Bcl11b: A Master Transcription Factor Controlling Human NK Cell Development

项目总结/摘要 自然杀伤（NK）细胞是先天性淋巴细胞，其介导针对病毒感染的细胞毒性， 恶性细胞一旦激活，它们还释放趋化因子和细胞因子，帮助启动和协调细胞因子。 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，由于我们对人类NK细胞的了解有限，这一努力受到阻碍。 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，我们产生了大量的初步数据，全面表征了 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)血清反应阳性的献血者为了补充这些分析，我们进行了基于流式细胞术的分析， 在分选的外周血NK细胞亚群上的转录因子表达和ChIP-seq以构建转录 这些因子网络可能调节NK细胞分化的关键步骤。这项工作的高潮揭示了 Bcl11b的核心作用。这是完全出乎意料的，因为在小鼠身上进行的几项详细研究表明， Bcl11b的表达仅限于T、NKT和ILC2谱系，并起到加强T细胞身份的作用。我们 初步结果还揭示了Runx2和Bcl 11b之间的相互关系，Runx2调节Bcl 11b的表达， 在未成熟NK细胞中富集的一组转录因子和在整个NK细胞中富集的Bcl 11b调节基因 在典型NK细胞成熟和表现出T细胞样基因表达特征的适应性NK细胞中。在这 应用程序，我们将测试假设Runx2是一个主要的枢纽，执行表观遗传身份的不成熟 CD56bright NK细胞，而Bcl11b抑制Runx2指导的转录程序，以驱动经典NK细胞 细胞分化、成熟和细胞毒性效应子功能。我们还证实，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。 我们提出了两个独立的目标来测试我们的假设。在第一个目标中，我们将使用过表达 和敲除策略，以确定Bcl 11b和Runx2在典型NK细胞亚群中的作用。 NK细胞分化。这些结果将指导后续的实验，以确定是否Bcl11b 在造血祖细胞（HPCs）中的过表达促进NK细胞成熟和效应子功能， 在体外和过继转移到已建立肿瘤的小鼠中后。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验证假设 高水平的Bcl 11 b沿着活化受体和细胞因子受体刺激， 分化和成熟。我们还将检验适应性NK细胞介导上级抗肿瘤作用的假设。 相对于典型NK细胞的体内功能。从这个提议产生的结果将定义Bcl11b的作用 在促进典型和适应性NK细胞分化中，确定Bcl11b过表达是否增强 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.