Transcriptomic diversity and overlapping clonality across subsets of antibody-secreting and memory B cells from spontaneous germinal centers

Affinity matured self-reactive antibodies are found in autoimmune diseases like systemic lupus erythematous. Here we used fate-mapping reporter mice and single cell transcriptomics coupled to antibody repertoire analysis to characterize the post-germinal center (GC) B cell compartment in a new mouse model of autoimmunity. Antibody secreting cells (ASCs) and memory B cells (MemBs) from spontaneous GCs grouped into multiple subclusters. ASCs matured into two terminal clusters, with distinct secretion, antibody repertoire and metabolic profiles. MemBs contained FCRL5+ and CD23+ subsets, with different in vivo localization in the spleen. GC-derived FCRL5+ MemBs share transcriptomic and repertoire properties with atypical B cells found in aging and infection and localize to the marginal zone, suggesting a similar contribution to rapid recall responses. While transcriptomically diverse, ASC and MemB subsets maintained an underlying clonal redundancy. Therefore, self-reactive clones could escape subset-targeting therapy by perpetuation of self-reactivity in distinct subsets. Chimeras were produced by transferring bone marrow from 564Igi and AicdaCreERT2-EYFP reporters (autoimmune) and AicdaCreERT2-EYFP and WT (immunized) into irradiate WT hosts. The latter were immunized with NP-OVA in alum, and boosted with NP-OVA. Fate-mapped AID-EYFP cells were isolated from spleens by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq.

在系统性红斑狼疮等自身免疫疾病中，可检测到亲和力成熟的自身反应性抗体。本研究借助命运图谱报告基因小鼠、单细胞转录组学（single cell transcriptomics）联合抗体库分析（antibody repertoire analysis），对一种新型自身免疫小鼠模型的生发中心后（germinal center, GC）B细胞区室进行了表征。源自自发性生发中心的抗体分泌细胞（Antibody secreting cells, ASCs）与记忆B细胞（memory B cells, MemBs）可分为多个亚簇。抗体分泌细胞可分化为两个终末亚簇，二者在分泌功能、抗体库及代谢特征上均存在显著差异。记忆B细胞包含FCRL5阳性（FCRL5+）与CD23阳性（CD23+）两个亚群，二者在脾脏内的定位存在差异。生发中心来源的FCRL5+记忆B细胞与衰老及感染状态下出现的非典型B细胞具有相似的转录组学与抗体库特征，且定位于脾脏边缘区，提示二者在快速回忆应答中发挥相似的作用。尽管抗体分泌细胞与记忆B细胞的亚群在转录组层面存在多样性，但二者仍保留了潜在的克隆冗余性。因此，自身反应性克隆可通过在不同亚群中维持自身反应性，逃避免针对亚群的靶向治疗。本研究构建了嵌合体小鼠：将来自564Igi与AicdaCreERT2-EYFP报告基因小鼠（自身免疫模型组）以及AicdaCreERT2-EYFP与野生型（wild type, WT）小鼠（免疫组）的骨髓细胞，移植至辐照后的野生型宿主小鼠体内。免疫组小鼠经明矾佐剂乳化的硝基苯卵清蛋白（NP-OVA）进行初次免疫，并以NP-OVA进行加强免疫。研究人员通过荧光激活细胞分选术（Fluorescence-activated cell sorting, FACS）从脾脏中分离出经命运图谱标记的AID-EYFP阳性细胞，并采用单细胞RNA测序（single-cell RNA sequencing, scRNAseq）进行分析。