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

项目摘要 有效抗体（Ab）应答的产生严重依赖于幼稚B细胞的募集 进入次级淋巴器官，并在抗原激活后，它们协调分化为胚细胞 中心（GC）B细胞到记忆B细胞和产生Ab的浆细胞。了解哪些机械因素 然而，具有Ab-增强或-减弱活性的治疗目标的对照B细胞亚群转变仍然是 不太了解。来自我们实验室的令人兴奋的新发表数据突出了 人幼稚、GC和记忆B细胞亚群的糖组学特征，特征为i-线性或I-分支 聚-N-乙酰基乳糖胺（聚-LacNAc）。而GC B细胞主要表达I-分支的聚-LacNAc， 幼稚/记忆B细胞主要显示i-线性多聚LacNAc，其能够与免疫球蛋白牢固结合。 半乳糖凝集素（Gal）-9。Gal-9结合引起下游细胞增殖、活化和增殖的抑制。 与BCR接合相关的信号传导，同时有趣的是，引起促存活活性。鉴于选择性 Gal-9与幼稚/记忆B细胞结合，我们推测Gal-9作为外周B细胞的生理“调谐器 细胞活化和B细胞反应性。关于Gal-9在“反应性”淋巴结（LN）中分布的其他关键数据- 类似扁桃体组织显示，虽然幼稚B细胞表达内源性Gal-9，但Gal-9表达在 高内皮微静脉（HEV）。由于HEV启动与循环幼稚/记忆B细胞的粘附接触 并且密集地堆积在邻近B细胞滤泡的皮质中，HEV策略性地准备好引发Gal-9- 依赖性粘附/调节。Gal-9对淋巴细胞中B细胞的空间、细胞和功能调控 器官仍然是未知的，并且是半乳糖凝集素免疫学领域的主要空白。我们的指导假设是加尔- HEV上的9可以结合循环中的幼稚/记忆B细胞，并帮助将它们招募到LN中， 当B细胞穿过内皮并进入B细胞滤泡时，调节信号。在这个探索性的提议中， 我们将研究人内皮细胞（EC）衍生的Gal-9对人幼稚B细胞粘附的功能，并 免疫调节，并将在存在/不存在Gal-9的情况下测定B细胞归巢至外周LN。的 具体目标是：（1）研究Gal-9依赖的血管EC - B细胞粘附相互作用和2.）到 分析由精氨酸活化的人EC表达的Gal-9对人的免疫调节作用。 幼稚B细胞。我们将利用我们独特的经验和创新的原代人类细胞模型， 测定、突变小鼠、研究EC上的Gal-9是否支持幼稚B细胞粘附的基因编辑方法， LN归巢以及触发免疫调节活性。这些探索性研究直接挑战了当前的 半乳糖凝集素在很大程度上起免疫调节剂的作用，为Gal-9在促进B中提供了替代作用 细胞-EC粘附、LN归巢和B细胞活性的原位外周控制。重要的是，我们的发现将 揭示了调节B细胞免疫的新靶点，并为更大范围的资助提供了必要的数据。

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.