Ⅰ型干扰素介导Tfh细胞促进T细胞依赖性抗体反应的作用研究

TypeⅠinterferon(IFN) could enhance T cell-dependent antibody reactions, but the mechanisms remain not completely clear. Our studies have shown that IFN-a as a adjuvant could induce the proliferation and differentiation of T follicular helper(Tfh) cells and promote formation of germinal centers(GC)during the recombinant foot and mouth desease virus(FMDV) vaccine response. Tfh cells have been fully defined as a novel T cell subset in recent years, which specifically help B cell humoral immune response. Moreover, Tfh cells also play a potent role in the T cell-dependent antibody reactions. Collectively, these observations led us to hypothesize that typeⅠIFN enhances T cell-dependent antibody responses by acting as a natural adjuvant for the generation of Tfh cells.Therefore, this project going to investigate its mechanisms through in vivo test. For this purpose, We will be using flow cytometry, Laser confocal microscopy, real-time RT-PCR to analyze how typeⅠIFN induces Tfh proliferation thereby providing help to B cells immunization.To illustrate the mechanisms by which typeⅠIFN improves the T cell-dependent antibody reactions, we will block the IL-21/IL-21R and ICOS/ICOSL signaling pathways to disrupt the function of Tfh cells. The aim is to clarify the mechanisms of the Tfh cells on T cell-depentent antibody responses induced by typeⅠIFN.To further explore the typeⅠIFN could enhance T cell-dependent antibody responses by a new way. To determine the mechnisms of typeⅠIFN enhancing effective immune responses through the novel strategies as an adjuvant.

Ⅰ型干扰素能增强T细胞依赖性抗体反应，但具体机制不十分清楚。我们研究证实IFN-α佐剂在口蹄疫重组疫苗免疫应答中能诱导滤泡辅助性T细胞(Tfh细胞)的分化及促进生发中心形成。Tfh细胞是近年被明确定义的一种全新的辅助B细胞的T细胞亚群，在T细胞依赖性抗体反应中发挥重要作用。我们设想Ⅰ型干扰素是否通过介导Tfh细胞促进T细胞依赖性抗体反应。为此，本项目拟通过小鼠体内试验，利用流式细胞术、激光共聚焦显微术、Real-timeRT-PCR等方法研究Ⅰ型干扰素在Tfh细胞辅助B细胞免疫应答中发挥的免疫调控作用。通过阻断Tfh细胞与B细胞间的IL-21/IL-21R和ICOS/ICOSL信号通路后，观察Ⅰ型干扰素对T细胞依赖性抗体反应的影响，明确Tfh细胞在Ⅰ型干扰素增强T细胞依赖抗体反应中的作用地位，进一步探讨Ⅰ型干扰素增强T细胞依赖抗体反应的新途径，为Ⅰ型干扰素发挥佐剂效应机制提供新的理论基础。

猪IFN-α是抗A/O 型口蹄疫（foot-and-mouth disease，FMD）重组腺病毒疫苗的良好佐剂，可以长期保护动物不受口蹄疫病毒的感染。我们前期研究结果也显示IFN-α 佐剂能够辅助口蹄疫重组腺病毒疫苗提高免疫效果。研究认为，Ⅰ型干扰素可与T 细胞直接发生作用，在T细胞的分化、成熟、维持等方面起到非常重要的作用。滤泡辅助性T细胞（Follicular helper T cells，Tfh细胞）是一种全新的T细胞亚群，Tfh 细胞在 B 细胞的发育、抗体形成、抗体类别转换，分泌高亲和力抗体，为机体清除外来病原体，建立长期体液免疫等过程中扮演主要角色。我我们设想Ⅰ型干扰素是否通过介导Tfh 细胞促进T 细胞依赖的抗体反应。为此，本研究通过小鼠体外实验，利用磁珠分选小鼠脾脏CD4+T细胞，通过重组小鼠IFN-α刺激CD4+T细胞，观察重组小鼠IFN-α对小鼠Tfh细胞分化的影响。然后，通过小鼠体内实验，阻断影响Tfh细胞分化的关键通路IL-21/IL-21R和ICOS/ICOSL信号通路后，观察IFN-α对Tfh细胞分化的影响。最后，利用重组IFN-α 佐剂与FMDV 重组腺病毒rAd5EGS 联合免疫小鼠，通过观察IFN-α 佐剂增强重组疫苗诱导Tfh 细胞增殖分化及促进Tfh 细胞辅助B 细胞免疫应答的作用。结果显示，IFN-α作为佐剂能促进Tfh细胞的分化，并通过调控IL-21/IL-21R和ICOS/ICOSL信号通路影响Tfh细胞的分化，能促进生发中心的形成，促进GC B细胞的分化，诱导抗体的产生。研究结果为Ⅰ型干扰素发挥佐剂效应机制提供新的理论基础。