测来源于HCA587的HLA-A2限制性表位肽免疫HLA-A2转基因小鼠后产生细胞应答的水平,筛选出体内具有免疫原性的HLA-A2限制性表位肽。方法将来源于HCA587的候选HLA-A2限制性肽与MHC-Ⅱ类限制性肽HBV-core128-140、不完全弗氏佐剂、Cp G ODN1826联合应用皮下免疫HLA-A2转基因小鼠,用酶联免疫斑点实验(ELISpot)检测小鼠脾细胞表位肽特异性细胞免疫应答的水平,流式细胞术检测表位肽特异性的CD8+T细胞比例。结果在检测的4个表位肽中,p248-256诱导HLA-A2转基因小鼠产生的细胞免疫应答最强,HLA-A2-H-2Kb-p248-256四聚体染色进一步表明p248-256免疫后可产生p248-256特异性的CD8+T细胞。结论来源于HCA587的HLA-A2限制性表位肽中,p248-256具有较强的体内免疫原性。     

T cell receptor usage of virus-specific CD8 cells and recognition of viral mutations during acute and persistent hepatitis B virus infection

T cells specific for a single viral epitope, but using different T cell receptors, should have flexibility in their epitope recognition to protect the infected host against the emergence of viral escape mutants. Therefore, polyclonality of the hepatitis B virus (HBV)-specific cytotoxic T lymphocyte response has been hypothesized to be a major determinant in the control of infection. We analyzed the Vbeta chain composition of the core 18-27-specific CD8 cells in acute and persistently HBV-infected patients using HLA-A2 tetrameric complexes and a panel of Vbeta antibodies. Different T cell receptors were utilized by core 18-27-specific CD8 cells both in patients with acute and chronic infection. The functional ability of these epitope-specific T cells to respond to potential viral mutations was then tested. The polyclonal HBV-specific CD8 response present in patients with acute hepatitis displayed a limited efficiency to recognize mutations introduced within the epitope. The ability of core 18-27-specific CD8 to tolerate epitope mutations was found only during persistent HBV infection. The data suggest that although a clonally heterogeneous CD8 response can be largely inhibited by the occurrence of single epitope mutations in primary HBV infection, preferential selection of T cells able to counteract the emergence of viral mutations can occur during persistent infection.     

HBcAg-specific CD8 T cells play an important role in virus suppression,and acute flare-up is associated with the expansion of activated memory T cells

We analyzed the prevalence and longitudinal fluctuation of hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection using an HLA-A2–HBc18-27 tetramer. Thirty-five HLA-A2-positive patients with chronic HBV infection were divided into 17 HBe antigen (HBeAg)-positive and 18 anti-HBe antibody (anti-HBe)-positive patients. Five HLA-A2-positive normal subjects, five HLA-A2-negative patients with chronic HBV infection, and two HLA-A2-positive patients with acute HBV infection were included as controls. HBc18-27-specific CD8 T cells (c18-27-CD8Ts) were detected at a significantly higher prevalence in patients with anti-HBe (6/18) than in those with HBeAg (1/17), and their frequency reached 0.28% of the total CD8 T cells. The prevalence was significantly higher in patients with HBV DNA below 4.0 log genome equivalents (LGE)/ml (5/12) than in those with HBV DNA above 4.0 LGE/ml (2/23). The frequency of c18-27-CD8Ts was consistently higher in liver-infiltrating lymphocytes, ranging from 0.18 to 1.28%, than in autologous peripheral blood lymphocytes. Longitudinal analysis of patients with acute flare-up demonstrated that the elevation of alanine aminotransferase (ALT) was intimately associated with the expansion of c18-27-CD8Ts. Phenotypic analysis revealed that most c18-27-CD8Ts during acute flare-up expressed HLA-DR and CCR5, while those during low-ALT periods showed low expression. Furthermore, most liver-infiltrating c18-27-CD8Ts were positive for HLA-DR and CCR5, suggesting selective recruitment of activated c18-27-CD8Ts into the liver. In conclusion, HBV-specific CD8 T cells play an important role in the suppression of virus replication, and acute flare-up is associated with the expansion and activation of HBV-specific memory cells.     

