TOX, A Novel Regulator of Thymocyte Selection

描述（由申请人提供）：越来越清楚的是，胸腺的工作比最初认识到的要复杂得多。这个器官不仅是CD4和CD8 T细胞的产生器，而且也是自然杀伤T细胞（NKT）、上皮内T淋巴细胞、调节性T细胞（Treg）和先天样CD8 T细胞高度特化谱系发育的场所。控制这些不同的T细胞亚谱系从共同前体分化的详细分子机制仍有待确定。在这里，我们重点关注TOX（胸腺细胞选择相关的HMG盒子蛋白）在这种情况下的作用，这是一种核dna结合蛋白，在我们的实验室中首次被确定为胸腺选择事件的调节剂。我们制造了缺乏TOX的小鼠，证明了TOX是典型CD4 T细胞谱系以及NKT和Treg细胞发育所必需的。在这些突变动物中受影响的T细胞发育的特定阶段的抑制以前没有报道过，这使得对这一过程有了新的认识。此外，我们发现TOX在胸腺之外也发挥作用，包括在NK细胞发育和淋巴结器官发生中发挥强制性作用。我们在此提出三个具体目标来描述TOX在免疫系统发育中的作用和作用机制。首先，我们将研究TOX与胸腺中CD4和ThPOK表达的调节之间的关系，后者是CD4谱系的主要承诺因子。此外，我们提出了一种新的方法来鉴定参与建立CD4谱系基因程序的TOX依赖基因，并将比较TOX在不同T细胞谱系发育中的作用。其次，我们将分析TOX在淋巴结器官发生发展中的作用，重点关注TOX调节淋巴组织诱导细胞发育的潜力。我们还在培养系统中复制了缺乏TOX对NK细胞发育的抑制作用。我们建议详细分析TOX在这种情况下的作用，包括TOX是否可以调节另一个与TOX具有某些功能特性的核因子。第三，我们提出了最先进的方法来识别TOX的直接基因靶点和蛋白质结合伙伴，在体内CD4 T细胞发育的背景下。TOX在啮齿类动物和人类之间高度保守，其表达模式也是如此。因此，这些研究也应该对人类免疫系统的运作提供深入的了解。TOX定义了一个小的蛋白质亚家族，包括其他三个成员，其中一个成员与乳腺癌易感性位点有关，而TOX本身在一些癌细胞系和肿瘤中高度表达。因此，了解TOX的作用机制可能会影响人类的健康和疾病。

DESCRIPTION (provided by applicant): It has become increasingly clear that the workings of the thymus include greater complexity than first appreciated. Not only is this organ a generator of CD4 and CD8 T cells, but it is also a site for development of highly specialized lineages of natural killer T (NKT) cells, intraepithelial T lymphocytes, regulatory T (Treg) cells, and innate-like CD8 T cells. The detailed molecular mechanisms that control the differentiation of these distinct T cell sublineages from common precursors remain to be determined. Here we focus on the role of TOX (thymocyte selection-associated HMG box protein) in this context, a nuclear DNA-binding protein first identified in our laboratory as a regulator of thymic selection events. We produced TOX-deficient mice, demonstrating that TOX is required for development of the canonical CD4 T cell lineage as well as NKT and Treg cell development. Inhibition of the specific stage of T cell development that is affected in these mutant animals has not been reported previously, allowing novel insights into the process. In addition, we have found that TOX plays roles outside the thymus, including an obligatory role in NK cell development and lymph node organogenesis. We propose here three specific aims to delineate the role and mechanism of action of TOX in development of the immune system. First, we will investigate the relationship between TOX and regulation of CD4 and ThPOK expression in the thymus, the latter a dominant CD4 lineage commitment factor. In addition, we propose a novel approach to identify TOX-dependent genes involved in establishment of the CD4 lineage gene program, and will compare the role of TOX in development of different T cell lineages. Second, we will analyze the role of TOX in development of lymph node organogenesis, focusing on the potential for TOX to regulate lymphoid tissue inducer cell development. We also have replicated the inhibition of NK cell development in the absence of TOX in a culture system. We propose detailed analysis of the role for TOX in this context, including whether TOX might function to regulate another nuclear factor that shares some functional properties with TOX. Third, we propose state-of-the-art approaches to identify direct gene targets and protein binding partners of TOX, in an in vivo context of CD4 T cell development. TOX is highly conserved between rodents and humans, as is its expression pattern. Thus, these studies should provide insight into workings of the human immune system as well. TOX defines a small protein subfamily that includes three other members, one of which has been implicated as a breast cancer susceptibility locus, while TOX itself is highly expressed in some cancer cell lines and tumors. Understanding the mechanism of action of TOX is therefore likely to impact both human health and disease. PUBLIC HEALTH RELEVANCE: Development of T lymphocytes in the thymus is a complex process, but one that is key to creating a protective cellular arm of the immune system. This research program is directed at understanding the function of a small family of nuclear proteins, highly conserved in rodents and humans, in regulating this process. This knowledge will aid in future development of methods to reconstitute the immune system where necessary due to disease, and also may have future impact in treatment for breast cancer, since a gene encoding one member of this protein family has been mapped as a disease susceptibility locus.