Fc receptor-like 6 (FCRL6) defines pre-BCR dependent and independent pathways of natural antibody selection

B-1a cells produce 'natural' antibodies (Abs) to neutralize pathogens and clear neo self-antigens, but the fundamental selection mechanisms that shape their polyreactive repertoires are poorly understood. Here, we identified a B cell progenitor subset defined by Fc receptor-like 6 (FCRL6) expression, harboring innate-like defense, migration, and differentiation properties conducive for natural Ab generation Compared to FCRL6- pro B cells, the repressed mitotic, DNA damage repair, and signaling activity of FCRL6+ progenitors, yielded VH repertoires with biased distal Ighv segment accessibility, constrained diversity, and hydrophobic and charged CDR-H3 sequences. Beyond nascent autoreactivity, VH11 productivity, which predominates phosphatidylcholine-specific B-1a B cell receptors (BCRs), was higher for FCRL6+ cells as was pre-BCR formation, which was required for Myc induction and VH11, but not VH12, B-1a development. Thus, FCRL6 revealed unexpected heterogeneity in the developmental origins, regulation, and selection of natural Abs at the pre-BCR checkpoint with implications for autoimmunity and lymphoproliferative disorders. FCRL6+ and FCRL6- pro B (AA4.1+Lin-CD43+B220+CD19-IgM-) cells derived from the fetal livers of embryonic day 18 and bone marrow of adult 8-12 week old mice were sorted by flow cytometry from BALB/cJ strain mice. Single cell suspensions from leg bones were pooled from 5-10 adult male and female mice and 7-10 fetal livers were pooled from embryonic day 18 fetuses isolated from pregnant mothers according to the timing of vaginal plug formation. Data are from a total of eight samples that were isolated in duplicate from four independent sorts. Each sort yielded two samples per cell type (FCRL6+ and FCRL6- pro B cells).B-1a细胞可产生"天然"抗体（antibodies, Abs）以中和病原体并清除新生自身抗原（neo self-antigens），但调控其多反应性抗原受体库（repertoire）的核心选择机制仍未被充分阐明。本研究鉴定出一类以Fc受体样6（Fc receptor-like 6, FCRL6）表达为特征的B细胞祖细胞亚群，其携带有利于天然抗体生成的先天样防御、迁移及分化特性。相较于FCRL6阴性的前B细胞（pro B cells），FCRL6阳性祖细胞的有丝分裂、DNA损伤修复及信号转导活性受到抑制，由此产生的重链可变区（VH）抗原受体库呈现出偏倚的远端Ighv基因片段可及性、受限的多样性，以及带有疏水性与带电性的互补决定区H3（Complementarity-determining region 3 heavy chain, CDR-H3）序列。除初始自身反应性外，主导磷脂酰胆碱（phosphatidylcholine）特异性B-1a细胞B细胞受体（B cell receptor, BCR）的重链可变区11（VH11）的生成效率在FCRL6+细胞中更高，前B细胞受体（pre-B cell receptor, pre-BCR）的形成亦更为显著；而pre-BCR的形成是Myc原癌基因诱导及VH11型（而非VH12型）B-1a细胞发育所必需的。综上，FCRL6揭示了前BCR检查点处天然抗体的发育起源、调控与选择过程中存在未被预期的异质性，该发现对自身免疫性疾病（autoimmunity）及淋巴增殖性疾病（lymphoproliferative disorders）的研究具有重要启示。实验所用的FCRL6+与FCRL6-前B细胞（表型为AA4.1阳性、谱系标记阴性、CD43阳性、B220阳性、CD19阴性、IgM阴性的pro B细胞），均通过流式细胞术（flow cytometry）从BALB/cJ品系小鼠中分选得到，其来源为胚胎第18天（embryonic day 18）的胎肝以及成年8-12周龄小鼠的骨髓。实验将5-10只成年雌雄小鼠的腿骨制备的单细胞悬液（single cell suspensions）混合，同时将7-10只胚胎第18天胎鼠的胎肝混合——胎鼠的胎龄通过观察孕鼠阴道栓（vaginal plug）形成的时间确定。本研究数据共来自8个样本，均通过4次独立分选获得重复样本；每种细胞类型（FCRL6+与FCRL6-前B细胞）每次分选可获得2个样本。