Dynamic regulation of interleukin-7 receptor alpha chain

描述(申请人提供)：IL-7受体a链(IL-7ra)是IL-7和胸腺基质淋巴生成素的细胞因子受体亚单位。IL-7R1信号在淋巴细胞生成和T细胞反应中起重要作用。此外，IL-7ra的表达受到动态调节，以适应不同细胞类型的功能需求。而IL-7ra在造血干细胞(HSCs)上不表达，它是HSC来源的共同淋巴祖细胞(CLPs)的重要标志蛋白。IL-7ra在CLP分化为Pro-B细胞的过程中保持表达，但在骨髓中的未成熟B细胞和外周成熟B细胞中被关闭。在胸腺细胞发育过程中，最不成熟的双阴性胸腺细胞表达IL-7ra，而双阳性胸腺细胞则不表达IL-7ra。IL-7ra在单个阳性的CD4+和CD8+胸腺细胞中再次表达上调，并维持在外周的初始T细胞上。当遇到外来抗原时，初始T细胞被激活，并伴随着IL-7ra的下调而成为效应性T细胞。当抗原被清除后，部分效应性T细胞产生长时间的记忆T细胞，其IL-7ra的表达水平较高。已知GA结合蛋白和PU.1能激活IL-7ra基因的转录，但在分子水平上如何实现IL-7ra基因表达的动态变化却鲜为人知。本研究旨在确定IL-7ra基因座上的顺式调控模块。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素，这些因素有助于动态调节IL-7ra。重要的是，在淋巴细胞发育和T细胞反应过程中，许多基因如Notch反应基因Deltex 1、Wnt效应转录因子TCF1/LEF1显示出与IL-7ra同步的基因表达变化。IL-7ra基因调控的确切分子机制可以外推到其他同步表达的基因，从而为HSCs向T和B系分化以及从初始T细胞向效应T细胞和记忆性T细胞的转变提供了新的见解。这一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。特定目标。识别在T和B细胞发育过程中IL-7ra的动态表达以及T细胞对抗原刺激的反应的顺式调节模块。我们将使用包含整个IL-7ra基因座的细菌人工染色体(BAC)，通过在第一个IL-7ra编码外显子中插入GFP报告基因来产生报告转基因小鼠。结合Cre重组酶介导的使用不相容的Lox变异体的随机重组，我们将获得系统缺失调控区域/模块的报告转基因小鼠。通过建立3个转基因方正细胞系，我们将把200kb的调控区域缩小到1-2kb的调控序列，这些调控序列可以在HSC向CLP和B系细胞分化、胸腺细胞发育和T细胞反应过程中上调或下调IL-7ra的表达。 公共卫生相关性：白细胞介素7受体a链(IL-7ra)是一种重要的细胞表面蛋白，随着造血干细胞分化为对抗T和B细胞的病原体而发生动态变化。IL-7ra在维持幼稚T细胞和记忆性T细胞的动态平衡方面也起着重要作用。该项目将深入了解IL-7ra的表达变化如何适应这些细胞的功能需求。这些知识将为调控造血干细胞分化和免疫反应的结果提供科学依据，这对于了解T细胞恶性肿瘤，设计T细胞发育或功能缺陷的基因治疗，以及开发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.