Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates. Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions. Overall design: Human naive B cells (CD19+CD27-IgG-IgD+) are sorted from healthy donor PBMCs and cultured on a feeder layer of human CD40L-expressing mouse fibroblasts and restimulated after six days. Cytokines typically expressed by follicular T cells (Tfh) (IL-21, IL-4) are added to mimic Tfh help for B cell differentiation. The dynamics of human naive B cell differentiation were investigated in detail by single-cell RNA sequencing. Cultured cells were single-cell sorted on day 11 based on the expression of CD27 and CD38 to obtain a faithful representation of cells at varying stages of B cell to ASC differentiation. Next, cells were processed for single-cell RNA sequencing using the SMARTseq2 method.

B细胞向抗体分泌细胞（antibody-secreting cells, ASCs）的分化是介导保护性体液免疫的核心过程。深入解析调控抗体分泌细胞分化的信号线索，对于开发调控抗体生成的相关策略具有重要意义。本研究利用单细胞RNA测序技术，解析了人类初始B细胞向抗体分泌细胞分化的完整轨迹。通过对比体外分化模型中不同分化阶段B细胞的转录组与离体（ex vivo）获取的B细胞及抗体分泌细胞，本研究首次发现了淋巴组织中天然存在的新型抗体分泌细胞前体（pre-ASC）群体。本研究首次从人类初始B细胞的体外培养体系中鉴定出类生发中心（germinal center, GC）群体，该群体或可通过一条非经典分化途径转化为记忆B细胞群体，从而在体外重现了人体内的生发中心反应过程。本研究成果为解析健康与疾病状态下人类B细胞向抗体分泌细胞或记忆B细胞的分化过程，提供了更为精细的表征基础。

实验设计：从健康志愿者的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）中分选得到人类初始B细胞（CD19+CD27-IgG-IgD+），将其接种于表达人源CD40配体的小鼠成纤维细胞饲养层上进行培养，并于培养第6天进行二次刺激。添加滤泡辅助性T细胞（follicular T helper cell, Tfh）特异性分泌的细胞因子IL-21与IL-4，以模拟滤泡辅助性T细胞对B细胞分化的辅助作用。本研究通过单细胞RNA测序技术，对人类初始B细胞的分化动态过程进行了精细解析。于培养第11天，根据细胞表面CD27与CD38的表达水平对培养细胞进行单细胞分选，以精准获取处于B细胞向抗体分泌细胞分化不同阶段的细胞群体。随后采用SMARTseq2方法对分选得到的细胞进行单细胞RNA测序。