L21R-AS1对肾移植抗体介导排斥反应的Cis和Trans调控及机制研究

IL21, secreted by Tfh cells, induces IL21 autocrine itself and germinal center B cells differentiation through Tfh and B cell IL21 receptor (IL21R) binding respectively, and plays an important role in antibody-mediated rejection (AMR) after renal transplantation. It has been revealed in our former work that the predominant subtype of mature IL21r mRNA in murine spleen had switched into UC009JPE.2 after kidney transplantation, whereas the mechanism remains veiled. IL21R-AS1 partly overlaps IL21R by a reverse pattern. After IL21 stimulation, both nuclear and cytosolic IL21R-AS1 signals, and IL21R-AS1 binding snRNP70 increase significantly, therefore IL21R-AS1 is supposed to participate in the IL21 signal response through both Cis and Trans acting mechanisms. To validate the hypothesis, T-cell-dependent antibody (TD) response will be setup on Tfh/B cell co-culture system by keyhole limpet haemocyanin (KLH) administration in vitro. Dual nickase SpCas9 (spCas9n)-induced CRISPR genomic editing and rAAV- or LV-carried CAG-promoted overexpression are induced for IL21R-AS1 functional knock-down and knock-in. The role of IL21R-AS1 in alternative splicing of IL21R mature mRNA and TD response, furthermore in AMR after kidney transplantation will be evaluated by multiple methods.

Tfh细胞通过分泌IL21激活自身以及B细胞IL21R，促进B细胞向浆细胞转化，是诱导肾移植AMR的重要事件。课题组前期发现肾移植小鼠脾脏中优势Il21r的mRNA亚型发生改变，但机制不明。人IL21R基因3’端外显子与IL21R-AS1基因部分逆向重叠，课题组对Tfh细胞进行外源IL21刺激后发现IL21R-AS1与snRNP70结合度增高，且胞质中信号增强，因此推测IL21R-AS1存在Cis和Trans双重作用，参与IL21R的选择性剪接和IL21应答。本项目将利用离体Tfh/B细胞共培养系统，对IL21R-AS1进行基因过表达或敲除，构建相关功能域上调或缺失的细胞群落；在异种蛋白刺激下，评估IL21R-AS1对IL21R的剪接修饰以及对免疫相关蛋白的调控，探讨其在TD免疫应答中的角色和潜在机制；并在肾移植模型中探究其作为AMR治疗靶点的可行性和有效性。

IL21是Tfh细胞与B细胞交互作用的重要细胞因子。IL21-IL21R轴的活化，是Tfh细胞与B细胞交互作用的关键事件，IL21与IL21R特异性结合后，Tfh细胞IL21分泌功能得以维持，B细胞则向浆细胞成熟转变。Tfh细胞中IL21R-AS1长链非编码RNA与L21R的表达密切相关，通过建立Tfh和B细胞分选和共培系统，克隆全长IL21R-AS1的CDS，构建全长、350-1570、1580-2190、2190-2614段缺失段RNA，并转染B细胞，对IL21R-pre-mRNA丰度调控水平测定，初步确定1580-2190段存在针对IL21R mRNA的降解诱导结构域，因此证明具有Cis作用；在Tfh-B细胞共培养体系中给予匙孔血蓝蛋白刺激后，对于浆细胞（PC）的增殖、分化水平进行离体测定，结果显示IL21R-AS1的过表达可下调体系中B细胞激活以及PC的分化与功能；靶向B细胞的IL21R-AS1 mRNA处理可增加B细胞凋亡水平，提示IL21R-AS1在B细胞阳性筛选具有潜在功能需进一步展开研究；对临床外周血样本进行IL21R-AS1靶向RNA-蛋白质免疫共沉淀，显示组蛋白乙酰化相关信号通路相关蛋白HDAC-1,2,3亚型均有富集，提示在IL21R-AS1的Trans调控模式中，通过组蛋白乙酰化信号通路影响B细胞激活相关基因活化；肾移植ABMR模型小鼠中，通过构建B220+细胞靶向提送RNA体系，将装载IL21R-AS1全长mRNA靶向纳米颗粒静脉输注，结果显示IL21R-AS1 mRNA组可显著降低ABMR所造成的移植肾损伤。综上，通过本课题研究已初步证明，IL21R-AS1既可通过Cis作用对IL21R mRNA表达丰度进行调节，也可通过Trans作用对组蛋白乙酰化信号通路产生影响，从而参与B细胞激活。