HBcAg‐Specific IL‐21‐Producing CD4+ T Cells are Associated with Relative Viral Control in Patients with Chronic Hepatitis B

Function exhaustion of specific cytotoxic CD8+ T cell in chronic virus infection partly results from the low levels of CD4 help, but the mechanisms by which CD4 help T cell required to control hepatitis B virus infection are not well understood. In this study, we investigated the role of interleukin‐21‐producing CD4+ T cell response in viral control of hepatitis B virus infection. HBcAg‐specific interleukin‐21‐producing CD4+ T cells in blood were detected in patients with hepatitis B virus infection. Patients with acute hepatitis B had greater HBcAg‐specific interleukin‐21‐producing CD4+ T cells in blood compared with chronic hepatitis B patients, and there was no statistical significance between immune active chronic hepatitis B patients and inactive healthy carrier patients for these cells, whereas frequencies of these cells negatively correlated with HBV DNA levels but positively correlated with HBc18‐27‐specific IFN‐γ‐producing CD8+ T cells. Moreover, interleukin‐21 sustained HBc18‐27‐specific CD8+ T cells in vitro, and interleukin‐21 production by HBcAg‐specific IL‐21‐producing CD4+ T cells of acute hepatitis B patients enhanced IFN‐γ and perforin expression by CD8+ T cells from chronic hepatitis B patients. Our results demonstrate that HBcAg‐specific interleukin‐21‐producing CD4+ T cell responses might contribute to viral control by sustaining CD8+ T cell antiviral function.     

Frequencies of epitope-specific cytotoxic T lymphocytes in active chronic viral hepatitis B infection by using MHC class I peptide tetramers

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are thought to play key roles in viral control and liver damage. We have used HLA-A*02 tetramer complex to human HBV core 18-27 (Tc 18-27), envelope 183-191 (Te 183-191), envelope 335-343 (Te 335-343), and polymerase 575-583 (Tp 575-583) epitopes to characterize HLA class I-restricted CD8+ T cells in active chronic HBV infection. The frequencies of specific epitopes circulating tetramer+ cells were determined in whole-blood samples by analysis of flow cytometry. The correlation of HBV epitope-specific CTL, between viral replication and liver damage, was analyzed by multiple regression. Our data shown that HBV-specific CD8+ T cells can be easily detected in peripheral blood of active chronic HBV infections. No significant correlation was found between either the frequency of HBV-specific CD8+ T cells and the viral load, or the frequency of HBV-specific CD8+ T cells and the levels of alanine transaminase. These results suggest that the existence of epitope-specific HBV CTLs are not directly correlated to hepatocyte injury, and the frequencies of HBV-specific T cells are not determinant of immune-mediated protection in chronic HBV infection.     

The high prevalence of the I27 mutant HBcAg18-27 epitope in Chinese HBV-infected patients and its cross-reactivity with the V27 prototype epitope

HBcAg18-27 (FLPSDFFPSV, V27 epitope) is a dominant HLA-A2-restricted epitope in hepatitis B virus (HBV)-infected patients. So far, the occurrence of the epitope has not been assessed in China, where the prevalence of chronic HBV infection is high. In this report, we sequenced the HBV core gene in 105 Chinese patients with chronic HBV infection. Approximately 93.3% (98/105) of the core genes that were sequenced contained mutations with amino acid substitution at position 27 of the core protein: a mutation from a valine to an isoleucine (V27I). The mutant peptide (FLPSDFFPSI, I27) was found to bind to the HLA-A2 molecule with high affinity and elicit specific cytotoxic T lymphocyte (CTL) responses in acutely infected hepatitis B patients. In CTL assays using I27-specific pentamer staining, the V27 epitope showed a cross-reactive T cell response specific for the I27 epitope, but not vice versa. These findings provide important insights for the design of HBcAg18-27-based vaccines in the future.     

乙肝病毒感染不同阶段抗原肽特异性细胞毒性T 淋巴细胞的初步研究

目的:为了阐明HBV 感染不同阶段机体免疫功能的不同。方法:分别构建了我国最高频等位基因HLA鄄A*0201 三种常见抗原肽表位(HBVcore18-27、pol575-583、env335-343)四聚体复合物,并用这些四聚体检测HBV 感染外周血单个核细胞(PBMCs)中HLA-A*0201 限制性抗原特异性CD8+ T 细胞的频率、功能及CD127 的表达。结果:在大多数自限性HBV感染者的PBMCs 中抗原特异性CD8+ T 细胞的频率和增殖能力都高于慢性HBV 感染者。在低病毒载量的免疫低复制期抗原特异性CD8+ T 的数量和在高病毒载量、肝脏损害免疫清除期及免疫耐受期抗原特异性CD8+ T 细胞频率是相似的,同病毒定量及ALT 水平无显著相关性。慢性HBV 感染者抗原特异性CD8+ T 细胞增殖能力和病毒滴度呈反比。在自限性HBV 感染患者抗原特异性CD8+ T 细胞分泌IFN 的功能明显高于慢性HBV 患者,在免疫耐受期基本丧失产生细胞因子的能力;HBV 感染后不同免疫状态的HBsAg 水平不同,免疫耐受期最高,其次为免疫清除期、再活动期,低复制期的最低。而且随着HBsAg 水平下降,同HBV DNA 的相关性也逐渐下降。慢性HBV 感染者中CD8+ CD127+ T 细胞要低于对照组及自限性感染组,特别是在HBeAg 阳性的免疫耐受期及免疫清除期组患者中CD8+ CD127+ T 细胞比例更低。结论:慢性HBV 感染中抗原特异性CD8+ T细胞的频率不是决定免疫应答的唯一因素,慢性乙肝患者体内记忆性的抗原特异性CD8+ T 细胞并未被完全清除或缺失,这为治疗性疫苗和免疫恢复治疗提供了可行性。     

