Elucidating the role of EOMES in Memory-Like NK cell Differentiation

阐明 EOMES 在记忆样 NK 细胞分化中的作用

• 批准号: 10721878
• 负责人: Jennifer Tran
• 金额: $ 3.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10721878
• 关键词: ATAC-seq; Acute Myelocytic Leukemia; B-Lymphocytes; Binding; Biological Assay; CD8-Positive T-Lymphocytes; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cell Differentiation process; Cell Nucleus; Cell Reprogramming; Cell Therapy; Cell division; Cell physiology; Cells; Cellular biology; Chromatin; Clinic; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Cytokine Activation; DNA Binding; Data; Development; Disease; Disease remission; Epigenetic Process; Exhibits; Future; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Haptens; Hematologic Neoplasms; Hematopoietic stem cells; Heterogeneity; Host Defense; Human; IL18 gene; In Vitro; Interleukin-12; Interleukin-15; Intervention; Knock-out; Ligation; Lymphocyte; Lymphoid Cell; Malignant Neoplasms; Measures; Mediating; Memory; Modification; Molecular; Mus; NK cell therapy; Natural Killer Cells; Patients; Phase; Phase I Clinical Trials; Phenotype; Play; Process; Production; Proteins; Receptor Activation; Refractory; Relapse; Role; Running; Safety; Signal Transduction; T-Lymphocyte; Techniques; Testing; Transcript; Up-Regulation; Virus Diseases; cancer cell; clinical application; clinical efficacy; comparison control; cytokine; cytotoxicity; early phase clinical trial; improved; in vivo; insight; knock-down; multiple omics; neoplastic cell; novel; novel strategies; nuclease; prevent; programs; response; single-cell RNA sequencing; transcription factor; transcriptional reprogramming; transcriptome sequencing; tumor

Project Summary Natural killer (NK) cells are innate lymphoid cells that are crucial for host defense from viral infections and tumor cells. The ability of NK cells to recognize and kill malignant cells provides a strong premise to advance them as a cellular therapy in the clinical setting. While NK cell-based therapies are being explored in clinical trials, issues with persistence and in vivo functionality have been identified as limitations to their clinical application. Our lab pioneered cytokine-induced memory-like (ML) NK cells that are generated after brief activation with IL-12, IL-15, and IL-18. This overcomes many of the shortcomings of conventional NK cells as a cellular therapy. ML NK cells exhibited enhanced functionality, cytotoxicity, and persistence in a phase I clinical trial for acute myeloid leukemia (AML), with many patients achieving complete remissions from this novel intervention. Despite the translational advancement, there is little known about the underlying mechanisms of ML reprogramming in NK cells. Clarifying the molecular programs that drive the ML phenotype will have broad implications for optimizing NK cells as a cellular-based therapy for hematological malignancies. The proposed project aims to better understand the mechanisms of ML NK cell differentiation with a focus on the role of EOMES. EOMES is a T-box transcription factor (TF) that is essential for NK cell development from hematopoietic stem cells and has been implicated in generating memory in CD8+ T cells. Data from our lab demonstrated that EOMES deletion prior to activation with cytokines abrogated ML NK cell differentiation, suggesting that EOMES plays a key role in ML reprogramming. We hypothesize that activation with IL-12/15/18 triggers a molecular reprogramming of conventional NK cells to ML NK cells mediated by EOMES to induce a unique transcriptional profile and poised epigenetic state, which facilitates the enhanced functionality of ML NK cells upon restimulation by cytokines or a tumor cell. Aim 1 of this proposal focuses on determining the memory differentiation phase that requires EOMES. Specifically, we will assess the requirement of EOMES during initiation (prior to IL-12/15/18 activation) and differentiation (after IL-12/15/18 activation) using CRISPR/Cas9 deletion. Aim 2 focuses on defining the transcriptional and epigenetic landscape induced by EOMES in the different phases of ML reprogramming through CRISPR/Cas9 knockdown, single-cell RNAseq, and single-nuclei ATACseq, as well as defining the genes regulated by EOMES in ML NK cells using Cut&Run. Together, these results will reveal mechanisms of ML NK cell differentiation driven by EOMES, further advancing the potential of ML NK cell therapies and enhancing our understanding of ML NK cell biology.

项目摘要 自然杀伤（NK）细胞是先天性淋巴细胞，对于宿主防御病毒感染至关重要 和肿瘤细胞。NK细胞识别和杀伤恶性细胞的能力为推进 在临床环境中作为细胞疗法。虽然基于NK细胞的疗法正在临床上探索， 试验、持久性和体内功能性问题已被确定为其临床限制 应用程序.我们的实验室开创了尼古丁诱导的记忆样（ML）NK细胞， 用IL-12、IL-15和IL-18活化。这克服了传统NK细胞作为免疫调节剂的许多缺点。 细胞疗法ML NK细胞在I期临床试验中表现出增强的功能性、细胞毒性和持久性。 急性髓性白血病（AML）的临床试验，许多患者从这种新的治疗中获得完全缓解。 干预尽管翻译的进展，很少有人知道的基本机制， NK细胞中的ML重编程。阐明驱动ML表型的分子程序将具有广泛的 优化NK细胞作为血液恶性肿瘤的细胞疗法的意义。 该项目旨在更好地了解ML NK细胞分化的机制，重点是 关于EOMES的作用EOMES是一种T盒转录因子（TF），对NK细胞从 在造血干细胞中，它与在CD 8 + T细胞中产生记忆有关。来自我们实验室的数据 证明了在用细胞因子活化之前EOMES缺失消除了ML NK细胞分化， 这表明EOMES在ML重编程中起关键作用。我们假设IL-12/15/18的激活 触发由EOMES介导的常规NK细胞向ML NK细胞的分子重编程，以诱导 独特的转录谱和稳定的表观遗传状态，这有助于增强ML NK的功能 细胞因子或肿瘤细胞再刺激后的细胞。本提案的目标1侧重于确定内存 分化阶段需要EOMES。具体而言，我们将在 使用CRISPR/Cas9的起始（在IL-12/15/18活化之前）和分化（在IL-12/15/18活化之后） 删除。目的2的重点是定义转录和表观遗传景观诱导的EOMES中， 通过CRISPR/Cas9敲低、单细胞RNAseq和单核细胞进行ML重编程的不同阶段 ATACseq，以及使用Cut&Run定义ML NK细胞中由EOMES调节的基因。所有这些 结果将揭示由EOMES驱动的ML NK细胞分化的机制，进一步推进 ML NK细胞疗法和增强我们对ML NK细胞生物学的理解。