Thymocyte integrin regulation by plexinD1

描述（由申请人提供）：适应性T细胞免疫应答基于区分自身与非自身的能力。这种功能性源于胸腺内选择，其消除具有自身反应性T细胞受体（TCR）特异性的胸腺细胞，同时促进具有有用TCR的胸腺细胞的发育。库的形成依赖于胸腺细胞在离散发育阶段通过胸腺上皮细胞（TEC）呈递的自身肽-MHC复合物的手套的迁移，从而实现适当的阴性（凋亡）和阳性（存活）TCR选择过程。然而，在双阳性（DP）阶段，胸腺细胞表现出迁移特性，不容易解释简单的趋化因子梯度的反应，这表明存在额外的指导线索。我们已经表明，DP胸腺细胞plexinD 1调节表面表达施加控制他们的迁移在皮质向髓质方向在选择过程中。通过在TCR转基因背景下建立Plxnd 1条件性敲除（CKO）小鼠，我们现在发现，在Plxnd 1缺失后DP胸腺细胞的异常皮质定位严重影响阴性选择。plexinD 1如何在分子方面发挥这种控制作用是目前提案的重点。我们证明，plexinD 1持有B1-整合素在DP胸腺细胞的活性状态，允许坚持TEC表达的VCAM-1和层粘连蛋白。plexinD 1与其可溶性髓质配体sema 3E的相互作用释放粘附，而Plxnd 1 CKO DP胸腺细胞的相互作用是组成性缺陷的。引人注目的是，粘附和它的调节sema 3E是动态的，只有在力下表现为发生在细胞迁移。通过生物膜力探针（BFP）对单个a4 b1-VCAM-1键的询问表明这些是捕获键，并且sema 3E与丛蛋白D1的相互作用释放整联蛋白捕获键以减弱动态粘附。我们的研究结果代表了在任何生理系统中捕获键调节的第一次识别。在 目标1我们将使用高分辨率dSTORM图像分析和BFP技术来表征影响亲和力的纳米级粘连片中plexinD 1与b1-和b2-整联蛋白的关系。此外，我们将表征单个整合素分子的构象及其与特定配体的功能键。在目标2中，我们将确定与丛蛋白D1相互作用的蛋白质，包括调节GTP酶的相互作用组成分（如介导与整合素广泛串扰的GAP和GEF）和稳定整合素与肌动蛋白细胞骨架相互作用的蛋白质。将确定这些蛋白相互作用的plexinD 1上的对接位点。在目标3中，我们将使用可行的胸腺切片测定来探测在迁移过程中DP胸腺细胞整合素的plexinD 1调节，然后将这些结果扩展到导致自身免疫的Plxnd 1 CKO和种系Sema 3e-/-小鼠中的阴性选择的体内分析。这些结果将为控制胸腺细胞迁移和分化过程中整合素粘附奠定新的基础。我们的研究结果对整合素功能的调节具有普遍意义，包括轴突寻路、血管生成和肿瘤转移。

DESCRIPTION (provided by applicant): The adaptive T cell immune response is predicated upon the ability to discriminate self from non-self. This functionality arises from intrathymic selection that eliminate thymocytes bearing autoreactive T cell receptor (TCR) specificities while fostering development of thymocytes with useful TCRs. Repertoire formation is dependent on migration of thymocytes at discrete developmental stages through a gauntlet of thymic epithelial cell (TEC) presented self-peptide-MHC complexes enabling appropriate negative (apoptosis) and positive (survival) TCR selection processes. At the double positive (DP) stage, however, thymocytes exhibit migratory properties not easily explained by responses to simple chemokine gradients, suggesting the existence of additional guidance cues. We have shown that regulated surface expression of plexinD1 on DP thymocytes exerts control over their migration in the cortical to medullary direction during selection. By creating Plxnd1 conditional knockout (CKO) mice on a TCR transgenic background, we now show that aberrant cortical location of DP thymocytes following loss of Plxnd1 severely impacts negative selection. How plexinD1 exerts this control in molecular terms is the focus of the current proposal. We demonstrate that plexinD1 holds b1- integrins in an active state on DP thymocytes, allowing adherence to TEC-expressed VCAM-1 and laminin. The interaction of plexinD1 with its soluble medullary ligand, sema3E, releases adhesion, while that of Plxnd1 CKO DP thymocytes is constitutively defective. Strikingly, adhesion and its regulation by sema3E is dynamic, only being manifest under force as occurs upon cell migration. Interrogation of the individual a4b1-VCAM-1 bonds by biomembrane force probe (BFP) indicates that these are catch bonds, and that the sema3E interaction with plexinD1 releases the integrin catch bonds to attenuate dynamic adhesion. Our results represent the first identification of catch bond regulation in any physiological system. In Aim 1 we will use high resolution dSTORM image analysis and BFP techniques to characterize the relationship of plexinD1 to b1- as well as b2-integrins in nanoscale adhesion patches influencing avidity. Additionally, we shall characterize conformation of individual integrin molecules and their functional bonds with specific ligands. In Aim 2 we will identify the proteins interacting with plexinD1 including those interactome components regulating GTPases (such as GAPs and GEFs that mediate extensive crosstalk with integrins) and those stabilizing integrin interactions with the actin cytoskeleton. The docking sites on plexinD1 for these protein interactions will be determined. In Aim 3 we will use viable thymic slice assays to probe plexinD1 regulation of DP thymocyte integrins during migration, and then extend these results to in vivo analysis of negative selection in Plxnd1 CKO and germline Sema3e-/- mice leading to autoimmunity. These results will lay a new foundation for control of integrin adhesion during thymocyte migration and differentiation. Our findings have implications for regulation of integrin function generally, including in axon pathfinding, angiogenesis and tumor metastasis.