A Thymus - Bone Marrow Feedback Loop for Prothymocytes

胸腺 - 前胸腺细胞的骨髓反馈环路

• 批准号: 7061194
• 负责人: IRVING GOLDSCHNEIDER
• 金额: $ 35.4万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7061194
• 关键词: T lymphocyte; antibody; athymic mouse; binding sites; bone marrow; bromodeoxyuridine; capillary; cell adhesion molecules; cell migration; cell proliferation; chemical kinetics; chemoattractants; chemokine; developmental immunology; extracellular matrix; gene expression profiling; genetically modified animals; green fluorescent proteins; hematopoietic stem cells; interleukin 7; lymphopoiesis; northern blottings; passive immunization; thymectomy; thymus

DESCRIPTION (provided by applicant): Having identified a regulatory feedback loop in adult mice that coordinates the cyclical exportation of prothymocytes from the bone marrow with their gated importation by the thymus, we now plan to define the underlying mechanisms for this thymus-BM feedback loop. By analogy with the fetal thymus, we postulate that the cyclical generation and mobilization of prothymocytes in the adult mouse is due to the periodic release of chemoattractive factors from the thymus. We further postulate that gate-opening reflects the expression of a unique pattern of chemokine/adhesion/ECM molecules by PCVs in the C-M junction of the thymus; and that gate-closing is initiated and maintained by TN1 and TN2 thymocytes, possibly by an IL-7 dependent mechanism. Four specific aims are proposed to test these hypotheses: 1) to determine if the periodic release of prothymocytes from the BM is associated with their cyclical generation; 2) to determine if prothymocyte generation and mobilization is affected by diffusible chemotactic factors from the thymus; 3) to identify and characterize the intrathymic gates and niches for prothymocytes; 4) to determine which stages of early thymocytopoiesis are associated with gate opening and closing. The kinetics of prothymocyte proliferation in BM will be determined by BrdU-induced suicide and by DNA staining of purified prothymocytes with Hoechst 33342. The influence of the thymus on the kinetics of prothymocyte generation and mobilization will be studied in nu/nu and adult thymectomized, as well as normal, mice; as will the effects of thymus-derived chemoattractant factors and antibodies thereto. Purified prothymocytes from GFP transgenic mice will be used to identify binding sites (putative IMV-gates) along PCVs in the thymus of sublethally-irradiated mice, as well as the localization of these cells in putative IMN-niches. The gene expression profile of chemokine/adhesion/ECM molecules of purified high endothelial cells (HECs) from the prothymocyte-receptive LNs of Oncostatin-M and LIF transgenic mice will be determined by RNA hybridization. The involvement of candidate molecules in prothymocyte recruitment will be verified by antibody-staining/blocking studies, and the phenotype of these altered LN HEVs will be compared with that of the PCVs in the receptive thymus. The minimal cellular requirements for IMV-gate opening and closing, and the possible role of IL-7, wilt be determined by Lv. and Lt. adoptive transfer of prothymocytes from CD3 transgenic, RAG.-/-. and IL-7Ra -/- mice, and by the i.t. transfer of purified thymocyte subsets from WT mice. These results will provide basic information into the thymus-BM regulatory loop for prothymocytes, and may also have therapeutic implications regarding selective or enhanced T cell reconstitution, gene therapy, and classification/treatment of immunodeficiency disorders.

描述（由申请人提供）：确定了成年小鼠的调节反馈回路，该反馈环协调了从骨髓中的蛋白细胞周期性输出以及胸腺的门控进口，我们现在计划定义此Thymus-BM反馈回路的基本机制。通过与胎儿百里香的类似，我们假设成年小鼠中泼刺细胞的周期性产生和动员是由于定期从胸腺中的趋化因子释放。我们进一步假设，开门反映了PCV在胸腺的C-M交界处的趋化因子/粘附/ECM分子的独特模式的表达。闭合闸门是由TN1和TN2胸腺细胞启动和维持的，可能是由IL-7依赖机制。提出了四个特定的目的来检验这些假设：1）确定从BM中定期释放蛋白细胞是否与其周期性产生有关； 2）确定促炎细胞的产生和动员是否受到胸腺的扩散趋化因子的影响； 3）识别和表征胸膜细胞的胸膜内门和壁ni； 4）确定早期胸腺po虫的阶段与门开口和关闭有关。 BM中促炎细胞增殖的动力学将由BRDU诱导的自杀和Hoechst 33342纯化的蛋白生成细胞的DNA染色。胸腺衍生的化学因子因子和抗体的作用也将如此。来自GFP转基因小鼠的纯化的螺蛋白细胞将用于鉴定沿着含辐照的小鼠胸腺中PCV的结合位点（推定的IMV-GATE），以及这些细胞在促成的IMN-NICHES中的定位。从科斯替汀 - M和LIF转基因小鼠的蛋白细胞诱导的LN中，纯化高内皮细胞（HEC）的趋化因子/粘附/ECM分子的基因表达谱将通过RNA杂交确定。候选分子参与促蛋白细胞募集的参与将通过抗体染色/阻断研究来验证，并且将将这些改变的LN HEV的表型与接受性胸腺中的PCV进行比较。 IMV门开口和关闭的最小细胞需求以及IL-7的可能作用，可以通过LV确定。和中尉从CD3转基因rag。和IL-7RA - / - 小鼠，以及I.T.从WT小鼠中转移纯化的胸腺集。这些结果将为促蛋白细胞的胸腺-BM调节环提供基本信息，并且还可能对选择性或增强的T细胞重构，基因治疗以及免疫缺陷障碍的分类/治疗具有治疗意义。