Reciprocal changes of naïve and effector/memory CD8+ T lymphocytes in chronic hepatitis B virus infection

Persistent viruses, such as cytomegalovirus or human immunodeficiency virus, cause major perturbations of CD8+ T-lymphocyte subpopulations. To test whether chronic infection with hepatitis B virus (HBV) could also be responsible for such modifications, we analyzed the expression of CD27, CD28, CCR7, and perforin in blood CD8+ T lymphocytes. In comparison to healthy subjects and patients recovering from acute hepatitis B, chronic hepatitis B patients showed higher percentages of na?ve CD8+ T lymphocytes (CD45RA+CD27+CD28+), and lower percentages of intermediately-differentiated CD27+CD28?CD8+ T cells. The late differentiated CD45RA+CD27?CD28? subset was also present in a large percentage in some patients, but no statistically significant difference with healthy controls was observed. Removal from the circulation of intermediately-differentiated CD8+ T lymphocytes may occur during chronic HBV infection, favoring the recruitment of na?ve cells. This may result in impairment of the generation of functionally-competent memory cells, and an inability to achieve control of HBV replication.     

HBV特异性CTLs表面CD244和PD-1共表达与慢性乙型肝炎严重程度的相关性

 目的：探讨慢性乙型肝炎（CHB）患者外周血乙型肝炎病毒（HBV）特异性细胞毒性T淋巴细胞（CTLs）表面CD244与程序性死亡蛋白1（PD-1) 的共表达模式及其与疾病进展的关系。方法：采集81例CHB患者和14例健康者外周血,分离外周血单个核细胞（PBMC）;利用主要组织相容性复合体I（MHC-I）-肽五聚体技术标定HBV表位特异性CTLs;荧光抗体标记细胞表面CD244与PD-1分子,流式细胞术检测。结果：CHB患者CD244与PD-1在HBV特异性CTLs表面共表达较总CD8⁺ T细胞表面明显升高（67.48%±17.16% vs 14.01%±7.97%,P<0.01）;HBV感染的不同临床类型中,轻中度的CHB患者HBV特异性CTLs表面CD244与PD-1共表达水平较免疫耐受者明显升高(72.05%±16.86% vs 53.11%±18.05%,P<0.05),肝衰竭组CD244与PD-1共表达水平较轻中度CHB下调（63.11%±13.87% vs 72.05%±16.86%,P<0.05）并伴随干扰素γ（IFN-γ）分泌功能的恢复（30.95%±20.29% vs 13.63%±10.46%,P<0.01）。结论：CHB患者HBV特异性CTLs细胞表面高共表达CD244与PD-1分子,CD244与PD-1共表达在CHB不同临床类型中有差异,并与IFN-γ分泌水平呈负相关,提示CD244可能与CHB患者HBV特异性CTLs功能低下及CHB疾病进展相关。     

Hepatitis B virus core antigen epitopes presented by HLA-A2 single-chain trimers induce functional epitope-specific CD8+ T-cell responses in HLA-A2.1/Kb transgenic mice

The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward core antigen has been shown to affect the outcomes of hepatitis B virus (HBV) infection. Since single-chain trimers (SCT) composed of peptide epitope beta2-microglobulin (beta2m) and major histocompatibility complex (MHC) class I heavy chain covalently linked together in a single molecule have been shown to stimulate efficient CTL responses, we investigated the properties of human leucocyte antigen (HLA)-A2 SCTs encoding the HBV core antigen (HBcAg) epitopes C(18-27) and C(107-115). Transfection of NIH-3T3 cells with pcDNA3.0-SCT-C(18-27) and SCT-C(107-115) leads to stable presentation of HBcAg epitopes at the cell surface. HLA-A2.1/Kb transgenic mice vaccinated with the SCT constructs, either as a DNA vaccine alone or followed by a boost with recombinant vaccinia virus, were shown to generate HBcAg-specific CTL responses by enzyme-linked immunospot assay (ELISPOT) and in vitro interferon-gamma release experiments. HBcAg-specific CTLs from vaccinated HLA-A2.1/Kb transgenic mice were able to inhibit HBV surface and e antigen expression as indicated by HepG2.2.15 cells. Our data indicate that a DNA vaccine encoding a human HLA-A2 SCT with HBV epitopes can lead to stable, enhanced HBV core antigen presentation, and may be useful for the control of HBV infection in HLA-A2-positive HBV carriers.