Translational control of regulatory T cell induction

描述（由申请人提供）：外周诱导抗原特异性Foxp3+CD4 T细胞或调节性T细胞（Tregs）是治疗自身免疫性疾病的潜在途径。它也代表了免疫反应的不良结果，它限制了慢性感染和恶性肿瘤的恢复。早期的研究表明，次优或低剂量的抗原刺激有利于诱导Treg。最近，我们和其他人已经证明抗原激活期间mTOR阻断有利于外周Treg诱导。同样，抗原活化过程中细胞代谢应激的其他方式，如l -色氨酸剥夺，也会导致anergic和Treg表型的诱导。这些例子的一个共同特征是抑制帽依赖性mRNA翻译。当然，众所周知，一小部分基因实际上可以通过利用翻译起始的替代机制在代谢应激时增加其表达。该建议的中心假设是基因表达的翻译控制在Treg表型诱导的早期阶段起着关键作用。为了验证这一想法，我们已经使用翻译分析进行了一些初步实验，翻译分析是一种基于核糖体含量的密度将mRNA分层为重和轻组分的技术。较重的部分包含翻译最活跃的转录本。我们的初步结果表明，雷帕霉素治疗导致Foxp3 mRNA转录物的数量急剧增加，但没有明显转变为重组分。因此，我们的研究结果表明，在雷帕霉素治疗下Foxp3蛋白表达的增加主要是由其转录率的增加驱动的。然而，我们也测量了Stat5的翻译谱，Stat5是Foxp3表达必需的已知转录因子之一。值得注意的是，我们的数据显示Stat5转录本全部转移到重部分，而Stat5 mRNA的总量没有显著增加。因此，Stat5的表达似乎完全符合我们的中心假设。在本提案的第一个目标中，我们将重点关注Stat5作为案例研究。我们将扩展我们的初步结果，并记录Stat5翻译的动力学，总Stat5和磷酸化Stat5的表达水平，以及它与foxp3基因的结合。在第二个目标中，我们将执行全基因组翻译分析，以确定参与驱动Treg诱导的其他候选分子。这种Treg诱导机制代表了该领域的一种新范式，它将补充目前主要关注T细胞分化中染色质重塑的其他模型。显然，增加对Treg诱导机制的理解对于未来开发新的治疗策略至关重要，这些策略可用于治疗从自身免疫到癌症等多种疾病。公共卫生相关性：CD4 T细胞通过协调免疫反应对抗感染和癌症，在免疫中发挥关键作用。然而，CD4 T细胞的一个亚群，称为调节性T细胞，抑制免疫反应。这些对于预防自身免疫性疾病至关重要。诱导抗原特异性调节性T细胞的能力可能有助于开发针对多种疾病的新治疗策略。我们正在开展一项新的机制研究，通过专门研究细胞中蛋白质翻译控制的作用，来研究调节性T细胞是如何被诱导的。

DESCRIPTION (provided by applicant): Peripheral induction of antigen-specific Foxp3+CD4 T cells, or regulatory T cells (Tregs), is a potential pathway to treat autoimmune diseases. It is also represents an undesirable outcome of the immune response, which limits recovery from chronic infections and malignancies. Early work has shown that Treg induction is favored by suboptimal or low-dose antigen stimulation. More recently, others and we have demonstrated that mTOR blockade during antigen activation favors peripheral Treg induction. Similarly, other means of cellular metabolic stress during antigen activation, e.g., L-tryptophan deprivation, also lead to induction of anergic and Treg phenotypes. One common feature of these examples is inhibition of cap-dependent mRNA translation. Of course, it is well recognized that a small fraction of genes can actually increase their expression in times of metabolic stress by utilizing alternative mechanisms of translation initiation. The central hypothesis of this proposal is that translational control of gene expression plays a critical role in early stages of Treg phenotype induction. In order to test this idea, we have done some preliminary experiments using translational profiling, a technique which uses density-based stratification of mRNA into heavy and light fractions based on ribosome content. The heavy fractions contain transcripts undergoing translation most actively. Our preliminary results indicate that rapamycin treatment results in dramatic increase in the amount of Foxp3 mRNA transcripts, but without a significant shift into the heavy fractions. Therefore, our results indicate that increased Foxp3 protein expression seen with rapamycin treatment is driven primarily by increased rate of its transcription. However, we also measured the translational profile of Stat5, one of the known transcription factors essential for Foxp3 expression. Remarkably, our data show that Stat5 transcripts are all shifted into the heavy fractions, while the total amount of Stat5 mRNA isn't significantly increased. Therefore, Stat5 expression appears to fit perfectly our central hypothesis. In the first aim of this proposal we will focus on Stat5 as a case study. We will extend our preliminary results and document the kinetics of Stat5 translation, expression levels of total and phosphorylated Stat5, and its binding to the foxp3 gene. In the second aim we will perform genome-wide translational profiling to identify additional candidate molecules that are involved in driving Treg induction. This mechanism of Treg induction represents a new paradigm in the field, which will compliment other current models that focus primarily on chromatin remodeling in T cell differentiation. Clearly, increased mechanistic understanding of Treg induction is critical to future development of new therapeutic strategies in treatment of a great variety of diseases ranging from autoimmunity to cancer. PUBLIC HEALTH RELEVANCE: CD4 T cells play critical roles in immunity by orchestrating the immune responses against infections and cancer. However, a subset of CD4 T cells, called regulatory T cells, inhibits immune responses. These are critical for preventing autoimmune diseases. Ability to induce antigen-specific regulatory T cells could help to develop new therapeutic strategies for a wide range of diseases. We are starting a novel mechanistic investigation of how regulatory T cells get induced by looking specifically at the role of protein translation control in the cells.