Molecular Analysis and Lineage Tracing of Influenza-Specific, Lung-Resident Memory B Cells

摘要 我们最近发表的数据表明，流感感染引起的记忆B细胞存在于淋巴细胞和巨噬细胞中。 组织和肺（Allie et al Nat Immunol 2019）。记忆B细胞在肺部不循环， 特性，包括广泛的交叉反应性，使其与淋巴对应物区分开来。我们称之为 这些单元是肺驻留存储器B单元或BRM单元。流感特异性BRM细胞迅速定植于肺， 它们在肺组织和肺气道中停留数月。攻毒感染后，肺BRM 细胞原位分化并成为有助于二级保护的抗体分泌细胞（ASC）。 我们的数据还表明，肺中的抗原沉积对于BRM细胞的形成是必不可少的，这可能是因为 BRM细胞在诱导型支气管相关类支气管组织（iBALT）中局部产生，或者因为BRM 在淋巴结中产生的前体需要在肺中重新遇到抗原，以便在肺中驻留。 那个位置总之，这些数据表明BRM细胞是免疫的重要组成部分， 然而，我们对BRM细胞的来源、它们是如何被感染的， 选择，它们与什么抗原反应，以及它们在接种疫苗或二次接种后如何被召回（或不召回） 感染我们的中心假设是，肺中的BRM细胞是由一个不同的亚群形成的， 应答性B细胞，被独特地选择用于广泛的反应性，并且仅对呼吸道抗原应答。 为了验证这一假设，我们将利用单细胞方法，使我们能够确定个别B细胞 通过转录组（单细胞RNseq）、BCR克隆型（单细胞BCRseq）、DNA条形码化的 表面标志物抗体（CITEseq）和克隆和表达的BCR的亲和力/特异性/交叉反应性 作为重组抗体（单细胞克隆）。使用这些方法来比较流感人群- 随时间推移和攻毒感染后肺、引流淋巴结、脾和血液中的特异性B细胞，或 通过接种疫苗，我们将能够确定各种组织（特别是肺）中的记忆B细胞是如何被激活的。 它们之间的相互关系，它们选择的深度，它们交叉反应的程度，以及它们如何被 通过接种疫苗或感染召回。这些信息将为我们提供一条清晰的道路， 这些疫苗可以引发广泛反应的BRM细胞，这些细胞可以回到肺部，并提供针对 各种各样的流感亚型。

ABSTRACT Our recently published data show that memory B cells elicited by influenza infection reside in both lymphoid tissues and the lung (Allie et al Nat Immunol 2019). Memory B cells in the lung do not recirculate and have properties, including broad cross-reactivity, that distinguish them from their lymphoid counterparts. We termed these cells lung-resident memory B cells or BRM cells. Influenza-specific BRM cells rapidly colonize the lung, where they reside in both the lung tissue and the lung airways for months. Upon challenge infection, lung BRM cells differentiate in situ and become antibody-secreting cells (ASCs) that contribute to secondary protection. Our data also show that antigen deposition in the lung is essential for BRM cell formation – perhaps because BRM cells are generated locally in inducible Bronchus-Associated Lymphoid Tissue (iBALT) or because BRM precursors generated in lymph nodes need to re-encounter antigen in the lung in order to become resident in that location. Taken together, these data suggest that BRM cells are an important component of immunity to influenza, however we have only a rudimentary understanding of where BRM cells come from, how they are selected, what antigens they react with and how they are recalled (or not) after vaccination or secondary infection. Our central hypothesis is that BRM cells in the lung are formed from a distinct subset of responding B cells, are uniquely selected for broad reactivity, and respond exclusively to respiratory antigens. To test this hypothesis, we will take advantage of single cell methods that allow us to define individual B cells by a combination of transcriptome (single cell RNseq), BCR clonotype (single cell BCRseq), DNA-barcoded antibodies to surface markers (CITEseq) and affinity/specificity/cross-reactivity of BCRs cloned and expressed as recombinant antibodies (single cell cloning). Using these methods to compare populations of influenza- specific B cells in the lung, draining lymph node, spleen and blood over time and after challenge infection or vaccination, we will be able to determine how memory B cells in various tissues (particularly the lung) are related to one another, the depth of their selection, the extent of their cross-reactivity and how they can be recalled by either vaccination or infection. This information will give us a clear path forward in designing vaccines that elicit broadly reactive BRM cells that home to the lung and provide long-lived immunity against a wide variety of influenza subtypes.