Translational control of regulatory T cell induction

描述(申请人提供)：外周诱导抗原特异性Foxp3+CD4T细胞，或调节性T细胞(Tregs)，是治疗自身免疫性疾病的一种潜在途径。它也代表了免疫反应的不良结果，限制了从慢性感染和恶性肿瘤中恢复。早期的研究表明，次优或低剂量的抗原刺激有利于Treg的诱导。最近，其他人和我们已经证明，在抗原激活过程中阻断mTOR有利于外周Treg的诱导。类似地，抗原激活过程中细胞代谢应激的其他方式，如L-色氨酸剥夺，也会导致无能和Treg表型的诱导。这些例子的一个共同特征是抑制帽依赖的mRNA翻译。当然，人们很好地认识到，在代谢应激时期，一小部分基因实际上可以通过利用替代的翻译启动机制来增加它们的表达。这一建议的中心假设是，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，我们使用翻译图谱进行了一些初步实验，这是一种基于密度的mRNA分层技术，根据核糖体含量将mRNA分为重组分和轻组分。重质组分包含最活跃地进行翻译的文本。我们的初步结果表明，雷帕霉素处理导致Foxp3mRNA转录本的数量显著增加，但没有显著转移到重组份。因此，我们的结果表明，雷帕霉素治疗后Foxp3蛋白表达的增加主要是由其转录速度的增加所驱动的。然而，我们也测量了Stat5的翻译图谱，Stat5是已知的Foxp3表达必不可少的转录因子之一。值得注意的是，我们的数据显示，Stat5转录本都被转移到重组份，而Stat5 mRNA的总量并没有显著增加。因此，Stat5的表达似乎完全符合我们的中心假设。在这项提案的第一个目标中，我们将重点放在STAT5作为一个案例研究。我们将扩展我们的初步结果，并记录Stat5翻译的动力学，总的和磷酸化的Stat5的表达水平，以及它与Foxp3基因的结合。在第二个目标中，我们将进行全基因组的翻译图谱分析，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诱导机制的了解对于未来开发治疗从自身免疫到癌症的各种疾病的新治疗策略至关重要。公共卫生相关性：CD4T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，一种被称为调节性T细胞的CD4T细胞亚群抑制免疫反应。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细胞的能力可以帮助开发针对广泛疾病的新治疗策略。我们正在开始一项新的机制研究，通过特别关注蛋白质翻译控制在细胞中的作用，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.