膜性肾病中M型磷脂酶A2受体的T细胞抗原表位研究

Primary membranous nephropathy is an autoimmune kidney disease, with M type phospholipase A2 receptor (PLA2R) as the major target antigen. Epitope spreading process exists in disease development and has close associations with disease severity and clinical remission. We hypothesize that the initial epitope of spreading process, which could be recognized both by autoreactive T cells and by B cells, may be the crucial player in disease mechanism. After epitope recognition, CD4+ T cells stimulate and induce antibody production by B cells, which later on undergo epitope spreading process and lead to podocyte damage, electron dense deposits, and glomerular basement membrane thickening of primary membranous nephropathy. T cell infiltration may also participate in the inflammation injury of kidney by secreting different cytokines. Thus, T cell epitope mapping is essential for the investigations on pathogenesis. Our previous studies found two risk alleles, HLA-DRB1*1501 and DRB1*0301, which were susceptible for primary membranous nephropathy by encoding arginine13 and alanine71, and lysine71 on DRβ chain of HLA molecule for antigen presentation. We further predicted the possible T cell epitopes on PLA2R which may be presented by the susceptible HLA molecules and confirmed some of them by examinations. In this project, we aim to define the PLA2R T cell epitopes presented by the susceptible HLA molecules, identify the initial epitope by mutual T/B cell recognition and the critical amino acids in it, and elucidate the autoreactive T cell profile against each epitope and their roles in disease pathogenesis.

原发性膜性肾病是自身免疫性肾脏病，磷脂酶A2受体（PLA2R）是主要的靶抗原。在发病中存在B细胞抗原表位的扩展过程，且与疾病的发生发展密切相关。我们推测：该过程的初始阶段，自身免疫性T细胞和自身抗体共同识别的线性抗原表位，可能是疾病的启动因素。CD4+ T细胞识别抗原表位后，不仅能够诱导B细胞产生自身抗体，发生抗原表位扩展，导致疾病发生；还可以直接浸润到肾组织，释放细胞因子，参与肾小球的炎症损伤。因此，明确PLA2R的T细胞抗原表位是阐明该病发病机制的关键环节。我们的前期工作明确了疾病的易感HLA II类基因型及其编码的HLA分子上的关键氨基酸，并对其递呈的PLA2R的T细胞抗原表位进行了预测和初步验证。申请人拟从抗原递呈复合物入手，明确易感HLA分子递呈的PLA2R的T细胞抗原表位，确定T/B细胞共同识别的初始表位及其关键氨基酸，阐明识别各抗原表位的T细胞亚群组成及其在发病机制中的作用。

本课题的方向是膜性肾病的抗原表位研究，目标是阐明膜性肾病的主要靶抗原---磷脂酶受体（PLA2R）---的T细胞抗原表位的精确氨基酸序列。课题组顺利圆满完成了计划的研究内容。设计合成了一组相互重叠的线性肽段，共123条，包含了PLA2R的10个结构域，全长1378个氨基酸。首先检测了膜性肾病的易感HLA分子DRB1*1501和DRB1*0301所递呈的肽段，发现17条肽段能够与两种易感HLA分子均呈高亲和力结合，进而递呈给T细胞识别。收集了膜性肾病患者的外周血单个核细胞，发现10条肽段能够被患者的CD4+ T细胞识别，出现显著的增殖反应。增殖的T细胞分泌多种细胞因子，以IL-6、IL-9、IL-10、IL-17F和TNF-α为主，能够辅助自身反应性B细胞活化，产生自身抗体，启动膜性肾病的发生机制。上述研究，通过HLA递呈、T细胞增殖、T细胞效应，三方面的研究，最终确定了PLA2R上的10条肽段为膜性肾病的T细胞抗原表位，分别是CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1和CTLD7-2。该研究阐明了膜性肾病主要靶抗原PLA2R的精细的T细胞抗原表位，为深入理解该病的细胞免疫紊乱机制，进而研发新型的特异性免疫治疗手段，提供了重要的依据。已经发表了标注本课题基金资助的SCI论文17篇，包括肾脏病领域论著Top1的杂志Kidney Int（IF 19.0）2篇，J Am Soc Nephrol（IF 15.0）4篇。获得国家发明专利授权1项，申请2项。