鸡pMHCI-CD8复合体分子互作机制与抗病毒CTL免疫应答机理的研究

Cytotoxic T lymphocytes (CTL) immune response mediated by chicken CD8+ T cell plays a crucial role in the process of eliminating virus infection. However, till now, researches of chicken immunology are rarely reported, especially about the structural studies on the pMHCI and pMHCI-CD8. Chicken B15 is one of the most popular strains raised in the world. Seldom about the mechanism of antigen presentation, including virus epitopes and new epitope vaccine, has been reported in B15. In order to promote the structural studies of chicken MHCI-CD8 and functional analysis of CTL immune response, we will try to identify B15 restricted MDV-CTL epitopes and determine pMHCI and pMHCI-CD8 crystal structures. These will help us to understand the antigen presentation mechanism and CTL immune responses in chicken. .In order to identify MDV-CTL and AIV-CTL epitopes, we will screen peptides which can bind stably with BF2*1501 from MDV and AIV. Firstly, we will make the B15MDV-tetramer and AIV-tetramer, and prepare the Mouse anti chicken antibodies of MHCI and CD8 of chicken. Then, we will identify MDV-CTL and AIV-CTL immunodominant epitopes through the refolding of pMHCI in vitro. Generally speaking, effective immune adjuvant play a pivotal role in stimulating strong T-cell immune response, so we will choose chicken HSP108-N333 protein as an MDV-CTL and AIV-CTL epitopes vaccine adjuvant. High purity chicken HSP108-N333 proteins will be prepared by molecular cloning, protein expression and purification techniques. And then, HSP108-N333 protein and MDV-CTL epitope peptides complex will be used to immunize SPF chickens. The proportion of specific CTL cells in peripheral blood lymphocytes will be detected. Then, we will use the antibodies and tetramer combining the flow cytometry technology to value their immunological activity in vivo. In order to explore MDV-CTL epitope immune protection effect, SPF chickens immunized with the MDV-CTL epitope peptide and HSP108-N333 complex antigen at 1-day-old and 7-day-old, respectively, and infected MDV at 12 days. Chickens mortality and tumor incidence were recorded for 6 weeks after infection. All chicken liver, spleen, and kidney were taken for pathological analysis. Finally, we will try to crystallize and determine the structures of chicken pMHCI and pMHCI-CD8. And then we will analyze the interaction mechanism between MHCI and CD8..In conclusion, these research results will give insight into the immunological synapse and interact between the MHCI and CD8 at atom level. It will help us to understand the mechanism of CTL responses to virus infection, immune gene repertoire and pathways for anti-virus function. All these will promote the development of avian immunology.

CD8+T细胞介导的细胞毒性T细胞（CTL）免疫应答在清除鸡病毒感染过程中起着十分重要的作用。但是迄今为止，有关鸡CTL免疫的报道极少，尤其是B15即SPF鸡的pMHCI以及鸡pMHCI-CD8复合体结构以及涉及的CTL表位的鉴定、表位呈递机制和位苗的研究还尚处于起步阶段。为了推进鸡CTL免疫应答的分子间结构和功能研究，本项目拟解析鸡B15 品系pMHCI以及pMHCI-CD8复合体的三维结构，在阐释pMHCI呈递抗原表位特征的基础上，进一步阐明pMHCI与CD8分子间互作的结构基础。并制备鸡CTL检测用四聚体和鼠抗鸡CD8和MHC I 单抗，用多肽复性法和圆二色谱筛选MDV 全病毒表位肽库，挑选出多肽进行免疫。研究结果将阐明鸡CD8二聚体与pMHCI分子间的互作机制，进一步揭示鸡pMHCI抗原呈递、CTL免疫应答抗MDV效果以及鸡CD8+ T细胞的信号传导、调控通路相关的科学问题。

MHCI是细胞毒性T细胞（CTL）免疫应答的核心分子。MHCI主要递呈病毒CTL表位、通过免疫微突触内多肽-MHCI-CD8-TCR分子间相互作用、引发特异性CTL的增殖。CTL细胞再通过限制性识别，杀灭、清除被病毒感染的细胞，保护脊椎动物机体免受病毒等内源性微生物的侵害。但是迄今为止，有关鸡CTL免疫的报道极少，尤其是B15单倍型鸡的pMHCI、pMHCI-CD8复合体的结构以及涉及的CTL表位的鉴定、表位呈递机制和位苗的研究还尚处于起步阶段。为了推进鸡CTL免疫应答的分子间结构和功能研究，本项目筛选出了MDV和AIV病毒的CTL表位，制备了鸡CTL检测用四聚体和鼠抗鸡CD8和MHCI单抗，验证了表位的特异性。解析了鸡BF2*1501-PA123-130复合体的精细结构，阐明该表位被择优递呈的分子机制。成功解析了鸡cCD8αα/pBF2*15:01和cCD8αα/pBF2*04:01复合体的三维结构，阐述了鸡pMHCI-CD8复合物的两种结合模式，“抗体样”模式和“面对面”的模式。迄今为止仅在鸡中观察到“面对面”模式。与“抗体样”模式相比，“面对面”结合模式改变了cCD8αα同型二聚体与pMHCI的结合方向，这可能有助于丰富γδ T细胞结合鸡中有限的BF2等位基因呈递的多种肽。揭示了鸡pMHCI抗原呈递、CTL免疫、应答抗MDV效果以及鸡CD8+ T细胞的信号传导、调控通路相关的科学问题。