MECHANISMS OF RESISTANCE TO FAS-INDUCED APOPTOSIS IN HUMAN THYMOCYTES

Fas （APO-1或CD95）是免疫系统细胞中与凋亡细胞死亡调控相关的细胞表面受体。Fas蛋白在人胸腺细胞发育过程中的表达和功能，但不参与细胞死亡信号传导。了解Fas在人类胸腺细胞发育中的特定作用不仅可以阐明这些细胞如何通过该受体逃避凋亡死亡信号，而且可能为通过Fas介导的凋亡信号失调的疾病提供潜在的免疫调节疗法。人类胸腺细胞抵抗fas诱导的凋亡的机制将在三个并行和重叠的方法中进行探讨。首先，人类胸腺细胞特异性缺乏的凋亡信号步骤将通过测试与其他凋亡刺激共享的远端Fas信号通路的能力来定位。TNFalpha、TRAIL和FAS/FLICE嵌合体将被用于在涉及或短路FADD的条件下触发人胸腺细胞凋亡。其次，从人胸腺细胞中克隆的Fas受体将被测试其结合Fas配体和FADD多聚的内在能力，以及它们作为野生型Fas的显性阴性抑制剂的能力。第三，通过“死亡陷阱”方法筛选具有抗凋亡功能的人胸腺细胞cDNA文库。这些实验将补充本项目其他项目中其他信号功能的研究，以确定指示人类胸腺免疫细胞发育的信号因子。

Fas (APO-1 or CD95) is a cell surface receptor associated with the regulation of apoptotic cell death in cells of the immune system. The expression and function of Fas protein in developing thymocytes in the human thymus, but is not function in signaling cell death. An understanding of the human species specific role of Fas in developing thymocytes would not only illuminate how these cells escape apoptotic death signaling through this receptor, but might suggest potential immunomodulating therapies for diseases in which apoptotic signaling through Fas is dysregulated. The mechanism(s) which are responsible for the resistance of human thymocytes to Fas-induced apoptosis will be explored in three concurrent and overlapping approaches. First, apoptotic signal steps which are specifically deficient in human thymocytes will be localized by testing for the competence of the distal Fas signal pathway which is shared with other apoptotic stimuli. TNFalpha, TRAIL, and FAS/FLICE chimeras will be employed to trigger apoptosis in human thymocytes under conditions which involve or short circuit FADD. Second, Fas receptors cloned from human thymocytes will be tested for their intrinsic competence to bind Fas ligand and to multimerize FADD, and for their ability to serve as dominant negative inhibitors of wild type Fas. Thirdly, a human thymocyte cDNA library will be screened for proteins with anti- apoptotic function through a "death trap" approach. These experiments will complement studies of other signal function in other projects of this program project to define the signaling factors which instructed the development of immune cells in the human thymus.