Definition of follicular stromal cell subset interactions with B cells

描述(由申请人提供)：淋巴组织内免疫反应的启动涉及到激活的淋巴细胞在一个精心设计的静止基质细胞网络中的移动。淋巴组织结构由可区分的显微解剖“邻域”组成，这些“邻域”以基质细胞的复杂排列为基础。由于基质细胞亚群分泌不同的趋化因子组合，B细胞滤泡内的趋化因子指纹可与其邻近的T细胞区区分开来。通过激活后趋化因子受体表达的变化，参与抗原的B和T细胞重新定位自己，聚集在新的利基环境中，促进它们的同源相遇，并使关键效应器分化的关键步骤成为可能。尽管在宏观范围内，NA？ve B细胞在整个卵泡中的均匀分布可能暗示着在 在固定的结构基础上，有许多基质细胞亚集位于毛囊内。除了滤泡树突状细胞(FDC)，B细胞小叶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位于滤泡边缘最靠近T细胞区的内缘，包膜下窦壁是多种细胞类型的集合，包括边缘网状细胞，树突向下生长。尽管在最初的激活事件中，间质细胞在确定淋巴细胞取向的里程碑中起着关键作用，但B细胞与滤泡间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B细胞和构成毛囊基础设施的基质细胞亚群之间的相互作用。目的1：明确B细胞滤泡的基质细胞骨架。使用荧光骨髓嵌合体的活体显微镜，可以确定血管和淋巴管相对于FDCs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位置、间距和相关细胞类型的一致性。目的2：通过活体显微镜，确定原始和活化B细胞与基质细胞亚群相互作用的倾向。与基质接触的B细胞之间的形态动力学和推进方式将与缺乏基质的区域内的B细胞进行比较，并确定血液或淋巴携带的抗原/佐剂对这一行为的影响。间质间隙内的原始和激活B细胞的运动参数将被量化，以检验这种运动是瞬时的和从一个间质接触到另一个间质接触的定向转移的假设。目的3：原位检测B细胞与间质细胞亚群接触过程中潜在信号事件的细胞内指示物。与基质细胞相邻的突触形态的B细胞将通过组织学检查Notch、BMP和BCR介导的信号与基质细胞亚群接触的细胞内指标。为了验证这种接触支持BCR衍生的紧张性信号的假设，我们将对在荧光BM嵌合体的LNS中导入NFAT-EGFP逆转录病毒构建的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.