滤泡辅助T细胞在乙肝疫苗免疫应答中的表型和功能分析及调控机制研究

Hepatitis B is a major global public health problem. Hepatitis B vaccine is the most effective way to prevent hepatitis B virus (HBV) infection and its related chronic sequences. Approximately 5%-20% vaccinees, however, are yet susceptible to HBV due to no efficient protective antibody (Ab) response induction to hepatitis B vaccine. Mechanisms underlying failure of hepatitis B vaccine-induced Ab response in healthy adult opulation are poorly understood. In this program, three study groups with different anti-HBs Ab levels were screened from adults who had received three doses of hepatitis B vaccine and evaluated by age and gender. Peripheral vein blood samples were collected at baseline (T0) and 30-45 day (T1).postvaccination for evaluation of immune response. Radioimmnoassay were used to test anti-HBs. Vaccine normal responders, vaccine lower responders, and vaccine nonresponders were comparable at T0 and T1 in T follicular helper cells (Tfh) immnophenotypic characteristics. At T1, three groups developed an expansion of phenotypic and functional memory peripheral Tfh (pTfh) cells co-culturing with memory B cells and effect B cells, respectively, and ex vivo hepatitis B vaccine-stimulated IgG Ab-secreting cells in an ELISPOT assay. BCL6-related specific microRNAs (miRNAs) were screened after performing the method of miRNAs array and bioinformatics. These specific miRNAs is expected to test in vitro. Compared with characteristics and function of pTfh cells between pre- and post-miRNA transduction, further results would be achieved on how it have effects on cell apoptosis, proliferation, activity and cytokine secreting function of pTfh. Thus, miRNA activated pTfh cells may compromise vaccine-induced antibody response, for which a mechanism to improve humoral immunity of miRNA-activated pTfh differentiation is postulated, which may provide some beneficial clues to understand function and mechanisms of pTfh in the regulation of hepatitis B.vaccine-induced Ab responses.

乙型病毒性肝炎（乙肝）是全球公共卫生问题，注射疫苗可有效预防乙肝。但接种后仍有5%-20%因不具有效抗体而继续为乙肝病毒易感者，其分子机制不明。在本研究中，从全程接种乙肝疫苗对象中随机选择年龄和性别等因素在三组（正常、弱、无免疫应答组）间无差异的研究对象，抽取接种前后外周静脉血，利用流式染色术和酶联免疫斑点术分析与抗体反应相关pTfh细胞亚群的表型和功能；通过microRNAs（miRNAs）芯片技术和生物信息学技术筛选出免疫弱无应答组pTfh细胞BCL6基因相关miRNAs；再在体外验证特异性miRNA对pTfh细胞活力、增殖、凋亡及其分泌细胞因子功能的影响，初步探讨miRNA在pTfh辅助B细胞产生抗体过程中的可能调控机制，尝试从促进pTfh分化角度阐明pTfh细胞增强体液免疫应答的分子机制，为乙肝疫苗接种后有效提高抗体滴度提供科学的潜在靶目标。

高危成年人群接种乙肝疫苗是预防和阻断乙肝病毒感染的最有效途径。但是，在慢性丙肝患者接种乙肝疫苗后的体液免疫应答失败率却显著提高。滤泡辅助T细胞（T follicular helper cells, Tfh）是CD4+辅助性T细胞亚群中的一种，可辅助B淋巴细胞增殖分化形成生发中心（Germinal center, GC），在抗体类别转换重组，亲和力提高等过程中扮演重要作用。以往研究发现，接种疫苗后，外周循环滤泡辅助T细胞（Peripheral blood Tfh, pTfh）参与了体液免疫产生的抗体过程。在本项目中，对慢性丙肝患者进行自愿全程接种乙肝疫苗，按照乙肝抗体表达水平分为：弱免疫应答组与免疫应答失败组。选择健康成人自愿全程接种乙肝疫苗后的正常免疫应答者为正常免疫应答对照组。通过观察接种乙肝疫苗后的cTfh表型以及含量变化，确定pTfh与体液免疫应答之间的依存关系，初步探索与体液免疫应答水平相关pTfh细胞亚群的分布和pTfh细胞各亚群在辅助B细胞产生特异性高亲和性抗体的功能，初步推断pTfh细胞亚群与体液免疫应答关系。在无免疫应答的乙肝疫苗接种者中，其外周循环血中同免疫应答正常的乙肝疫苗接种者一样表达滤泡辅助T细胞（以表达CXCR5、ICOS、CD40L等膜表面分子为特点），但是，外周循环血中的滤泡辅助T细胞占CD4+细胞比例要显著低于弱免疫应答者，(P=0.015），也显著低于正常免疫应答者（P=0.019）；弱免疫应答者的外周循环中的滤泡辅助T细胞占CD4+细胞比例与正常免疫应答者之间差异不具有统计学意义（P>0.05）。在此研究基础上，进一步利用流式技术分析了应答免疫者体内的滤泡辅助T细胞亚群的表型主要以分泌IFN-γ和IL-4为主，但不以IL-17A为主。说明正常免疫应答组滤泡辅助T细胞以TH1型和TH2型为主；弱、无免疫应答组滤泡辅助T细胞以TH1型为主；三组免疫应答均不以TH17型为主。该项研究提示滤泡辅助T细胞在乙肝疫苗接种后的体液免疫应答过程中的潜在的重要作用，有望通过提高疫苗设计激发无免疫应答者体内的滤泡辅助T细胞的比例，从而使无免疫应答者获得成功免疫应答。