Regulation of lymphocyte survival by NF-kB proteins

描述(由申请人提供)：淋巴细胞的发育和功能严重依赖于细胞凋亡对其生存的调节。最近几年的研究表明，肿瘤坏死因子受体超家族的成员(如肿瘤坏死因子受体1和Fas)在激活的成熟淋巴细胞中起着关键的诱导凋亡作用。然而，目前尚不清楚这些凋亡途径是否也对调节发育中的淋巴细胞的生存起着重要作用。这项研究的目的是为了更好地了解淋巴细胞存活过程中涉及的促凋亡和抗凋亡机制。具体地说，我们将利用基因敲除的小鼠模型，研究调节凋亡的核因子-kB转录因子在调节发育中的B淋巴细胞存活中的作用。1)我们发现，将REIA-/-胎肝细胞过继转移到受照射的淋巴细胞缺陷的Rag1-/-小鼠体内后，淋巴细胞的生成受到了损害。在联合缺乏Re1a和肿瘤坏死因子受体1(TNFR1)的情况下，受损的淋巴细胞生成在很大程度上得到了挽救，这表明Re1a的一个关键功能可能是阻止TNFR1诱导的对发育中的淋巴细胞的杀伤。在这里，我们将研究肿瘤坏死因子诱导相对发育的B细胞杀伤的机制，包括caspase蛋白酶和活性氧物种(ROS)在肿瘤坏死因子杀伤中的潜在参与。2)为了了解导致肿瘤坏死因子敏感的机制，我们研究了潜在重要的抗凋亡基因的调控。我们发现，一个关键的抗凋亡基因BCI-2在Re1a-/-B细胞中的结构性表达显著减少。在这里，我们将进一步研究Re1a在调节潜在重要的抗凋亡基因中的可能作用。利用逆转录病毒转导系统，我们将在RelA-/-B细胞中重新表达表达受损的抗凋亡基因，以确定它们对肿瘤坏死因子杀伤的影响。还将确定逆转录病毒转导的亲属/胎肝造血前体细胞在恢复Rag1/-小鼠淋巴细胞生成方面的能力。3)我们还确定了成熟T细胞在调节发育中的淋巴细胞存活方面的潜在重要作用。我们的结果提示，成熟的T细胞可能提供信号，可以抵消TNFR1的细胞毒效应。在这里，我们将确定所涉及的T细胞亚群，以及负责这种保护作用的T细胞衍生因子的性质。尤其令人感兴趣的是CD4OL和IL-3，它们是这种T细胞保护作用的中介。在此，我们将确定CD4OL和IL-3在体外抑制肿瘤坏死因子杀伤Re1a-/-发育中的B细胞以及在体内挽救淋巴系生成中的作用。这些研究还可能为在治疗环境下增强淋巴生成的策略的开发提供洞察力。

DESCRIPTION (provided by applicant): The development and function of lymphocytes is critically dependent upon regulation of their survival by apoptosis. Studies 'carried-out over the last several years have shown that members of the TNF receptor superfamily (e.g., TNF receptor 1 and Fas) function as key inducers of apoptosis in activated mature lymphocytes. However, it is not known whether these apoptotic pathways 'are also important for regulating survival of developing lymphocytes. The goal of this investigation is to provide a better understanding of pro-apoptotic and anti-apoptotic mechanisms involved in developing lymphocyte survival. Specifically, we will study the role of the apoptosis-regulating NF-kB transcription factor in regulating survival of developing B-lymphocytes, using knock-out mouse models. 1) We show here an impairment of lymphopoiesis following adoptive transfer of ReIA-/- fetal liver cells into irradiated lymphocyte-deficient Rag1-/- mice. Impaired lymphocyte generation was largely rescued in the combined absence of Re1A and TNF receptor 1 (TNFR1), indicating a key function of Re1A may be to prevent TNFR1-induced killing of developing lymphocytes. Here we will investigate the mechanisms by which TNF induces killing of RelA-/- developing B cells, including the potential involvement of caspase proteases and reactive oxygen species (ROS) in TNF killing.2) To understand mechanisms responsible for susceptibility of RelA-/- B cells to TNF, we have studied regulation of potentially important anti-apoptotic genes. We have found that constitutive expression of one key anti-apoptotic gene, BcI-2, was 'significantly reduced in Re1A-/- B cells. Here we will further investigate a possible role for Re1A in regulation of potentially important anti-apoptotic genes. Using a retroviral transduction system, we will re-express anti-apoptotic genes showing impaired expression in RelA-/- B cells to determine their effect on TNF killing. The ability of retrovirus-transduced RelA-/- fetal liver hematopoietic precursors in restoring lymphocyte generation in Rag1-/- mice will also be determined.3) We have also identified a potentially important role for mature T cells in regulating survival of developing lymphocytes. Our results suggest that mature T cells may provide signals, which can counteract the cytotoxic effects of TNFR1. Here we will identify T cell subsets involved, and the nature of T cell derived factors responsible for this protective effect. Of particular interest as mediators of this T cell protective effect are CD4OL and IL-3. Here we will determine the role of CD4OL and IL-3 in inhibition of TNF killing of Re1A-/- developing B cells in vitro, and in rescuing Iymphopoiesis in vivo. These studies may also provide insights into development of strategies for enhancing lymphopoiesis under therapeutic settings.