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）：在成年小鼠中确定了调节反馈环路，该环路协调骨髓前胸腺细胞的周期性输出与胸腺的门控输入，我们现在计划定义这种胸腺-BM反馈环路的基本机制。通过与胎儿胸腺进行类比，我们假设成年小鼠中前胸腺细胞的周期性生成和动员是由于胸腺周期性释放化学吸引因子。我们进一步假设，开门反映了胸腺 C-M 连接处 PCV 表达的趋化因子/粘附/ECM 分子的独特模式；并且门关闭是由 TN1 和 TN2 胸腺细胞启动和维持的，可能是通过 IL-7 依赖性机制。提出了四个具体目标来检验这些假设：1）确定原胸腺细胞从 BM 的周期性释放是否与其周期性生成相关； 2) 确定前胸腺细胞的生成和动员是否受到胸腺扩散性趋化因子的影响； 3) 识别和表征前胸腺细胞的胸腺内门和生态位； 4) 确定早期胸腺细胞生成的哪些阶段与门的打开和关闭相关。 BM 中前胸腺细胞增殖的动力学将通过 BrdU 诱导的自杀和使用 Hoechst 33342 对纯化的前胸腺细胞进行 DNA 染色来确定。胸腺对前胸腺细胞生成和动员动力学的影响将在 nu/nu 和成人胸腺切除中进行研究，以及正常小鼠；胸腺衍生的趋化因子及其抗体的作用也是如此。来自 GFP 转基因小鼠的纯化前胸腺细胞将用于识别亚致死辐射小鼠胸腺中 PCV 的结合位点（假定的 IMV 门），以及这些细胞在假定的 IMN 生态位中的定位。来自制瘤素-M和LIF转基因小鼠的前胸腺细胞接受性LN的纯化高内皮细胞(HEC)的趋化因子/粘附/ECM分子的基因表达谱将通过RNA杂交测定。候选分子参与前胸腺细胞募集将通过抗体染色/封闭研究进行验证，并且这些改变的 LN HEV 的表型将与接受胸腺中的 PCV 的表型进行比较。 IMV 门打开和关闭的最低细胞要求以及 IL-7 的可能作用将由 Lv 决定。和Lt.来自CD3转基因的前胸腺细胞的过继转移，RAG.-/-。和 IL-7Ra -/- 小鼠，并由 i.t.从 WT 小鼠中转移纯化的胸腺细胞亚群。这些结果将为原胸腺细胞的胸腺-BM 调节环路提供基本信息，并且还可能对选择性或增强 T 细胞重建、基因治疗和免疫缺陷性疾病的分类/治疗具有治疗意义。