CNS antigen-specific control of early B-lineage development in the meninges

In this work we report that early developing B cells present in the meninges of mice at all ages. Single cell RNA-sequencing (scRNA-seq) analysis revealed a consecutive trajectory of meningeal developing B cells in mice and non-human primates (NHPs). Parabiosis together with lineage tracing of hematopoietic stem cells (HSCs) showed that meningeal developing B cells are continuously replenished from the HSC-derived progenitors via a circulation-independent route. Importantly, autoreactive immature B cells which recognize myelin oligodendrocyte glycoprotein (MOG), a central nervous system (CNS)-specific antigen, are eliminated from the meninges but not BM. Furthermore, genetic deletion of MOG tolerated the development/presence/residence of self-reactive B cells in the meninges. Thus, we propose that meninges function as a unique reservoir for B cell development, allowing in situ negative selection of CNS-antigen-autoreactive B cell clones to ensure a local non-self-reactive immune repertoire. Overall design: Flow-sorted murine meningeal cells (CD45–CD11b–), which were pre-labeled with hashtag antibodies (Biolegend), were pooled and loaded at 30,000 cells per channel. Flow-sorted macaque immune cell population (CD45–CD11b–CD3–) were loaded at 8,000 cells per channel. The cells were then partitioned into the GemCode instrument, where individual cells were lysed and mixed with beads carrying unique barcodes in individual oil droplets. The products were subjected to reverse transcription, emulsion breaking, cDNA amplification, and sample index attachment. Libraries were pair-end (150+ 150 bp) sequenced on a NovaSeq (Illumina).本研究报道，各年龄段小鼠脑膜中均存在早期发育的B细胞（B cell）。单细胞RNA测序（single cell RNA-sequencing, scRNA-seq）分析显示，小鼠与非人灵长类（non-human primates, NHPs）的脑膜发育B细胞存在连续分化轨迹。联体共生实验结合造血干细胞（hematopoietic stem cells, HSCs）谱系示踪结果表明，脑膜发育B细胞可通过不依赖血液循环的途径，持续由造血干细胞衍生的祖细胞补充。值得注意的是，识别中枢神经系统（central nervous system, CNS）特异性抗原髓鞘少突胶质细胞糖蛋白（myelin oligodendrocyte glycoprotein, MOG）的自身反应性未成熟B细胞会在脑膜中被清除，但不会在骨髓（bone marrow, BM）中被清除。此外，敲除MOG基因会使脑膜中允许自身反应性B细胞的发育、存在与定植。据此我们认为，脑膜可作为B细胞发育的独特储库，能够对中枢神经系统抗原反应性B细胞克隆实施原位阴性选择，从而维持局部免疫组库的非自身反应性特性。整体实验设计：经流式分选的小鼠脑膜细胞（CD45–CD11b–）预先经Biolegend公司的hashtag抗体（hashtag antibodies）标记，混合后以每通道30000个细胞的密度上样；流式分选的猕猴免疫细胞群（CD45–CD11b–CD3–）则以每通道8000个细胞的密度上样。随后将细胞置于GemCode仪器中进行分区：单个细胞在独立的油滴中完成裂解，并与带有独特条形码的磁珠混合。所得产物依次进行逆转录、破乳、cDNA扩增及样本索引标签连接。构建的测序文库采用Illumina NovaSeq测序平台进行双端（150+150 bp）测序。