B淋巴细胞介导超抗原SEC2活化鸡T淋巴细胞及其促进体液免疫的机制研究

Recently, all countries have gradually started to limit part or all of the antibiotics used in livestock farming due to the problems of food safety and antibiotic resistance, and these problems promote the study of immunopotentiator for livestock and poultry in the post-antibiotic era simultaneously. Our preliminary research findings showed that superantigen SEC2 could significantly enhance the chicken vaccine effect, which suggested SEC2 could enhance the chicken humoral immune response by activating chicken T cells, but the detailed mechanism is still unknown. Based on the previous results, we suppose that chicken B cells could play the part of superantigen presenting cells during the process of SEC2 induced T cells activation, which also provide the signal for the activation of B cells; meanwhile the cytokines secreted by activated chicken T cells could enhance the effect of B cells activation. In order to testify our hypothesis, the detailed procedures are designed and listed as follows. Firstly, the proliferation activity of chicken spleen lymphocytes, peripheral blood mononuclear cells and lymphocyte subtypes triggered by SEC2 are tested in vitro, and the secretion of cytokines and other associated bioactive mediators are measured either. Subsequently, chicken T cells and B cells are purified by flow cytometry, and SEC2-EGFP fusion protein will be used to testify if chicken B cells could play the part of superantigen presenting cells during the process of SEC2 induced T cells activation. Thirdly, based on the signals needed for the B cells activation, we further testify the effect of CD40/CD40L complex and associated cytokines on the process of SEC2 induced chicken B cells activation. Finally, the SEC2 induced enhancement of chicken immunity and vaccine effect are analyzed.The aim of this project is to clarify the role of B cells on activating chicken T cells by SEC2 and their effect on promoting the humoral immune response, and provide support for the application of superantigen as a new immunopotentiator on the poultry farming.

家禽抗生素滥用引起的食品安全及细菌耐药性问题推动了家禽免疫增强剂的研究。前期研究发现超抗原SEC2可显著增强鸡疫苗效价，表明其可通过活化鸡T细胞来促进体液免疫，但具体机制尚不明确。基于前期研究结果我们推测：鸡B细胞能通过行使超抗原递呈功能激活T细胞的同时为自身活化提供信号，并协同T细胞分泌的细胞因子等放大B细胞活化效应，促进体液免疫。本项目首先拟通过分子生物学方法明确SEC2对鸡淋巴细胞的增殖活性及细胞因子分泌，筛选具有显著变化的活性因子；随后流式分选鸡T、B细胞，利用SEC2-EGFP融合蛋白验证B细胞是否通过行使超抗原递呈功能来激活T细胞；进而通过正向激活和反向抗体抑制研究CD40、CD40L及相关活性因子在SEC2活化B细胞中的作用；并验证SEC2增强鸡体免疫力及疫苗效价。其结果重在阐明SEC2活化鸡T细胞后促进体液免疫反应的作用机制，为开发金葡菌超抗原类家禽免疫增强剂提供科学依据。

超抗原能够以与普通抗原不同的方式，与MHC II结合，只需要极低的剂量即能激活大量具有特异Vβ片段的T淋巴细胞，释放大量细胞因子，强化细胞毒T淋巴细胞活性，最终产生极强的免疫应答效应。本项目重在验证“鸡B细胞能否通过行使超抗原递呈功能激活T细胞的同时为自身活化提供信号，并协同T细胞分泌的细胞因子等放大B细胞活化效应，促进体液免疫”。但随后研究发现SEC2刺激鸡源淋巴细胞增殖效果不稳定，需应用较大剂量才可在体外刺激鸡源淋巴细胞显著增殖，但大剂量SEC2的体内注射不仅导致鸡体出现明显副作用（如食欲不振，活动量减少等），并且对家禽食用的安全性也存在风险。因此，本项目更换实验动物为小鼠来验证上述假设，实验结果初步表明SEC2未通过B细胞行使超抗原递呈功能激活相对应T细胞，但B淋巴细胞数量及活性均显著上调，推测是通过其它APC细胞激活T淋巴细胞后间接引起的，具体机制正在研究中。随后，研究发现SEC2与SEB不同，在其激活T淋巴细胞时无需CD28/CD86-CD80辅助信号通路，部分解释为何SEC2超抗原活性低于SEB。进一步研究发现PI3K/PLC信号通路在SEC2超抗原活性的发挥中有重要作用，相关机制正在研究中。此外，为明确不同亚型超抗原激活淋巴细胞的机制是否具有一致性，本项目克隆并纯化获得致呕吐毒性较低的金葡菌类肠毒素SElQ及SElK，并对其生物学活性及激活淋巴细胞的机制是否符合本项目假设进行验证。而对SElQ及SElK（与SEC2比较）的研究结果也表明二者具有高效低毒的特性，在临床上（抗肿瘤生物制剂）具备潜在开发价值。上述实验结果不仅丰富了金葡菌肠毒素类超抗原活性的发挥机制研究，同时为临床上免疫增强剂的筛选提供更多选择。