Determining the role of NLRP3 on the placement and protective potential of CD73+ BRMs post influenza infection

项目摘要 关于抗原（Ag）特异性记忆B（BMEM）细胞，在公共卫生相关的背景下知之甚少。 病原体我们的研究清楚地解决了非循环、流感特异性、常驻记忆B的子集 BRM细胞（BRM）。BRM的建立依赖于早期CD 40信号传导，并维持CD 73表达。 淋巴结（LN）中CD 73-表达下降，而淋巴结（LN）中CD 73-表达下降。 人口这就提出了CD 73+和CD 73-在形成和功能上的内在差异的问题。 BRM。CD 73是LN中的生发中心（GC）迁移物的标志物，并且LN中的记忆细胞驻留在 靠近捕获和转移抗原的巨噬细胞和可以提供帮助的T细胞;因此， 我们假设CD 73 + BRM将归巢于肺中有组织的淋巴样结构区域， 依赖于T细胞的帮助我们的RNASeq数据显示NLRP 3（NOD样受体蛋白3），一种先天危险 传感复合物，是最上调的基因在CD 73 + BRM。据我们所知，NLRP 3活性不是 与B细胞分化相关，这使得这一发现新颖，并可能开创先天性研究。 在适应性反应中类似于B细胞的功能。NLRP 3活性产生白细胞介素B（IL-1 B），IL-1 B是 一种众所周知的白细胞介素6（IL-6）上调剂，可促进GC和浆细胞分化。作为我们 CD 73 + BRM可能有更高的NLRP 3表达，我们假设NLRP 3在回忆过程中是活跃的， 产生GC和ASC，而CD 73-BRM可能是肺中的哨兵， ASCs，以应对返回的病毒。 为了验证这些假设，我们提出以下问题：1）CD 73+和CD 73- BRM细胞是否依赖于GC？如果 因此，GC特异性阻断应减少肺中的CD 73 + BRM并产生低亲和力BCR。2)做CD 73 + BRM优先驻留在IBALT中？如果是这样，使用组织学，我们应该观察到它们聚集在 而CD 73-BRM分散在实质中。3)CD 73+和CD 73- BRM细胞是否应答 在功能分析中的不同？我们将通过对BRM进行体外测定来回答这个问题 使用细胞因子混合物和分选的T细胞将细胞分化为ASC。4)NLRP 3复合体是否会成为 在BRM中被激活如果是这样，使用NLRP 3炎性小体报告小鼠，我们预计， CD 73 + BRM细胞，而不是CD 73- BRM细胞，以增加NLRP 3活性。5)NLRP 3是否监管 BRM的形成或功能？如果是这样的话，B细胞特异性NLRP 3缺乏应该改变以下比例： CD 73+和CD 73-BRM及其保护功能，在发病率和死亡率方面存在明显差异。 这项研究的结果，尽管结果将是极其重要的，以允许解决一个 用于有效疫苗设计靶群体和潜在分子靶。

Project Summary Little is known about antigen (Ag) specific memory B (BMEM) cells, in the context of a public health relevant pathogen. Our studies clearly resolve the subsets of non-circulating, influenza specific, resident memory B cells (BRM) in the lung. BRM establishment relied on early CD40 signaling and maintained a CD73 (ectoenzyme)+ and CD73- population, whereas the lymph node (LN) showed a decline in the CD73- population. This raised the question of the inherent differences in formation and function of CD73+ and CD73- BRMs. CD73 is a marker of germinal center (GC) emigrants in the LN and memory cells in the LN reside in niches close to macrophages that capture and transfer antigen and T cells which can provide help; therefore, we hypothesized that the CD73+BRMs will home to areas of organized lymphoid structures in the lung and be dependent on T cell help. Our RNASeq data shows NLRP3 (NOD-like receptor protein 3), an innate danger sensing complex, to be the most upregulated gene in CD73+BRMs. To our knowledge NLRP3 activity is not associated with B cell differentiation, which makes this finding novel and may inaugurate studies into innate like functions in B cells during adaptive responses. NLRP3 activity produces interleukin B (IL-1B) and IL-1B is a well-known up regulator of interleukin 6 (IL-6), which promotes GCs and plasma cell differentiation. As our CD73+BRMs may have higher NLRP3 expression, we hypothesize that NLRP3 will be active during recall and produce GCs and ASCs, while the CD73-BRMs may be sentinels in the lung which spontaneously convert to ASCs, in response to the returning virus. To test these hypotheses, we ask the following: 1) Are CD73+ and CD73- BRM cells dependent on GCs? If so, GC-specific blockade should reduce CD73+ BRMs in the lung and produce low affinity BCRs. 2) Do CD73+ BRMs preferentially reside in iBALT? If so, using histology, we should observe them congregating in the iBALT while the CD73-BRMs are scattered in the parenchyma. 3) Do CD73+ and CD73- BRM cells respond differently in functional assays? We will answer this question by performing in vitro assays for BRM differentiation to ASCs, using cytokine cocktails and sorted T cells. 4) Does the NLRP3 complex become activated in BRMs upon viral challenge? If so, using NLRP3 inflammasome reporter mice, we expect that CD73+ BRM cells, but not CD73- BRM cells to increase NLRP3 activity. 5) Does NLRP3 regulate the formation or function of BRMs? If so, B cell specific NLRP3 deficiency should alter the proportions of CD73+ and CD73-BRMs and their protective function with observable differences in morbidity and mortality. The findings from this study despite the outcome will be extremely important to allow for the resolution of a target population and potential molecular targets for an effective vaccine design.