T-bet regulates the maintenance and ASC differentiation potential of lymph node and lung effector memory B cell subsets

While human and mouse memory B cells (Bmem) can express T-bet, its role in regulating Bmem function is largely unknown. We characterized multiple transcriptionally distinct clusters of mature, somatically-mutated nucleoprotein (NP)-specific Bmem in LNs and lungs of influenza-infected mice. Although none of the Bmem expressed the plasma cell (PC) lineage commitment factors Blimp1, one cluster was enriched for Tbx21+ cells. Like the previously described human T-bet+ effector Bmem (eBmem) population, Tbx21+ mouse Bmem upregulated gene networks associated with effector metabolism, protein synthesis and the unfolded protein response. Using constitutive and inducible mouse models to ablate T-bet in B cells, we showed that T-bet expression by Bmem was required for persistence of LN and lung eBmem with rapid in vitro and in vivo PC differentiation potential. Thus, T-bet marked NP+ eBmem that were poised to differentiate and was required for maintenance of eBmem that respond in the lung following viral re-exposure. Overall design: On two separate days, on D30 following PR8 (H1N1) influenza infection mdLN from Tbx21 reporter were isolated and pooled. Influenza specifc (NP+) and non-specific (NPneg) memory B cells were isolated by fluorescence activated cell sorting (FACS) and stained with CITE-seq antibodies (ADT and HTO). GEX, VDJ, and ADT libraries were prepared using the 10X platform.

尽管人类与小鼠的记忆B细胞（memory B cell, Bmem）均可表达T-bet（T-bet），但其在调控记忆B细胞功能中的作用仍知之甚少。本研究对流感感染小鼠的淋巴结（lymph nodes, LNs）与肺脏内多个转录特征迥异的成熟体细胞突变核蛋白（nucleoprotein, NP）特异性记忆B细胞聚类开展了表征分析。尽管所有记忆B细胞均不表达浆细胞（plasma cell, PC）谱系定型因子Blimp1，但其中一个聚类显著富集Tbx21阳性细胞。与此前已报道的人类T-bet阳性效应记忆B细胞（effector Bmem, eBmem）群体特征一致，Tbx21阳性小鼠记忆B细胞会上调与效应代谢、蛋白质合成及未折叠蛋白反应相关的基因网络。本研究通过组成型与诱导型小鼠模型敲除B细胞中的T-bet，证实记忆B细胞的T-bet表达对于维持淋巴结与肺脏中具备快速体外（in vitro）及体内（in vivo）浆细胞分化潜能的效应记忆B细胞存活不可或缺。综上，T-bet可标记处于分化预备状态的NP阳性效应记忆B细胞，且对维持病毒再次暴露后肺脏内响应性效应记忆B细胞的稳态至关重要。

总体实验设计：于PR8（H1N1）流感病毒感染后的第30天，分别在两个独立日期获取Tbx21报告小鼠的内侧引流淋巴结（medial draining lymph nodes, mdLN）并混合样本。通过荧光激活细胞分选（fluorescence activated cell sorting, FACS）分离流感特异性（NP+）与非特异性（NPneg）记忆B细胞，并用CITE-seq抗体（ADT与HTO）进行标记。采用10X平台构建基因表达（GEX）、VDJ及ADT测序文库。