Analysis of vascular Galectin-9 as an immunomodulator of B-cell activity

项目摘要 有效抗体(Ab)反应的发展严重依赖于幼稚B细胞的招募 进入次级淋巴器官，在抗原激活时，它们协调分化为生发 将(GC)B细胞集中到记忆B细胞和产生抗体的浆细胞。了解哪些机械性因素 然而，以增强或减弱抗体活性为治疗目标的对照B细胞亚群转变仍然是 人们对此知之甚少。来自我们实验室的令人振奋的新公布的数据突出了在 以I-线性或I-分支为特征的人类幼稚、GC和记忆B细胞亚群的血糖特征 聚-N-乙酰乳糖胺(PolyLacNAcs)而GC B细胞主要表达I分支的聚LacNAcs， 幼稚/记忆B细胞主要表现为I-线性聚-LacNAcs，这使其能够与免疫- 调节剂，半乳糖凝集素(Gal)-9。Gal-9结合导致下游抑制细胞增殖、激活和 信号与BCR-参与有关，有趣的是，它唤起了支持生存的活动。考虑到精挑细选 Gal-9-与幼稚/记忆B细胞结合，我们推测Gal-9是外周B细胞的生理“调谐器” 细胞活化和B细胞反应性。Gal-9在“反应性”淋巴结(LN)中分布的其他关键数据- 如扁桃体组织显示，虽然幼稚的B细胞表达内源性Gal-9，但Gal-9在 高内皮微静脉(HEV)。由于HEV开始与循环中的幼稚/记忆B细胞进行粘连接触 并密集分布在B细胞滤泡附近的皮质中，HEV在战略上准备诱导Gal-9- 依赖黏附/调节。Gal-9对淋巴组织中B细胞的空间、细胞和功能调控 器官仍不为人所知，是半乳糖凝集素免疫学领域的一大空白。我们的指导性假设是盖尔- HEV上的9可以结合循环中的幼稚/记忆B细胞，帮助它们招募到LNS中并传递 当B细胞穿过内皮并进入B细胞滤泡时，调节信号。在这个探索性的提案中， 我们将研究人内皮细胞(EC)来源的Gal-9对人幼稚B细胞黏附和 在Gal-9存在或不存在的情况下，免疫调节和Will分析B细胞归巢到外周LNS。这个 具体目标是：1.)研究Gal-9依赖的血管内皮细胞-B细胞黏附相互作用。至 细胞因子激活的人内皮细胞表达Gal-9对人免疫调节作用的分析 幼稚的B细胞。我们将采用我们独特的体验和创新的原代人体细胞模型，粘附性 检测、突变小鼠、基因编辑方法以研究ECs上的Gal-9是否支持幼稚的B细胞黏附， 归巢以及触发免疫调节活动。这些探索性研究直接挑战了当前 信条认为Galectins主要作为免疫调节剂发挥作用，为Gal9在促进B细胞死亡方面提供了另一种作用 细胞-内皮细胞黏附、层粘连蛋白归巢和外周B细胞活性的原位调控。重要的是，我们的发现将 揭示调节B细胞免疫的新靶点，并为更大范围的赠款提供必要的数据。

PROJECT ABSTRACT Development of effective antibody (Ab) responses is critically dependent on the recruitment of naïve B cells into secondary lymphoid organs and, upon antigen activation, their coordinated differentiation into germinal center (GC) B cells to memory B cells and Ab-producing plasma cells. Knowing which mechanistic factors control B cell subset transition with the therapeutic goal of Ab-boosting or -attenuating activity, however, is still poorly understood. Exciting new published data from our laboratory highlight striking differences in the glycomic signatures of human naïve, GC and memory B cell subsets featured by either i-linear or I-branched poly-N-acetyllactosamines (poly-LacNAcs). Whereas GC B cells express mainly I-branched poly-LacNAcs, naïve/memory B cells display principally i-linear poly-LacNAcs, which enable robust binding to immuno- modulator, galectin (Gal)-9. Gal-9-binding causes downstream inhibition of cell proliferation, activation and signaling related to BCR-engagement while, interestingly, evoking a pro-survival activity. Given the selective Gal-9-binding to naive/memory B cells, we speculate that Gal-9 serves as a physiologic “tuner” of peripheral B cell activation and B cell reactivity. Other key data on the distribution of Gal-9 in “reactive” lymph node (LN)- like tonsil tissue reveal that, while naïve B cells express endogenous Gal-9, Gal-9 expression is strongest on high endothelial venules (HEV). Since HEVs initiate adhesive contact with circulating naïve/memory B cells and are densely packed in the cortex adjacent to B cell follicles, HEVs are strategically poised to elicit Gal-9- dependent adhesion/regulation. The spatial, cellular and functional control of Gal-9 on B cells in lymphoid organs is still unknown and a major gap in the field of galectin immunology. Our guiding hypothesis is that Gal- 9 on HEV can bind circulating naïve/memory B cells and help recruit them into LNs as well as transmit regulatory signals as B cells traverse the endothelium and enter B cell follicles. In this exploratory proposal, we will examine the function of human endothelial cell (EC)-derived Gal-9 on human naïve B cell adhesion and immunoregulation and will assay for B cell homing to peripheral LNs in the presence/absence of Gal-9. The Specific Aims are: 1.) To study Gal-9-dependent vascular EC – B cell adhesive interactions and 2.) To analyze immunoregulatory effects of Gal-9 expressed by cytokine-activated human ECs on human naïve B cells. We will employ our unique experience and innovative primary human cell models, adhesion assays, mutant mice, gene editing methods to study whether Gal-9 on ECs supports naïve B cell adhesion, LN-homing as well as trigger immunoregulatory activity. These exploratory studies directly challenge current dogma that galectins largely function as immunoregulators, offering an alternative role for Gal-9 in facilitating B cell-EC adhesion, LN-homing and in situ peripheral control of B cell activity. Importantly, our findings will reveal novel targets for modulating B cell immunity and provide necessary data for a grant larger in scope.