Antigen-driven affinity maturation of B cells in meningeal ectopic lymphoid tissue in a model of multiple sclerosis

在继发性进行性多发性硬化症（SP-MS）和实验性自身免疫性脑脊髓炎（EAE）（MS模型）中，已在脑膜中鉴定出富含B细胞的淋巴样细胞积聚。这些聚集体代表脑膜异位淋巴组织（mELT）。像次级淋巴组织一样，异位淋巴组织被认为含有生发中心（GC），其中B细胞增殖并经历其免疫球蛋白（IG）基因的体细胞超突变。这种抗原驱动的亲和力成熟过程可能导致B细胞受体和Ig对其靶抗原的亲和力增加，并可能增强致病潜力。尽管包括我们自己的研究在内的研究表明mELT含有GC，但其功能和与疾病进展的相关性仍然很差。在这里，我们建议研究mELT是否支持抗原驱动的亲和力成熟，并有助于中枢神经系统（CNS）自身免疫的小鼠EAE模型的疾病发病机制。我们将结合激光捕获显微镜和下一代深度免疫库测序，全面表征mELT中的B细胞库。将mELT组库与其他淋巴组织（包括CNS引流颈淋巴结）中的组库进行比较。同时，将分析mELT中选择的单个B细胞。匹配库测序数据与单细胞分析将提供一种新的方法，以确定配对的IG重链和轻链从过度代表的B细胞克隆与先验估计其“进化”的立场。假设它们在生物学上最相关，将克隆并表达这些配对的IG序列以测试它们的抗原结合亲和力，作为抗原驱动的亲和力成熟的量度。将在体外和体内评估其致病潜力。该项目将有助于理解B细胞和mELT在CNS自身免疫中的作用，并可能有助于确定治疗SP-MS的新治疗靶点。

In secondary progressive multiple sclerosis (SP-MS) and experimental autoimmune encephalomyelitis (EAE), a model of MS, B cell-rich accumulations of lymphoid cells have been identified in the meninges. These aggregates represent meningeal ectopic lymphoid tissue (mELT). Like secondary lymphoid tissue, ectopic lymphoid tissue is thought to contain germinal centers (GC), where B cells proliferate and undergo somatic hypermutation of their immunoglobulin (Ig) genes. This process of antigen-driven affinity maturation may result in B cell receptors and Igs with increased affinity to their target antigens and possibly enhanced pathogenic potential. Although studies, including our own, suggest that mELT contain GCs, their functionality and relevance to disease progression remains poorly characterized. Here, we propose to investigate whether mELT supports antigen-driven affinity maturation and contributes to disease pathogenesis in murine EAE models of central nervous system (CNS) autoimmunity. We will comprehensively characterize the B cell repertoire in mELT, combining laser capture microscopy and next-generation deep immune repertoire sequencing. mELT repertoires will be compared to repertoires in other lymphoid tissues, including CNS-draining cervical lymph nodes. In parallel, a selection of single B cells from mELT will be analyzed. Matching repertoire sequencing data with single cell analyses will provide a novel approach to identifying paired Ig heavy and light chains from overrepresented B cell clones with an a priori estimation of their “evolutionary” standing. Assuming that they are biologically most relevant, these paired Ig sequences will be cloned and express to test their antigen binding affinity as a measure of antigen-driven affinity maturation. Their pathogenic potential will be assessed in vitro and in vivo. This project will contribute to the understanding of the role of B cells and of mELT in CNS autoimmunity and may help identify novel therapeutic targets in treatment of SP-MS.