Regulatory T cell control of intestinal tumorigenesis

描述(申请人提供)：APC基因突变在许多癌症中都会发生，但在结直肠癌(CRC)中尤其常见。ApcMin/小鼠是一个被高度研究的肠道肿瘤发生的模型，因为APC基因突变导致Wnt信号通路的失调。我们最近利用IL-17A缺陷(KO)ApcMin/小鼠的研究发现，IL-17A是一种影响肿瘤发生的促炎细胞因子。野生型(WT)Tregs导入ApcMin/小鼠后肿瘤显著消退，而ApcMin/小鼠的Tregs不能，提示Tregs是ApcMin/Tregs缺失的肿瘤消退的重要因素。在ApcMin/小鼠中，Tregs和效应性T细胞中GATA-3的表达均降低。我们对Tregs和其他免疫细胞类型的分析发现，Wnt拮抗剂Dikkopf-1(Dkk1)是Tregs产生的最丰富的Wnt家族基因。重要的是，IL-17A有效地抑制了ApcMin/Tregs中Dkk1的表达，但不能抑制WT Tregs中Dkk1的表达。Dkk1可诱导ApcMin小鼠GATA-3和IL-10的表达下调。Dkk1也能刺激Treg的增殖，但不影响Treg的功能。在标准的小鼠炎症性肠病模型中，缺乏Treg来源的Dkk1阻断了Treg抑制炎症性肠病(IBD)的能力。基于这些结果，首先，我们假设Treg来源的Dkk1在体内平衡条件下抑制由IL-17A引发的肠炎症。第二，我们认为在ApcMin/Treg中Dkk1的表达缺失导致了IL-10的丢失，同时也未能调控控制ApcMin/肠道干细胞增殖的Wnt通路。第三，我们假设Th17细胞来源的Treg来源的Dkk1和IL-17A是肿瘤微环境的关键调节因子，直接靶向携带ApcMin/突变的ISCs。四个目标将集中检验这三个假说。首先，DKK-1缺陷的Treg将用于体内肿瘤退化实验和体外生物学研究，以确定Dkk1对Treg功能的贡献。Dkk1诱导Treg增殖的机制将从三个不同的信号通路进行研究。其次，利用体内和体外IL-17受体缺陷的Foxp3 ApcMin/Tregs，研究IL-17A下调ApcMin/Tregs中Dkk1的作用。并对IL-17A和Dkk1调节IL-10的机制进行了探讨。第三，IL-17A和Dkk1对携带APC基因突变的肠干细胞(ISC)的影响将被表征。在ApcMin/Backback中条件删除ISCs中的IL-17受体将在体内进行研究。我们将在ApcMin/小鼠体内将Dkk1送入肠道，测试Dkk1作为一种 有治疗作用。最后，我们将利用来自结肠活检组织的Treg/T细胞扩增技术将我们的发现扩展到人类FAP Treg，并将它们与散发性结肠癌患者和健康捐赠者的细胞进行比较。这些研究应该会给出重要的机制洞察，以了解可以破坏免疫系统细胞的特定炎症分子。这种机械论的洞察力应该为将这些结果转化为临床提供一种合理的方法。

DESCRIPTION (provided by applicant): Mutations in the Apc gene occur in many cancers but are especially frequent in colorectal cancer (CRC). The ApcMin/+ mouse is a highly studied model of intestinal tumorigenesis since the Apc gene mutation results in dysregulation of the Wnt signaling pathway. Our recent work using IL-17A deficient (KO) ApcMin/+ mice has identified IL-17A as a proinflammatory cytokine affecting tumorigenesis. Introduction of wildtype (WT) Tregs into ApcMin/+ mice regressed tumors markedly while Tregs from ApcMin/+ mice could not, suggesting an important factor from Tregs for tumor regression lacking in ApcMin/+ Tregs. In ApcMin/+ mice, the expression of Gata-3 was decreased in Tregs as well as effector T cells. Our analysis of Tregs and other immune cell types has identified the Wnt antagonist Dikkopf-1(Dkk1) as the most abundant Wnt family gene produced by Tregs. Importantly, IL-17A effectively inhibited Dkk1 expression in ApcMin/+ Tregs but not in WT Tregs. Dkk1 could induce Gata-3 and also IL-10, which was downregulated in ApcMin/+ mice. Dkk1 also stimulated Treg proliferation but did not impair Treg function. The lack of Treg-derived Dkk1 blocked the ability of Treg to suppress inflammatory bowel disease (IBD) in a standard murine IBD model. Based on these results, first we hypothesize that Treg-derived Dkk1 suppresses intestinal inflammation that is fueled by IL-17A under homeostatic conditions. Second, we argue that the loss of Dkk1 expression in ApcMin/+ Treg leads to the loss of IL-10, and also fails to regulate the Wnt pathway controlling ApcMin/+ intestinal stem cell proliferation. Third, we hypothesize that Treg-derived Dkk1 and IL-17A from Th17 cells are crucial regulators of the tumor microenvironment, directly targeting ISCs carrying the ApcMin/+ mutation. Four aims will focus on testing these three hypotheses. First, Dkk-1 deficient Treg will be used in in vivo tumor regression experiments and in in vitro biologic studies to define the contribution of Dkk1 to Treg function. The mechanism that Dkk1 induces Treg proliferation will be studied in three different signaling pathways. Second, the role of IL-17A to downregulate Dkk1 in ApcMin/+ Tregs will be studied by utilizing IL-17 receptor-deficient Foxp3+ ApcMin/+ Tregs in vivo and in vitro. The mechanism which IL-17A and Dkk1 regulates IL-10 will be studied as well. Third, the effects of IL-17A and Dkk1 on intestinal stem cells (ISC) carrying the mutation in the Apc gene will be characterized. Conditional deletion of the IL-17 receptor in ISCs in the ApcMin/+ background will be studied in vivo. We will deliver Dkk1 into the intestine in ApcMin/+ mice, testing the potential for Dkk1 as a therapeutic. Finally, we will extend our findings to human FAP Treg utilizing a Treg/T cell expansion technique from colon biopsies, and compare them with cells from sporadic colon cancer patients and healthy donors. These studies should give important mechanistic insight into the specific inflammatory molecules that can compromise cells of the immune system. This mechanistic insight should provide a rational approach to translation of these results to the clinic.