NF-KB REGULATION OF LYMPHOCYTE FUNCTION AND APOPTOSIS

在脊椎动物中，除了激活必要的先天防御机制外，核因子-kappaB/Rel被认为是适应性免疫的中央协调调节因子。然而，由于先天免疫系统细胞中的核因子-kappaB的伴随缺陷和核因子-kappaB复合体的冗余功能，通过对单个核因子-kappaB基因突变的小鼠的分析，不能直接评估核因子-kappaB在淋巴细胞中执行的广泛的关键功能。我们已经开发了一个模型系统，其中由于高度同源的亚基p50和p52的同时丢失，核因子-kappaB二聚体的谱系被深深地扰乱。我们建议使用这个模型来直接和系统地评估核因子-kappaB因子在B和T淋巴细胞的发育和功能中的生物学影响。该提案有三个具体目标。在目标1中，我们将讨论核因子-kappaB在B淋巴细胞生成中的作用。通过体内和体外相结合的方法，我们将确定NF-kappaB控制这一成熟步骤的机制(S)，并将首先评估先前提出的NF-kappaB在B细胞存活、激活和有丝分裂中所扮演的角色的生理学相关性。确定转录因子对B细胞选择的贡献也可能提供对自身免疫机制和治疗的洞察。在目标2中，我们将研究核因子-kappaB在T细胞动态平衡中的作用。先前提出的核因子-kappaB在共刺激和激活诱导细胞死亡(AICD)中的作用的生理学相关性将通过检测缺乏核因子-kappaB的T细胞在完整的先天免疫系统的指导下对其自然抗原的反应来确定。为了进一步评估核因子-kappaB在自身免疫和慢性炎症中的潜在广泛作用，还将检测这些细胞的自身抗原驱动的反应。在目标3中，我们试图识别和表征核因子-kappaB调控的抗细胞凋亡基因的一个子集。为了实现这一目标，我们成功地优化了哺乳动物文库功能筛选的实验方案。这是因为这些基因似乎是由B和T细胞中的共刺激途径激活的生存程序的完整效应器，并且可能直接参与成熟B细胞的产生和T细胞的动态平衡。此外，核因子-kappaB调控的促生存基因参与了肿瘤的发生、肿瘤细胞对化疗和电离辐射的抵抗，从而为癌症治疗提供了潜在的新靶点。

In vertebrates, in addition to activating essential innate defense mechanisms, NF-kappaB/Rel factors are believed to function as central coordinating regulators of adaptive immunity. However, the direct assessment of the broad spectrum of critical functions executed by NF-kappaB in lymphocytes could not be obtained by the analyses of mice harboring disruptions of individual NF-kappaB genes, because of the concomitant deficiency of NF-kappaB in cells of the innate immune system and the redundant functions of NF-kappaB complexes. We have developed a model system where, due to the simultaneous loss of highly homologous subunits p50 and p52, the repertoire of NF-kappaB dimers is profoundly perturbed. We propose to use this model in a direct and systematic evaluation of the biologic impact of NF- kappaB factors in the development and function of B and T lymphocytes. The proposal has three Specific Aims. In Aim 1, we will address the role of NF-kappaB in B lymphopoiesis. NF-kappaB complexes are required for the establishment of the long-lived peripheral B cell pool By combining an in vivo and an in vitro approach, we will determine the mechanism(s) through which NF- kappaB controls this maturation step, and will begin by evaluating the physiologic relevance of previously proposed roles of NF-kappaB in B cell survival, activation, and mitogenesis. Defining the contribution of the transcript factor to B cell selection may also offer insights into autoimmune mechanisms and treatments. In Aim 2, we will examine roles of NF-kappaB in homeostasis of T cells. The physiologic relevance of previously suggested roles of NF-kappaB in costimulation and activation- induced cell death (AICD) will be determined by examining responses of NF-kappaB deficient T cells to their natural antigen, when instructed by an intact innate immune system. To further assess potential broad roles of NF-kappaB in autoimmunity and chronic inflammation, self-antigen-driven responses of these cells will also be examined. In Aim 3, we seek to identify and characterize a subset of NF-kappaB regulated genes that act to antagonize apoptosis. To achieve this goal we have successfully optimized an experimental protocol for the functional screening of mammalian libraries. This is of interest since these genes appear to be integral effectors of survival programs activated by costimulatory pathways in B and T cells, and may be directly involved in the generation of mature B cells and T cell homeostasis. In addition, NF-kappaB regulated pro-survival genes have been implicated in tumorigenesis, and resistance of tumor cells to chemotherapy and ionizing radiation, thereby providing potential new targets for cancer therapy.