Dynamic regulation of interleukin-7 receptor alpha chain

描述（由申请人提供）：白细胞介素-7受体α链（IL-7 Ra）是IL-7和胸腺基质淋巴细胞生成素的细胞因子受体亚单位。IL-7 R1衍生的信号在淋巴细胞生成和T细胞应答中具有重要作用。此外，IL-7 Ra表达被动态调节以适应不同细胞类型中的功能需求。尽管IL-7 Ra在造血干细胞（HSC）上不表达，但它是HSC衍生的共同淋巴样祖细胞（CLP）的重要标志物蛋白。当CLP分化为前B细胞时，IL-7 Ra表达得以维持，但在骨髓中的未成熟B细胞和外周中的成熟B细胞中被关闭。在胸腺细胞发育过程中，大多数未成熟的双阴性胸腺细胞表达IL-7 Ra，但双阳性胸腺细胞不表达。IL-7 Ra表达在单阳性CD 4+和CD 8+胸腺细胞中再次上调，并维持在外周的幼稚T细胞上。在遇到外源抗原时，初始T细胞被激活并成为效应T细胞，伴随着IL-7 Ra的下调。在抗原被清除后，一部分效应T细胞产生具有更高表达IL-7 Ra的持久记忆T细胞。已知GA结合蛋白和PU. 1可激活IL-7 Ra基因的转录，但在分子水平上如何实现IL-7 Ra的动态表达变化还知之甚少。 本研究旨在鉴定IL-7 Ra基因位点的顺式调控模块。我们的长期目标是进一步确定关键的顺式调节元件和相关因子，有助于IL-7 Ra的动态调节。重要的是，在淋巴细胞发育和T细胞应答期间，许多基因如Notch应答基因Deltex 1、Wnt效应转录因子TCF 1/LEF 1显示与IL-7 Ra一致的同步基因表达变化。IL-7 Ra基因调控的精确分子机制可以外推到具有同步表达的其他基因，从而提供对HSC向T和B谱系分化以及幼稚T细胞向效应和记忆T细胞转变的见解。这些知识可能对免疫缺陷的治疗和疫苗/佐剂设计具有重要意义。 具体目标。鉴定在T和B细胞发育以及T细胞对抗原刺激的应答过程中促进IL-7 Ra动态表达的顺式调节模块。我们将使用含有整个IL-7 Ra基因座的细菌人工染色体（BAC），通过在第一个IL-7 Ra编码外显子中插入GFP报告基因来产生报告基因转基因小鼠。结合Cre重组酶介导的随机重组，使用不相容的Lox变体，我们将获得含有系统性缺失调控区/模块的报告基因转基因小鼠。通过建立少至3个转基因创始细胞系，我们将200 kb调节区缩小至1-2 kb调节序列，其可在HSC分化为CLP和B谱系细胞、胸腺细胞发育和T细胞应答期间赋予IL-7 Ra的上调或下调. 公共卫生相关性：白细胞介素-7受体α链（IL-7 Ra）是一种重要的细胞表面蛋白，其显示伴随造血干细胞分化为对抗病原体的T和B细胞的动态变化。IL-7 Ra还在维持初始和记忆T细胞的稳态中起关键作用。该项目将深入了解IL-7 Ra表达变化如何调节这些细胞的功能需求。这些知识将为操纵造血干细胞分化和免疫应答的结果提供科学依据，这对于理解T细胞恶性肿瘤，设计缺陷T细胞发育或功能的基因治疗以及制定更有效的疫苗和佐剂具有重要意义。

DESCRIPTION (provided by applicant): Interleukin-7 receptor a chain (IL-7Ra) is a cytokine receptor subunit for IL-7 and thymic stromal lymphopoietin. IL-7R1-derived signal has important roles in lymphopoiesis and T cell responses. In addition, IL-7Ra expression is dynamically regulated to tune into functional requirements in different cell types. Whereas IL-7Ra is not expressed on hematopoietic stem cells (HSCs), it is an important marker protein for HSC-derived common lymphoid progenitors (CLPs). IL-7Ra expression is maintained while CLPs differentiate to pro-B cells, but is turned off in immature B cells in bone marrow and mature B cells in the periphery. During thymocyte development, the most immature double negative thymocytes express IL-7Ra, but double positive thymocytes do not. IL-7Ra expression is upregulated again in single positive CD4+ and CD8+ thymocytes and maintained on naive T cells in the periphery. Upon encountering foreign antigens, naive T cells are activated and become effector T cells accompanied by down-regulation of IL-7Ra. After the antigens are cleared, a portion of effector T cells give rise to long lasting memory T cells with higher expression of IL-7Ra. GA binding protein and PU.1 are known to activate the transcription of IL-7Ra gene, however, little is known about how the dynamic expression changes of IL-7Ra are achieved on a molecular level. This research project aims to identify cis regulatory modules in the IL-7Ra gene locus. Our long-term goal is to further define critical cis regulatory elements and associated factors that contribute to the dynamic regulation of IL-7Ra. Importantly, during lymphocyte development and T cell responses, many genes such as Notch response gene Deltex 1, Wnt effector transcription factors TCF1/LEF1 show synchronized gene expression changes in concert with IL-7Ra. The precise molecular mechanism underlying IL-7Ra gene regulation could be extrapolated to other genes with synchronized expression and thus provides insights into differentiation of HSCs to T and B lineages and transition of naive T cells to effector and memory T cells. This knowledge may have important implications in treatment of immunodeficiency and vaccine/adjuvant designs. SPECIFIC AIM. To identify cis regulatory modules that contribute to the dynamic expression of IL-7Ra during T and B cell development and T cell responses to antigen stimulation. We will use a bacterial artificial chromosome (BAC) containing the entire IL-7Ra locus to generate reporter transgenic mice by inserting a GFP reporter in the first IL-7Ra-coding exon. In combination with Cre recombinase-mediated stochastic recombination using incompatible Lox variants, we will obtain reporter transgenic mice containing systematic deletion of regulatory regions/modules. By establishing as few as 3 transgenic founder lines, we will narrow down a 200 kb regulatory region to 1-2 kb regulatory sequences which can confer the up- or down- regulation of IL-7Ra during differentiation of HSCs to CLPs and B lineage cells, thymocyte development, and T cell responses. PUBLIC HEALTH RELEVANCE: Interleukin-7 receptor a chain (IL-7Ra) is an important cell surface protein that shows dynamic changes accompanying differentiation of hematopoietic stem cells to pathogen combating T and B cells. IL-7Ra also plays critical roles in maintaining homeostasis of naive and memory T cells. This project will generate insights into how IL-7Ra expression changes are tuned into functional requirements in these cells. The knowledge will provide a scientific basis for manipulating outcomes of hematopoietic stem cell differentiation and immune responses, which is important for understanding T cell malignancy, designing gene-therapy of defective T cell development or function, and formulating more effective vaccines and adjuvants.