Direct Epigenetic Reprogramming of T Cells

 描述(由申请人提供)：CD4效应器/记忆T细胞内的异质性对于我们处理不同病原体的能力至关重要。例如，促进针对细胞内感染(Th1细胞)、蠕虫感染(Th2细胞)和真菌感染(Th17细胞)的免疫防御需要专用的CD4T辅助细胞群。另一方面，这些分化状态中的每一种都与人类疾病有关：Th1和Th17细胞可以促进自身免疫，而Th2细胞可以促进过敏和哮喘。因此，了解和学习利用血统选择背后的机制对于理解和治疗免疫性和传染性疾病至关重要。我们感兴趣的是表观遗传对T细胞谱系承诺和T细胞记忆的调节。在这里，我们提出了一种新的策略来靶向Th细胞表观基因组，以改变其表型，促进或抑制炎性免疫反应。细胞表观基因组以DNA甲基化、组蛋白修饰和ncRNA为代表，被认为反映了细胞的分化历史并决定了其表型。与人类辅助T细胞合作，我们发现记忆CD4T细胞快速产生关键细胞因子的能力(快速回忆能力) 与表观遗传基因平衡相关：在静止的记忆细胞中，在几个调控元件上存在阳性的组蛋白修饰。基于这一观察，我们假设记忆T细胞谱系承诺是在表观基因组中编码的。作为证明这一假设(以及T细胞重新编程)的第一步，我们将尝试通过改变我们和其他人之前确定的几个关键元素的染色质标记来改变T细胞的表型。为了做到这一点，我们正在使用TALEMS-基于TAL的工程DNA结合域(DBD)与表观遗传修饰酶的融合蛋白。我们将使用TALEMS从IL4/13基因座先前发现的调控元件中去除阳性染色质标记(例如，H3K4甲基化)，以测试这些标记的存在是否确实是维持Th2表型和IL4/13基因诱导能力所必需的。我们还将测试阴性标记(如K9me2和DNA甲基化)的沉积是否足以逆转Th2分化。类似地，我们将测试这些元素上阳性标记的沉积是否足以强制在幼稚T细胞中诱导Th2细胞因子。最后，我们将测试这种表观遗传重编程是否会影响实验性哮喘小鼠的疾病表型。如果成功，这一策略可能会导致创建针对免疫性疾病的疗法和癌症的免疫疗法。在一种假设的情况下，对于哮喘，过敏原特异性炎性Th2细胞可以使用四聚体从患者的血液中纯化出来，增殖并重新编程为免疫抑制树突状细胞。然后，这些Treg细胞可以被送回患者体内，以恢复耐受，并有可能提供 一种解药。重要的是，过敏原特异性Tregs治疗实验性哮喘的能力已经在小鼠模型中得到证实。同样，肿瘤抗原特异的Treg细胞可以从肿瘤中纯化并重新编程为Th1效应器用于免疫治疗。通过这项研究获得的知识将使类似的应用在其他生物医学领域成为可能。

 DESCRIPTION (provided by applicant): The heterogeneity within the CD4+ effector/memory T-cell compartment is critical for our ability to deal with diverse pathogens. For example, dedicated populations of CD4+ T helper cells are required for promoting immune defense against intracellular infections (Th1 cells), helminth infections (Th2 cells), and fungal infection (Th17 cells). On the other hand, each of these differentiated states is associated with human disease: Th1 and Th17 cells can promote autoimmunity, while Th2 cells can promote allergy and asthma. Thus, understanding and learning to exploit the mechanisms that underlie lineage choice is vital for understanding and treatment of immunological and infectious diseases. We are interested in epigenetic regulation of T-cell lineage commitment and T-cell memory. Here, we propose a novel strategy to target the Th cell epigenome in order to modify their phenotype and promote or inhibit inflammatory immune response. The cellular epigenome, represented by DNA methylation, histone modifications and ncRNA, is believed to reflect the differentiation history of the cell and determine its phenotype. Working with human helper T cells, we have found that the ability of memory CD4 T cells to quickly induce key cytokines (rapid recall ability) is correlated with epigenetic gene poising: the presence of positive histone modifications at several regulatory elements in the resting memory cells. Based on this observation, we hypothesize that memory T-cell lineage commitment is encoded in the epigenome. As a first step toward proving this hypothesis (and to T-cell reprogramming), we will attempt to modify T cell phenotype by changing chromatin marks at several key elements that we and others have previously identified. To do so, we are using TALEMs - fusion proteins of TAL-based engineered DNA binding domains (DBDs) with Epigenetic Modifier enzymes. We will use TALEMs to remove positive chromatin marks (e.g., H3K4 methylation) from the previously identified regulatory elements in the IL4/13 locus to test whether the presence of such marks is indeed required for the maintenance of Th2 phenotype and IL4/13 gene inducibility. We will also test whether deposition of negative marks (e.g., K9me2 and DNA methylation) there will be sufficient to reverse Th2 differentiation. Similarly, we will test whether deposition of the positie marks at these elements is sufficient to force Th2 cytokine inducibility in naïve T cells. Finallywe will test, whether such epigenetic reprogramming can affect disease phenotype in a mouse model of experimental asthma. If successful, this strategy may potentially lead to creation of therapies for immunological diseases and immunotherapy of cancer. In one hypothetical scenario, for asthma, allergen- specific inflammatory Th2 cells could be purified from patient's blood using tetramers, propagated and reprogrammed into immunosuppressive Tregs. These Treg cells could then be returned back to patient to restore tolerance and, potentially, to provide a cure. Importantly, the ability of allergen-specific Tregs to cure experimental asthma has already been demonstrated in a murine model. Similarly, tumor antigen-specific Treg cells could be purified from tumors and reprogrammed into Th1 effectors for immunotherapy. Knowledge gained through this study would enable similar applications in other biomedical areas.