幽门螺杆菌Lpp20抗原引起的免疫优势CD4+T细胞应答及与胃部疾病的相关性

Previous investigations have demonstrated that CD4+ T cells play a crucial role in pathogenesis of Helicobacter pylori (H.pylori) infection and immunological defense and CD4+ T cell epitopes exert a key function in T cell activation. Immunodominant response is an important feature of anti-bacterial cellular response, but it is not well elucidated in the immune response against H.pylori infection. Our team and other research groups have proved that Lpp20 is one of major protective antigens of H.pylori, which induce immune responses after H.pylori invades host. Base on BALB/c mice model, our previous study has identified two H-2d restricted CD4+ T cell epitopes on Lpp20 which predominantly induced Th1 type cell response to mainly secret IFN-γ. Taking into consideration the fact that MHC restriction is different between human and mice and H-2d restricted epitopes are not feasible to the design of human vaccine, we will target H.pylori-infected patients, isolate and expand Lpp20 specific T cells from peripheral blood monocytic cell (PBMC) of H.pylori patients, identify immunodomiant epitopes on Lpp20 using overlapping technology, analyze HLA restriction and access its correlation with various gastritis (atrophic gastritis, superficial gastritis, antral gastritis, fulfill stomach gastritis) and ulcer(duodenal ulcer, gastric ulcer). This study will provide insight into understanding H.pylori pathogenesis and host defense mechanism, and facilitate the development of effective epitope vaccine.

已有证据显示宿主CD4+ T细胞反应在幽门螺杆菌（H.pylori）感染中发挥着重要作用。免疫优势应答是抗细菌感染免疫的一个主要特征，但在抗H.pylori感染中CD4+ T细胞免疫优势应答还没被完全阐述。本课题组及其他研究小组已证实Lpp20是H.pylori的一个主要保护性抗原， 前期我们以BALB/c小鼠为模型，鉴定了Lpp20的H-2d限制性CD4+ T细胞表位。 考虑到小鼠和人MHC的差异及H-2d限制性表位不适用于人疫苗的设计，本课题拟以H.pylori感染者为对象，分离其外周血单个核细胞中特异性T细胞并体外扩增，利用重叠肽方法，对Lpp20中激发CD4+ T细胞应答的免疫优势表位进行筛选，并分析其HLA限制性，从而进一步评价Lpp20特异性CD4+T细胞反应与不同胃炎和胃溃疡的相关性，此研究将有助于全面理解H. pylori感染及宿主免疫防御机制并为研制新的表位疫苗提供基础。

抗原特异性CD4+T细胞在H.pylori感染的免疫保护机制中发挥着重要作用。Lpp20是H.pylori一种保守的脂蛋白，已被认为是H.pylori主要保护性抗原。我们前期研究鉴定了2个Lpp20的H-2d限制性CD4+T细胞表位，以此构建的表位疫苗在预防和治疗接种中均保护小鼠抵抗H.pylori的感染。免疫优势CD4+T细胞反应是抗微生物感染的一个重要特性。尽管许多H.pylori抗原的HLA限制性的免疫优势CD4+T细胞表位已确定,但Lpp20 HLA限制性的免疫优势CD4+T细胞表位还尚未鉴定。在本研究中,用系统的方法鉴定H.pylori感染者中Lpp20特异性免疫优势CD4+T细胞表位。分离H.pylori感染者PBMC,用rLpp20刺激，体外扩增Lpp20特异性T细胞系，用27个覆盖Lpp20抗原的重叠18mer肽进行筛选，发现L55-72和L79-96是CD4+T细胞反应的免疫优势表位。然后用13mer肽对这两个18mer表位进一步筛选，确定核心序列分别是L57-69和L83-95。用PCR-SBT基因分型、抗体阻断及各种不同HLA等位基因的EB病毒转化的B淋巴母细胞(Epstein-Barr virus-transformed B lymphoblastoid cell lines，BLCLs)表位递呈实验测定表位肽的HLA限制性，L57-69和L83-95的限制性分别为HLA-DRB1*1501和HLA-DRB1*1602,能被rLpp20负载的自体DC和H.pylori全细胞裂解液刺激的DC识别，并且能促进H.pylori感染者CD4+T细胞的增殖。为进一步分析l57-69特异性的CD4+T细胞反应与H pylori引起的胃疾病的联系，测定表达HLA-DRB1*1501限制性的H.pylori感染者的L57-69特异性CD4+T细胞反应。胃炎组L57-69的CD4+T细胞百分比明显高于消化性溃疡组，消化性溃疡组高于胃癌组，非萎缩性胃炎组CD4+T细胞的百分比明显高于萎缩性胃炎组，胃窦胃炎组CD4+T细胞的百分比高于全胃炎组，十二指肠溃疡组CD4+T细胞的百分比高于胃溃疡组。因此，L57-69特异性CD4+T细胞反应对表达HLA-DRB1*1501限制性的H.pylori感染者具有保护作用，能抵抗严重胃疾病。