人类核抗原特异性B细胞的鉴定及其特征分析

Anti-nuclear antibody (ANA) produced from the nuclear antigen-specific B cell is an important index for the clinical diagnosis and prognosis of systemic lupus erythematosus (SLE), and also is one critical aspect for the study of SLE pathogenesis. However, the precise identification of nuclear antigen-specific B cell is a difficult question up to date. Nucleosome is the main self-antigen in SLE, which could exist in the bodies of SLE patients in free form. Our previous study found that anti-nucleosome antibody was in high level in serum samples of SLE patients or SLE mice models compared to health people or control mice, suggesting that nucleosome-specific B cells are the main type of nuclear antigen-specific B cells. Accordingly, we hypothesize one new high-throughput method to identify nuclear antigen-specific B cells based on the B cell receptor (BCR) specificity and flow cytometry. In the project, we will: 1) set up new method for the identification of nuclear antigen-specific B cells with GFP-labeled nucleosome; 2) analyze molecular phenotype and sequence feature of antibody genes of nuclear antigen-specific B cells from SLE patients; 3) study the correlation between the number and phenotype of this type of B cells and SLE clinical symptoms. The launch of the project will provide one new fast, effect and specific method for identifying nuclear antigen-specific B cells, which will supply theory evidence for the intensive study of these B cells as well as the development of novel therapy strategy for SLE by targeting nuclear antigen-specific B cells.

核抗原特异性B细胞产生的抗核抗体（ANA）是系统性红斑狼疮（SLE）临床诊断和预后的重要指标，也是研究SLE发病机理的一个关键。然而，核抗原特异性B细胞的准确鉴定一直是个难题。核小体是SLE的主要自身抗原，以游离形式存于SLE患者体内。我们前期工作发现，SLE患者和模型小鼠血清中均含有高水平抗核小体抗体，提示核小体特异性B细胞是一类主要的核抗原特异性B细胞。因此，我们提出建立一种以B细胞受体（BCR）特异性和流式细胞术为基础的、高通量鉴定核抗原特异性B细胞的新方法。本项目将：1）建立利用GFP标记的核小体鉴定核抗原特异性B细胞的方法；2）分析SLE患者中核抗原特异性B细胞的分子表型和抗体基因序列特征；3）研究这类B细胞数量和表型与SLE临床表征的相关性。本项目的实施将为鉴定核抗原特异性B细胞提供一种特异、高效和快速的新方法，为深入研究这群B细胞、研发以其为靶标的SLE疗法提供理论依据。

核抗原特异性B细胞产生的抗核抗体（ANA）是系统性红斑狼疮（SLE）临床诊断和预后的重要指标，也是研究SLE发病机理的一个关键科学问题。然而，核抗原特异性B细胞的准确鉴定一直是个难题。本研究拟建立一种以B细胞受体（BCR）特异性和流式细胞术为基础的、高通量鉴定核抗原特异性B细胞的新方法并分析这群B细胞的生物学特征和潜在功能。为了开展本项目研究，我们在研究前期开展了大量概念验证性的工作，包括抗核抗体阳性对照抗体的筛选，克隆表达和功能验证，血清抗核抗体亲和力测定平台的建立。在此基础上，本研究建立了利用标记的dsDNA鉴定DNA特异性B细胞的新方法，利用该方法从小鼠脾脏细胞纯化获得DNA特异性B细胞并分析了这群B细胞的生物学特征。另外，我们初步证实了高亲和力抗DNA抗体在抗感染免疫反应中发挥潜在保护作用。本项目的实施建立了一种鉴定和纯化核抗原特异性B细胞的特异、高效和便捷的技术平台，为深入研究核抗原特异性B细胞生物学特征，抗核抗体的起源及其潜在生物学意义提供有力的技术支持和理论参考。本项目开发的研究策略及部分研究数据已以学术论文形式发表于国际学术期刊Proceedings of the National Academy of Sciences of the United States of American(PNAS,2019 116(37) 18550-18560)和 Frontiers in Immunology (Front. Immunol., 10 June 2020)。