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，由流感感染引发的记忆B细胞驻留在两个淋巴中 组织和肺(Allie等人NAT免疫学2019年)。肺中的记忆B细胞不再循环， 性质，包括广泛的交叉反应，区别于它们的淋巴对应物。我们称之为 这些细胞驻留在肺内的记忆B细胞或BRM细胞。流感特异性BRM细胞迅速在肺部定居， 在那里它们同时存在于肺组织和肺部呼吸道中数月之久。在挑战感染时，肺BRM 细胞在原位分化，成为抗体分泌细胞(ASCs)，有助于二级保护。 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--可能是因为 BRM细胞在可诱导的支气管炎相关淋巴组织(IBALT)中局部产生，或者是因为BRM 在淋巴结中产生的前体需要再次遇到肺中的抗原才能驻留在 那个位置。综上所述，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细胞来自哪里，它们是如何形成的，我们只有一个初步的了解 选择，它们与什么抗原反应，以及在接种疫苗或二次接种后如何召回(或不召回) 感染。我们的中心假设是，肺中的BRM细胞是由不同的 反应性B细胞是唯一具有广泛反应性的细胞，仅对呼吸道抗原有反应。 为了验证这一假设，我们将利用单细胞方法来定义单个B细胞 通过转录组(单细胞RNseq)、bcr克隆型(单细胞BCRseq)、DNA条形码 表面标志物抗体(CITEseq)及其亲和力/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的种群- 肺部、引流淋巴结、脾和血液中的特定B细胞随着时间的推移以及在攻击感染或 接种疫苗后，我们将能够确定不同组织(特别是肺)中的记忆B细胞是如何 相互关联，他们选择的深度，他们交叉反应的程度，以及他们如何 因接种疫苗或感染而被召回的。这些信息将为我们在设计中指明前进的道路 诱导广泛反应的BRM细胞的疫苗，这些细胞归巢于肺部，并提供针对AFP的长期免疫力 流感亚型种类繁多。

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.