PlexinD1-regulated intrathymic movement

描述（由申请方提供）：胸腺细胞成熟和胸腺结构依赖于精确的胸腺内细胞迁移。然而，胸腺细胞运输的协调在分子水平上还没有得到很好的理解。最近，我们描述了在CD 4 + CD 8+双阳性（DP）胸腺细胞上高度调节的plexinD 1表达，其通过T细胞受体/辅助受体接合进一步修饰。通过其配体，脑信号蛋白3E（sema 3E）激活丛蛋白D1，抑制CXCR 4/CXCL 12信号在预选择DP胸腺细胞和CCR 9/CCL 25信号在CD 69+后选择DP胸腺细胞。使用Plxnd 1缺陷的胎肝细胞移植小鼠，plexinD 1的丢失导致CD 69+胸腺细胞留在皮质中，原位成熟形成异位单阳性（SP）胸腺细胞簇。因此，DP和SP胸腺细胞之间的边界在皮质髓质交界处被破坏和髓质结构的形式下的胸腺囊。这些结果表明，通过调节趋化因子梯度的生物反应，在指导成熟的胸腺细胞迁移的plexinD 1的重要性。在Plxnd 1-/-和Sema 3e-/-敲除小鼠中观察到的畸形皮质髓质结构进一步与这一观点一致。为了解决胸腺发育中Plxnd 1缺陷的细胞相互作用和功能后果，本提案将追求三个主要目标。首先，我们将集中于PlexinD 1在调节CCR 9/CCL 25和CXCR 4/CXCL 12途径中的作用，以及使用Ccr 9-/-和Cxcr 4条件性敲除小鼠的DP胸腺细胞的定向迁移。具体而言，将评估这些趋化因子信号传导途径与Plxnd 1缺陷之间的联系以及相关的延迟胸腺细胞向预选（CD 69-）和后选（CD 69+）DP胸腺细胞的髓质迁移。第二，我们将通过多光子激发显微镜分析胎肝移植小鼠中表达GFP的（绿色）Plxnd 1-/-胸腺细胞与肾包膜下胎胸腺植入物内的tdTomato（红色）皮质胸腺上皮细胞相互作用的动力学，使用表达CFP的（蓝色）野生型细胞作为内部对照。第三，我们将使用OT-I/OT-II TCR和RIP-OVA转基因小鼠和胎肝细胞移植来分析Plxnd 1缺陷对T细胞发育过程中的阴性选择的影响，特别是筛选移植有Plxnd 1-/-胎肝细胞的小鼠以获得自身免疫现象的证据。总的来说，我们的研究将提供深入了解plexinD 1介导的调节DP胸腺细胞运动的胸腺内的胸腺细胞分化和选择，可能会导致自身免疫病理学在成熟的免疫系统的影响的分子基础。 公共卫生相关性：T细胞功能对于哺乳动物宿主保护免受各种感染性疾病和癌症的侵害至关重要。T细胞来自胸腺。因此，了解正常胸腺结构、分化和成熟所需的过程将有助于产生更有效的免疫力。

DESCRIPTION (provided by applicant): Thymocyte maturation and thymic architecture is dependent upon precise intrathymic cell migration. The orchestration of thymocyte trafficking, however, is not well understood at the molecular level. Recently, we described the highly-regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes which is further modified by T cell receptor/co-receptor engagement. Activation of plexinD1 via its ligand, semaphorin 3E (sema3E), represses CXCR4/CXCL12 signaling in pre-selection DP thymocytes and CCR9/CCL25 signaling in CD69+ post-selection DP thymocytes. Using Plxnd1-deficient fetal liver cell-transplanted mice, loss of plexinD1 causes CD69+ thymocytes to remain in the cortex, maturing in situ to form ectopic single positive (SP) thymocyte clusters. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions is disrupted and medullary structures form under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients. The dysmorphic corticomedullary structures observed in Plxnd1-/- and Sema3e-/- knockout mice are further consistent with this view. To address the cellular interactions and functional consequences of Plxnd1 deficiency in thymic development, the present proposal will pursue three major aims. First, we will focus on the role of plexinD1 in regulation of CCR9/CCL25 and CXCR4/CXCL12 pathways and the directed migration of DP thymocytes using Ccr9-/- and Cxcr4 conditional knockout mice. Specifically, the links between these chemokine signaling pathways and Plxnd1 deficiency as well as the associated retarded thymocyte migration towards the medulla of pre-selected (CD69-) and post-selected (CD69+) DP thymocytes will be assessed. Second, we will analyze by multi-photon excitation microscopy the dynamics of GFP- expressing (green) Plxnd1-/- thymocytes in fetal liver-transplanted mice interacting with tdTomato (red) cortical thymic epithelial cells within renal subcapsular fetal thymic implants, using CFP-expressing (blue) wild type cells as an internal control. Third, we will analyze the impact of Plxnd1 deficiency on negative selection during T cell development using OT-I/OT-II TCR and RIP-OVA transgenic mice and fetal liver cell transplantation, in particular screening mice transplanted with Plxnd1-/- fetal liver cells for evidence of autoimmune phenomena. Collectively, our studies will offer insight into the molecular basis of plexinD1-mediated regulation of DP thymocyte movements within the thymus with consequential effects upon thymocyte differentiation and selection that may lead to autoimmune pathology in the mature immune system. PUBLIC HEALTH RELEVANCE: T cell function is critical for mammalian host protection against a wide variety of infectious disorders and cancers. T cells emanate from the thymus. Hence, understanding of the processes required for normal thymic architecture, differentiation and maturation will contribute to generation of more effective immunity.