LBP佐剂调控TLR/Blimp-1促进cDC2s诱导记忆性Tfh细胞参与FMD疫苗免疫机制研究

Follicular helper T cells (Tfh) represent the subpopulation of T cells that provides help for B cells to participate in humoral immunity.Therefore, how to effectively induce differentiation of Tfh cells, especially the memory Tfh cells, is a new strategy for vaccine design. Our study shows that Lycium barbarum polysaccharide (LBP) as the adjuvant can promote dendritic cells (DCs) inducing the differentiation of Tfh cells, but the mechanism is not yet clear. The latest research shows that Blimp-1-deficient cDCs preferentially induce Tfh cells. We speculate that LBP can induce the differentiation of Tfh cells by negative regulating Blimp-1 through Toll like receptor (TLR). In this study, the effects of Blimp-1 overexpression or silencing on the maturation of cDC2s induced by LBP were investigated by flow cytometry, qPCR, wester-blot and ELISA.Searching the key target for LBP regulation of TLR/Blimp-1 by blocking TLR-p38 MAPK/NF-κB signaling pathway, to elucidate the molecular mechanism of LBP adjuvant promoting cDC2s induced differentiation of Tfh cells by negative regulating of Blimp-1. Recombinant foot-and-mouth disease vaccine as antigen to explore the mechanism of LBP adjuvant promote cDC2s induced memory Tfh cells involved in the immune response to the vaccine, which contributing the theoretical basis for development of LBP as a vaccine adjuvant.

滤泡辅助性T细胞（Tfh）是专职辅助B细胞参与体液免疫的T细胞亚群，因此，如何有效诱导Tfh细胞，特别是记忆性Tfh细胞分化是疫苗设计的新策略。我们研究发现，枸杞多糖（LBP）佐剂能促进树突状细胞（DCs）诱导Tfh细胞分化，但机制尚不清楚。最新研究发现，DCs缺失Blimp-1后优先诱导Tfh细胞分化。我们推测，LBP可能通过Toll样受体（TLR）下调Blimp-1，诱导Tfh细胞分化。本研究拟通过体外过表达或沉默Blimp-1后，流式细胞术、qPCR、wester-blot及ELISA检测沉默或过表达Blimp-1后对LBP诱导cDC2s成熟的影响，明确其在LBP促进cDC2s诱导Tfh细胞分化中的作用；通过阻断TLR-p38MAPK/NF-κB通路后，寻找LBP调控TLR/Blimp-1的关键靶点，阐明LBP佐剂负向调控Blimp-1促进cDC2s诱导Tfh细胞分化的分子机制；最后，以口蹄疫重组疫苗为抗原，探讨LBP佐剂促进cDC2s成熟及其诱导记忆性Tfh细胞参与疫苗免疫应答的机制，为LBP开发为新型佐剂提供理论依据。

我们研究发现，枸杞多糖（LBP）能促进口蹄疫（FMD）重组腺病毒疫苗（rAd5VP1）诱导滤泡辅助性T细胞（T follicular helper cells，Tfh）的分化，Tfh细胞是专职辅助B细胞参与体液免疫的T细胞亚群，因此，探讨LBP如何诱导Tfh细胞分化的机制十分重要。我们研究证实，LBP体外能促进小鼠骨髓树突状细胞（DCs）的成熟，且能促进DC诱导Tfh细胞分化。小鼠体内研究发现，LBP能促进rAd5VP1诱导小鼠脾脏DC的成熟、促进Th2和Tfh细胞的分化。LBP显示良好的佐剂效应，但其促进DC成熟的具体机制尚不清楚，本研究以TLR4为切入点，探讨其促进DC成熟的分子机制。体外研究结果证实，LBP能增强DC中TLR4通路中关键分子NFκB、p38、Erk1、Erk2、JNK、Blimp1及DC亚型相关转录因子IRF4、IRF5、IRF8、Batf3 mRNA和磷酸化蛋白表达水平；TLR4通路中关键分子TLR4、NFκB、p38、Erk、JNK被阻断后，能有效干预LBP促进DC中Blimp-1的表达；沉默Blimp-1后可影响LBP诱导DC成熟，研究结果提示，Blimp-1在LBP经TLR4通路诱导DC成熟过程中发挥重要作用。小鼠体内研究结果证实，LBP增强小鼠脾脏CD11c+DCTLR4通路中p-NFκB、p-p38、p-Erk1/2、Blimp1分子和DC亚型相关分子IRF4、IRF8蛋白的表达。因此，本研究结果证实，LBP可通过TLR4-NFκB/Erk1/2-Blimp1信号通路促进DC成熟，并诱导cDC1s和cDC2s的分化，我们推测，LBP可促进cDC2s诱导Tfh细胞的分化。本研究结果为LBP开发为口蹄疫疫苗新型佐剂提供理论依据。