Fc receptor-like 4 (FCLR4) and Fc receptor-like receptor 5 (FCRL5) define and control human atypical memory B cells [RNA-Seq]

We performed NGS on Ig heavy chain V sequences amplified from RNA obtained from human tonsillar B cell subsets. From these NGS data, we reconstructed ongoing B cell responses as lineage trees, providing crucial in vivo developmental context. Each memory subset typically maintained its lineage, denoting mechanisms enforcing their phenotypes. FCRL4-FCRL5+ cells were the most represented memory subset in lineage trees, indicating robust participation in ongoing responses. Affinity maturation of both FCRL4-FCRL5+ and FCRL4+FCRL5+ atypical memory cells was stalled in chronic streptococcus infection, conceivably due to negative feedback from IgG immune complexes. We propose that FCRL4 and FCRL5 function as sensors of immune complexes, thus controlling atypical memory B cell responses. B cells were isolated from human tonsil cells and FACS-sorted into subsets on the basis of CD19-, IgD CD38, FCRL4, and FCRL5 surface expression. CD38-IgD- memory cells were sorted into three subsets: FCRL4- FCRL4+ (F5), FCRL4- FCRL5- (DN) and FCRL4+ FCRL5+ (DP). In addition, IgD+CD38- naïve/IgD+ memory cells(Naive), IgD-D38+ GC cells(GC) , and IgD-CD38++ plasmablasts (PB) subsets were also sorted . IgM, IgA or IgG HC variable regions were separately amplified from each sorted subset and these amplified samples were subjected to deep sequencing.

我们针对从人类扁桃体B细胞亚群的RNA扩增得到的免疫球蛋白重链可变区序列，开展了下一代测序（Next Generation Sequencing，NGS）。基于上述NGS数据，我们重构了正在进行的B细胞应答的谱系树，为体内发育过程提供了关键的背景信息。每个记忆细胞亚群通常维持其自身的谱系，这表明存在维持其表型的调控机制。Fc受体样蛋白4（FCRL4）-Fc受体样蛋白5（FCRL5）阳性细胞是谱系树中占比最高的记忆细胞亚群，提示其在当前免疫应答中发挥了活跃的参与作用。在慢性链球菌感染状态下，FCRL4-FCRL5+与FCRL4+FCRL5+非典型记忆细胞的亲和力成熟过程受阻，这一现象可能源于IgG免疫复合物介导的负反馈调控。我们提出，FCRL4与FCRL5可作为免疫复合物的感受器，进而调控非典型记忆B细胞的应答过程。我们从人类扁桃体细胞中分离B细胞，并基于CD19阴性、IgD、CD38、FCRL4及FCRL5的表面表达水平，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting，FACS）将其分为不同亚群。其中，CD38-IgD-记忆细胞被进一步分为三个亚群：FCRL4-FCRL5+（F5）、FCRL4-FCRL5-（DN）以及FCRL4+FCRL5+（DP）。此外，我们还分选了IgD+CD38-初始/IgD+记忆细胞（Naive）、IgD-CD38+生发中心（Germinal Center，GC）细胞以及IgD-CD38++浆母细胞（plasmablasts，PB）亚群。我们针对每个分选得到的亚群分别扩增了IgM、IgA或IgG重链可变区，并对这些扩增产物进行了深度测序。