Extrathymic T cell precursors: commitment and efficacy

描述（由申请方提供）：尽管已经描述了几种特征良好的祖细胞，但对T淋巴细胞生成最有效的胸腺移行细胞的性质尚不清楚。这些细胞中的一些可以产生淋巴和非淋巴谱系，其他的是淋巴限制的，还有其他的是T淋巴谱系限制的。最近，借助于前TCR 1控制的报告基因，在血液中描述了具有T谱系限制潜力的细胞，循环T细胞祖细胞（CTP）。在此，提出进一步表型表征CTP及其后代，并进一步详细分析其在T细胞生成和向T细胞谱系定型中的功效。此外，通过分析从Notch依赖性转录缺陷的前体产生具有CTP表面表型的细胞，将在具有Notch依赖性转录的可逆抑制的小鼠中解决T谱系定型的Notch依赖性。随后，在分离CTP并解除转录阻断后，将确定功能潜力。最后，将CTP的基因表达与多能（LSK）和胸腺内（ETP）前体的基因表达进行比较，试图将这些细胞置于分化途径中。将特别强调LRF基因的基因调控，拮抗Notch在淋巴细胞的早期前体。在第二个目的中，将通过在将骨髓或血细胞注射到IL-7 ra缺陷型受体中之前耗尽这些前体来比较几种充分表征的胸腺外前体产生T细胞的功效。在另外的研究中，通过IL-7 R1-cre的谱系命运追踪将用于确定表达IL-7 R1的前体对胸腺内前体如ETP的贡献。此外，通过在有限稀释条件下将胸腺内供体来源的细胞接种到含有OP 9-DL 1饲养细胞的培养物中，在注射细胞后24小时比较细胞归巢至胸腺的短期潜力。一旦建立了关于具有多潜能（原型LSK）、淋巴限制性潜能（原型CLP）或T谱系限制性潜能（原型CTP）的不同前体的信息，这些子集将通过已经建立的标记物进一步细分，以便尽可能精确地查明最相关的前体。总的来说，本项目旨在建立胸腺外T细胞系定型的机制，以及确定最有效的胸腺外T淋巴细胞生成前体。公共卫生相关性：该项目旨在鉴定和表征骨髓和血液中最有效的细胞，这些细胞在不同条件下允许重建T细胞免疫系统，以防止感染和恶性肿瘤。胸腺外T细胞前体的鉴定将有助于在通过X射线照射和/或导致T细胞耗竭的细胞毒性药物治疗恶性肿瘤后免疫系统的更快再生。

DESCRIPTION (provided by applicant): The nature of thymus immigrants that contribute most effectively to T lymphopoiesis is unknown even though several well-characterized progenitors have been described. Some of these cells can give rise to lymphoid and non-lymphoid lineages, others are lymphoid-restricted and yet others are T lymphoid lineage-restricted. Cells with T lineage-restricted potential, circulating T cell progenitors (CTP), have recently been described in the blood with the aid of a pre-TCR1- controlled reporter gene. Here it is proposed to further phenotypically characterize CTP and their progeny and to analyze their efficacy in T cell generation and commitment to the T cell lineage in further detail. Furthermore, the Notch dependence of T lineage commitment will be addressed in mice with reversible inhibition of Notch-dependent transcription by analyzing the generation of cells with a CTP surface phenotype from precursors that are deficient in Notch- dependent transcription. Subsequently, after isolation of CTP and relieving the transcriptional block, the functional potential will be determined. Finally, gene expression of CTP will be compared with that of multipotent (LSK) and intrathymic (ETP) precursors in an attempt to place these cells in a differentiation pathway. Special emphasis will be given to gene regulation by the LRF gene that antagonizes Notch in early precursors of lymphoid cells. In a second aim, the efficacy of several well-characterized extrathymic precursors to generate T cells will be compared by depleting such precursors prior to injecting bone marrow or blood cells into Il-7ra- deficient recipients. In additional studies lineage fate tracing by IL-7R1-cre will be used to determine the contribution of IL-7R1-expressing precursors to intrathymic precursors such as ETP. Furthermore, the short-term potential of cells to home to the thymus will be compared 24 hours after injection of cells by explanting intrathymic donor-derived cells under conditions of limiting dilution into cultures containing OP9-DL1 feeder cells. Once information on different precursors with either multipotential (prototype LSK), lymphoid-restricted potential (prototype CLP) or T lineage-restricted potential (prototype CTP) has been established these subsets will be further subdivided by already established markers in order to pinpoint the most relevant precursors as precisely as possible. Overall this project aims at establishing mechanisms of extrathymic T lineage commitment as well as identifying the most potent extrathymic precursors for T lymphopoiesis. PUBLIC HEALTH RELEVANCE: This project aims at the identification and characterization of the most effective cells in bone marrow and blood that under different conditions permit the reconstitution of the T cell immune system that protects from infection and malignancy. Identification of the extrathymic T cell precursors will help in the faster regeneration of the immune system after treatment of malignancy by x- irradiation and/or cytotoxic drugs that result in T cell depletion.