Bcl11b: A Master Transcription Factor Controlling Human NK Cell Development

项目概要/摘要 自然杀伤 (NK) 细胞是先天性淋巴细胞，可介导针对病毒感染和病毒感染的细胞毒性。 恶性细胞。激活后，它们还释放趋化因子和细胞因子，帮助启动和协调 适应性免疫反应。由于这些功能特性，人们对开发 NK 持续感兴趣 用于免疫治疗的细胞产品。然而，我们对人类 NK 细胞的了解有限，阻碍了这一努力 分化和调节这一过程的转录因子网络。在当前支持的工作中 K99/R00 资金，我们生成了大量的初步数据，全面表征 从巨细胞病毒分离的分选 NK 和 CD8 T 细胞亚群的转录和表观遗传景观 (CMV) 血清阳性供体。为了补充这些分析，我们进行了基于流式细胞术的分析 转录因子表达和 ChIP-seq 在分选的外周血 NK 细胞亚群上构建转录 潜在调节 NK 细胞分化关键步骤的因子网络。这部作品的巅峰之作揭晓 Bcl11b 的核心作用。这是完全出乎意料的，因为几项针对小鼠的详细研究表明 Bcl11b 表达仅限于 T、NKT 和 ILC2 谱系，并起到增强 T 细胞身份的作用。我们的 初步结果还揭示了 Runx2 和 Bcl11b 之间的相互关系，其中 Runx2 调节 一组在未成熟 NK 细胞中富集的转录因子，以及在整个细胞中富集的 Bcl11b 调节基因 典型的 NK 细胞成熟和适应性 NK 细胞表现出类似 T 细胞的基因表达特征。在这个 在应用程序中，我们将测试以下假设：Runx2 是强制未成熟表观遗传身份的主要枢纽 CD56bright NK 细胞，而 Bcl11b 抑制 Runx2 指导的转录程序以驱动典型 NK 细胞分化、成熟和细胞毒效应功能。我们还假设高水平的 Bcl11b 有助于 CMV 诱导的适应性 NK 细胞的 T 细胞样特征。 我们提出两个独立的目标来检验我们的假设。在第一个目标中，我们将使用过度表达 和敲低排序 NK 细胞亚群中的策略，以定义 Bcl11b 和 Runx2 在典型过程中的作用 NK 细胞分化。这些结果将指导后续的实验，以确定 Bcl11b 是否 造血祖细胞 (HPC) 中的过度表达可促进 NK 细胞成熟和效应功能 体外和过继转移到已形成肿瘤的小鼠体内后。在第二个目标中，我们将确定 Bcl11b 在促进 CMV 诱导的适应性 NK 细胞的 T 细胞相关特征中的作用并检验假设 高水平的 Bcl11b 以及激活受体和细胞因子受体刺激可驱动适应性 NK 细胞 分化和成熟。我们还将检验适应性 NK 细胞介导卓越抗肿瘤作用的假设 相对于典型 NK 细胞在体内发挥功能。该提案产生的结果将定义 Bcl11b 的作用 在促进典型和适应性 NK 细胞分化中，确定 Bcl11b 过度表达是否会增强 NK 细胞介导的抗肿瘤活性，并明确测试适应性 NK 细胞的抗肿瘤功能。

PROJECT SUMMARY/ABSTRACT Natural killer (NK) cells are innate lymphocytes that mediate cellular cytotoxicity against virally infected and malignant cells. Upon activation, they also release chemokines and cytokines that help prime and coordinate the adaptive immune response. Because of these functional properties, there is continued interest in developing NK cell products for immunotherapy. However, this effort is hindered by our limited understanding of human NK cell differentiation and the transcription factor networks that regulate this process. In work supported by current K99/R00 funding, we generated a considerable amount of preliminary data comprehensively characterizing the transcriptional and epigenetic landscapes in sorted NK and CD8+ T cell subsets isolated from cytomegalovirus (CMV) seropositive donors. To complement these analyses, we performed flow cytometry-based analyses of transcription factor expression and ChIP-seq on sorted peripheral blood NK cell subsets to construct transcription factor networks that potentially regulate key steps in NK cell differentiation. The culmination of this work revealed a central role for Bcl11b. This was completely unexpected given several detailed studies in mice showing that Bcl11b expression is restricted to the T, NKT, and ILC2 lineages and acts to enforce T cell identity. Our preliminary results also revealed a reciprocal relationship between Runx2 and Bcl11b, with Runx2 regulating a set of transcription factors enriched in immature NK cells and Bcl11b regulating genes enriched throughout canonical NK cell maturation and in adaptive NK cells exhibiting a T cell-like gene expression signature. In this application we will test the hypothesis that Runx2 is a major hub that enforces the epigenetic identity of immature CD56bright NK cells, while Bcl11b suppresses the Runx2-directed transcriptional program to drive canonical NK cell differentiation, maturation, and cytotoxic effector function. We also posit that high levels of Bcl11b contribute to the T cell-like features of CMV-induced adaptive NK cells. We propose two independent aims to test our hypotheses. In the first aim, we will use overexpression and knockdown strategies in sorted NK cell subsets to define the roles of Bcl11b and Runx2 during canonical NK cell differentiation. These results will guide subsequent experiments designed to determine whether Bcl11b overexpression in hematopoietic progenitor cells (HPCs) promotes NK cell maturation and effector function both in vitro and after adoptive transfer into mice with established tumors. In the second aim, we will determine the role of Bcl11b in promoting T cell-associated features of CMV-induced adaptive NK cells and test the hypothesis that high levels of Bcl11b along with activating receptor and cytokine receptor stimulation drives adaptive NK cell differentiation and maturation. We will also test the hypothesis that adaptive NK cells mediate superior antitumor function in vivo relative to canonical NK cells. Results generated from this proposal will define the role of Bcl11b in promoting canonical and adaptive NK cell differentiation, determine whether Bcl11b overexpression enhances NK cell-mediated antitumor activity, and definitively test the antitumor function of adaptive NK cells.