Direct Epigenetic Reprogramming of T Cells

 描述（由申请人提供）：CD 4+效应/记忆T细胞区室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，需要专门的CD 4 + T辅助细胞群来促进针对细胞内感染（Th 1细胞）、蠕虫感染（Th 2细胞）和真菌感染（Th 17细胞）的免疫防御。另一方面，这些分化状态中的每一种都与人类疾病有关：Th 1和Th 17细胞可以促进自身免疫，而Th 2细胞可以促进过敏和哮喘。因此，理解和学习利用谱系选择的基础机制对于理解和治疗免疫和感染性疾病至关重要。我们对T细胞谱系定型和T细胞记忆的表观遗传调控感兴趣。在这里，我们提出了一种新的策略，以目标Th细胞表观基因组，以修改其表型和促进或抑制炎症免疫反应。以DNA甲基化、组蛋白修饰和ncRNA为代表的细胞表观基因组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CD 4 T细胞快速诱导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：在静息记忆细胞中的几个调节元件处存在阳性组蛋白修饰。基于这一观察结果，我们假设记忆T细胞谱系承诺编码在表观基因组中。作为证明这一假设（以及T细胞重编程）的第一步，我们将尝试通过改变我们和其他人先前已经确定的几个关键元件的染色质标记来修改T细胞表型。为此，我们正在使用TALEM-基于TAL的工程化DNA结合结构域（DBD）与表观遗传修饰酶的融合蛋白。我们将使用TALEM去除阳性染色质标记（例如，H3 K4甲基化），以测试这些标记的存在是否确实是维持Th 2表型和IL 4/13基因诱导所必需的。我们还将测试是否沉积负标记（例如，K9 me 2和DNA甲基化）将足以逆转Th 2分化。同样，我们将检测这些元件上的阳性标记是否足以在初始T细胞中诱导Th 2细胞因子。最后，我们将测试这种表观遗传重编程是否能影响实验性哮喘小鼠模型的疾病表型。如果成功，这一策略可能会导致免疫性疾病和癌症免疫疗法的产生。在一种假设的情况下，对于哮喘，过敏原特异性炎性Th 2细胞可以使用四聚体从患者的血液中纯化，增殖并重编程为免疫抑制性T细胞。然后，这些Treg细胞可以返回到患者体内以恢复耐受性，并可能提供 解药了重要的是，过敏原特异性Tclase治愈实验性哮喘的能力已经在小鼠模型中得到证实。类似地，可以从肿瘤中纯化肿瘤抗原特异性Treg细胞，并将其重编程为用于免疫治疗的Th 1效应子。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。

 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.