PlexinD1-regulated intrathymic movement

描述（申请人提供）：胸腺细胞成熟和胸腺结构依赖于胸腺内细胞的精确迁移。然而，胸腺细胞运输的编排在分子水平上还没有得到很好的理解。最近，我们描述了plexinD1在CD4+CD8+双阳性（DP）胸腺细胞中的高度调控表达，并通过T细胞受体/共受体结合进一步修饰。plexinD1通过其配体信号蛋白3E （sema3E）激活，抑制预选择DP胸腺细胞中的CXCR4/CXCL12信号和CD69+后选择DP胸腺细胞中的CCR9/CCL25信号。用plxnd1缺失的胎肝细胞移植小鼠，plexinD1缺失导致CD69+胸腺细胞留在皮质，原位成熟形成异位单阳性（SP）胸腺细胞簇。因此，DP和SP胸腺细胞在皮质-髓质连接处的边界被破坏，胸腺被膜下形成髓质结构。这些结果表明，plexinD1通过调节趋化因子梯度的生物学反应，在指导成熟胸腺细胞迁移中的重要性。在Plxnd1-/-和Sema3e-/-敲除小鼠中观察到的皮质髓质结构畸形进一步与这一观点一致。为了解决Plxnd1缺陷在胸腺发育中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，我们将利用CCR9 -/-和CXCR4条件敲除小鼠研究plexinD1在CCR9/CCL25和CXCR4/CXCL12通路调控中的作用，以及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通路与Plxnd1缺陷之间的联系，以及相关的预先选择（CD69-）和后选择（CD69+） DP胸腺细胞向髓质的迁移迟缓。其次，我们将通过多光子激发显微镜分析胎儿肝移植小鼠中表达GFP（绿色）的Plxnd1-/-胸腺细胞与tdTomato（红色）皮质胸腺上皮细胞在肾包膜下胎儿胸腺植入物中相互作用的动力学，以表达cfp（蓝色）的野生型细胞作为内部对照。第三，我们将利用OT-I/OT-II TCR和rib - ova转基因小鼠和胎肝细胞移植，分析Plxnd1缺乏对T细胞发育过程中负选择的影响，特别是筛选移植了Plxnd1-/-胎肝细胞的小鼠，寻找自身免疫现象的证据。总的来说，我们的研究将深入了解plexind1介导的胸腺内DP胸腺细胞运动调控的分子基础，并对胸腺细胞分化和选择产生相应的影响，从而可能导致成熟免疫系统中的自身免疫病理。

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.