Definition of follicular stromal cell subset interactions with B cells

描述（由申请方提供）：淋巴组织内免疫应答的启动涉及活化淋巴细胞在复杂的静止基质细胞网络内精心协调的运动。类肉瘤组织结构由基于基质细胞复杂排列的可区分的微解剖学“邻域”组成。由于基质细胞亚群分泌独特的趋化因子组合，B细胞滤泡内的趋化因子指纹可与其相邻的T细胞区区分开。通过活化后趋化因子受体表达的变化，抗原接合的B和T细胞重新定位以聚集在新的小生境内，促进它们的同源相遇并使关键效应物分化的关键步骤成为可能。虽然在宏观上，幼稚B细胞在整个卵泡中的均匀分布可能表明在卵泡发育中也存在类似的均匀性。 固定的结构基础，而是有许多基质细胞亚群位于卵泡内。除了充分表征的滤泡树突细胞（FDC）外，B细胞滤泡还含有淋巴和血液内皮细胞，后者被周细胞覆盖。成纤维细胞网状细胞环绕在最靠近T细胞区的卵泡边缘的内侧边缘，其中被膜下窦衬里是更多细胞类型的集合，包括具有下降树突的边缘网状细胞。尽管在初始激活事件中基质细胞在定义淋巴细胞定向的标志中起着关键作用，但B细胞与滤泡基质相互作用的许多基本方面尚不清楚。在这里，我们建议检查之间的相互作用B细胞和基质细胞亚群，使滤泡的基础设施。目的1：明确B细胞滤泡的基质细胞结构。使用荧光骨髓嵌合体的活体显微镜检查，将确定血管和淋巴管维斯维斯FDC和其他滤泡内基质细胞的相对位置。我们将评估免疫前后它们的并列、间距和相关细胞类型的一致性。目的2：通过活体显微镜确定幼稚和活化B细胞与基质细胞亚群相互作用的倾向。将与基质接触的B细胞之间的形态动力学和推进模式与缺乏基质的区域内的那些进行比较，并确定血液或淋巴携带的抗原/佐剂对该行为的影响。运动参数将定量基质间隙内的原始和活化的B细胞，以检验这样的运动是短暂的和从一个基质接触到另一个基质接触的定向转移的假设。目的3：原位检测B细胞与基质细胞亚群接触期间潜在信号事件的细胞内指标。通过组织学检查与基质细胞相邻的具有突触形态的B细胞的Notch、BMP和BCR介导的信号传导的细胞内指示物，与基质细胞亚群接触一致。为了检验这样的接触支持BCR衍生的紧张性信号传导的假设，我们将在荧光BM嵌合体的LN内对用NFAT-eGFP逆转录病毒构建体转染的B细胞进行活体成像。

DESCRIPTION (provided by applicant): The initiation of immune responses within lymphoid tissue involves a carefully orchestrated movement of activated lymphocytes within an elaborate network of stationary stromal cells. Lymphoid tissue architecture is comprised of distinguishable microanatomic "neighborhoods" that are based on a complex arrangement of stromal cells. Due to the secretion of distinctive combinations of chemokines by stromal cell subsets, the chemokine fingerprint within B cell follicles is distinguishable from its neighboring T cell zone. Through shifts in chemokine receptor expression after activation, antigen engaged B and T cells reposition themselves to congregate within new niches, promoting their cognate encounters and enabling key steps in the differentiation of critical effectors. Although on a macro scale, the uniform distribution of na¿ve B cells throughout the follicle might suggest a similar uniformity in the stationary structural underpinnings, there are instead many stromal cell subsets positioned within follicles. In addition to the well characterized follicular dendritic cell (FDC), B cell folicles also harbor lymph and blood endothelial cells, the later covered by pericytes. Fibroblastic reticular cells rim the inside edge of the border of the follicle closest to the T cell zone, wheres the subcapsular sinus lining is a collection of yet more cell types including a marginal reticular cell with descending dendrites. Despite the critical role of stromal cells in defining the landmark for lymphocyte orientation during initial activation events, many fundamental aspects of B cell interactions with follicular stroma are unknown. Here we propose to examine the interactions between B cells and the stromal cell subsets that make up the follicular infrastructure. Aim 1: Define the stromal cell framework of B cell follicles. Using intravital microscopy of fluorescent bone marrow chimeras, the relative positioning of blood and lymph vessels vis-¿-vis FDCs and other intra-follicular stromal cells will be defined. We will assess the consistency of their juxtapositions, spacing and associated cell types before and after immunization. Aim 2: Define the propensity of na¿ve and activated B cells to interact with stromal cell subsets via intravital microscopy. The morphodynamics and mode of propulsion between B cells in contact with stroma will be compared to those within regions that lack stroma, and the impact of blood or lymph-borne antigen/adjuvants on this behavior determined. Motility parameters of na¿ve and activated B cells within stromal gaps will be quantified to test the hypothesis that such movement is transient and a directed shift from one stromal contact to another. Aim 3: Examine B cells in situ for intracellular indicators of potential signaling events during contacts with strmal cell subsets. B cells with synaptic morphology adjacent to stromal cells will be examined by histology for intracellular indicators of Notch, BMP and BCR mediated signaling coincident with stromal cell subset contacts. To test the hypothesis that such contacts support BCR derived tonic signaling, we will perform intravital imaging of B cells transfected with a NFAT-eGFP retroviral construct within LNs of fluorescent BM chimeras.