T-CELL ANTIGEN 4-1BB--SIGNALING AND FUNCTION

描述（改编自申请人摘要）：初始T细胞缺乏 蛋白4-1BB不仅在T细胞活化时被诱导， 也保留在活化的T细胞上。 4-1BB的天然配体也是 诱导并在活化的抗原呈递B细胞上发现， 巨噬细胞 当4-1BB与CD 3、IL-2和CD 4+细胞同时刺激时， 产生IFN-γ。 申请人的假设是4-1BB是一种 T细胞活化的共刺激蛋白， 4-1BB的结构域（4-1BBcy）及其结合蛋白（4-1BBbp） 在信号传导中。 此外，提出了4-1BB信令 途径是不同的，并导致产生更大范围的 细胞因子比CD 28信号通路。 以测定分子 根据4- 1BB介导的信号传导和4-1BB的功能， 该建议包括五个具体目标。 1.为了确定细胞质 4-1BB区域对信号传导很重要。 为了方便地测定 4- 1BB cy介导的信号传导并消除内源性4-1BB效应， 将制备表达4-1BBcy嵌合蛋白的C8.A3细胞。 的 嵌合蛋白将是hTNFRII-4-1BBcy（人肿瘤的胞外域 坏死因子受体-4-1BBcy）和hEpoR-4-1BBcy（坏死因子受体的胞外域， 人促红细胞生成素受体-4-1BBcy）。 IL-2和IFN-γ将被 在触发嵌合受体后测量。 2.分离和 表征与4-1BB信号相关的细胞内分子 转导 基于酵母的双杂交系统已被用于 获得编码结合4-1BBcy的蛋白质的基因。 cDNA克隆具有 已分离出可能与4-1BBcy相关的基因。 这些基因产物的表征将是重要的， 了解4- 1BB介导的信号传导。 3.为了比较和对比 由CD 28和/或4- 1BB触发诱导的细胞因子。 克隆人会 从4-1BB或CD 28敲除小鼠和重组CD 28产生， 4-1BB将单独和一起表示。 Th克隆体将用于 为了检测CD 28或4-1BB是否可以参与相同的T细胞克隆， 并产生不同的细胞因子表达模式。 4.审查 4-1BB KO小鼠的选择性B和T应答的缺陷。 到 确定4-1BB在淋巴细胞发育中可能发挥的潜在作用 和/或激活，将进行比较研究，以评估 免疫系统的不同参数和免疫反应， 4-1BB+/+、4-1BB+/-和4-1BB-/-小鼠。 5.为了比较免疫反应 在4-1BB KO、CD 28 KO和4-1BB/CD 28 KO小鼠中。 在这一目标中， 将检验存在额外共刺激因子的假设， 负责部分正常反应的途径，或者 在缺乏CD 28/B7相互作用的情况下，替代途径接管。

DESCRIPTION (Adapted from the applicant's abstract): Naive T cells lack the protein 4-1BB, which is not only induced upon T cell activation, but also remains on activated T cells. The natural ligand for 4-1BB is also induced and is found on activated antigen-presenting B cells and macrophages. When 4-1BB is stimulated simultaneously with CD3, IL-2 and IFN-gamma are produced. The applicant's hypothesis is that 4-1BB is a costimulatory protein for T cell activation and that the cytoplasmic domain of 4-1BB (4-1BBcy) and its binding protein (4-1BBbp) are involved in signal transduction. Furthermore, it is proposed that 4-1BB signaling pathway is distinct and results in production of a greater spectrum of cytokines than the CD28 signaling pathway. To determine the molecular basis of 4-1BB-mediated signaling and the function of 4-1BB, this proposal contains five specific aims. 1. To determine the cytoplasmic regions of 4-1BB important for signaling. To conveniently assay the 4-1BBcy-mediated signaling and eliminate the endogenous 4-1BB effect, C8.A3 cells expressing 4-1BBcy chimeric proteins will be prepared. The chimeric proteins will be hTNFRII-4-1BBcy (the ectodomain of human tumor necrosis factor receptor-4-1BBcy) and hEpoR-4-1BBcy (the ectodomain of human erythropoietin receptor-4-1BBcy). IL-2 and IFN-gamma will be measured after triggering the chimeric receptors. 2. To isolate and characterize the intracellular molecules involved with 4-1BB signal transduction. The yeast-based two hybrid system has been employed to obtain genes that encode proteins that bind 4-1BBcy. cDNA clones have been isolated which are potentially associated with 4-1BBcy. Characterization of these gene products will be important for understanding 4-1BB-mediated signaling. 3. To compare and contrast the cytokines induced by CD28 and/or by 4-1BB-triggering. Th clones will be produced from 4-1BB or CD28 knockout mice and recombinant CD28 and 4-1BB will be expressed singly and together. The Th clones will be used to examine whether CD28 or 4-1BB can be engaged on the same T cell clone and produce different patterns of cytokine expression. 4. To examine selected B and T responses of 4-1BB KO mice for deficiencies. To determine a potential role that 4-1BB may play in lymphocyte development and/or activation, comparative studies will be carried out to evaluate different parameters of the immune system and the immune responses in 4-1BB+/+, 4-1BB+/-, and 4-1BB-/- mice. 5. To compare immune responses among 4-1BB KO, CD28 KO, and 4-1BB/CD28 KO mice. In this Aim a hypothesis will be tested that there exist additional costimulatory pathways responsible for part of the normal responses, or that alternative pathways take over in the absence of the CD28/B7 interaction.