Regulatory T Cell Control of Intestinal Tumorigenesis

APC基因突变出现在许多癌症中，但在结直肠癌(CRC)中尤其常见。 自从APC基因突变导致APC基因突变以来，ApcMin/+小鼠是一个高度研究的肠道肿瘤发生模型 在Wnt信号通路的失调中。我们最近使用IL-17A缺陷(KO)ApcMin/+小鼠所做的工作已经 发现IL-17A是一种影响肿瘤发生的促炎细胞因子。野生型(WT)树种引种 进入ApcMin/+小鼠的肿瘤显著退化，而来自ApcMin/+小鼠的Tregs则不能，这表明 ApcMin/+Tregs缺失的Tregs在肿瘤消退中的重要作用在ApcMin/+小鼠中， GATA-3在Tregs和效应性T细胞中均降低。我们对树突状细胞和其他免疫细胞类型的分析 已经确定Wnt拮抗剂Dikkopf-1(Dkk1)是由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可以 诱导GATA-3和IL-10，在ApcMin/+小鼠中下调。Dkk1也刺激了Treg 增殖，但不损害Treg功能。缺乏Treg衍生的Dkk1阻碍了Treg的能力 在标准的小鼠炎症性肠病模型中抑制炎症性肠病。基于这些结果，我们首先 假设Treg来源的Dkk1抑制由IL-17A刺激的肠道炎症 动态平衡状态。其次，我们认为在ApcMin/+Treg中Dkk1表达的缺失导致了 IL-10的丢失，也不能调节控制ApcMin/+肠道干细胞的Wnt通路 扩散。第三，我们假设Th17细胞的Treg来源的Dkk1和IL-17A是至关重要的 肿瘤微环境的调节者，直接靶向携带ApcMin/+突变的ISCs。四 AIMS将重点检验这三个假说。首先，DKK-1缺陷的Treg将用于体内肿瘤 回归实验和体外生物学研究，以确定Dkk1对Treg功能的贡献。这个 Dkk1诱导Treg增殖的机制将从三个不同的信号通路进行研究。第二, 利用IL-17受体缺陷研究IL-17A下调ApcMin/+Treg中Dkk1的作用 体内和体外Foxp3+ApcMin/+Tregs。IL-17A和Dkk1调节IL-10的机制将是 我也学过。第三，IL-17A和Dkk1对携带突变的肠道干细胞(ISC)的影响 将对APC基因进行表征。ApcMin/+背景下ISCs中IL-17受体的条件性缺失 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Treg/T细胞扩增技术将我们的发现扩展到人类FAP Treg 并将其与散发性结肠癌患者和健康捐赠者的细胞进行比较。 这些研究应该给出重要的机制洞察特定的炎症分子，可以 损害免疫系统的细胞。这种机械论的见解应该提供一种理性的方法来 将这些结果转化为临床应用。

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.