Investigating the role of Cdk5 and p35 in natural killer cell cytotoxicity

项目摘要/摘要 自然杀伤(NK)细胞是一种细胞毒性淋巴细胞，在杀死病毒感染者方面具有重要的免疫功能 细胞和癌细胞。NK细胞已被探索用于癌症免疫治疗，并具有优于T细胞的优势。 以治疗为基础。然而，它们的细胞毒性和肿瘤免疫监视功能通常在 癌症患者，很大程度上是由于转化生长因子-β水平升高，这是一种强大的免疫抑制细胞因子。周期蛋白- 依赖激酶5(CDK5)是一种CDK家族的丝氨酸/苏氨酸激酶。与基民盟的其他成员不同， 它的激酶活性主要依赖于辅活化子p35或p39的结合，其细胞周期尚不清楚。 角色。CDK5被认为主要在神经细胞中发挥作用，但最近的研究发现了CDK5的新作用 CDK5和p35在其他类型的细胞中表达，包括癌细胞和免疫细胞。我们第一次发现 CDK5和p35蛋白在NK细胞中均有表达，并在调节NK细胞中发挥重要作用 细胞毒性。此外，转化生长因子-β似乎以剂量依赖的方式诱导NK细胞p35的表达。 根据我们的初步数据，我们假设cdk5/p35激酶活性负向调节NK细胞。 并且是转化生长因子-β诱导的NK细胞功能障碍的关键介质，我们还假设CDK5/p35 抑制作用可用于加强NK细胞的免疫治疗。首先，我们将探讨CDK5/p35和转化生长因子是如何- β信号通路在NK细胞中重叠。利用基因工具敲除p35，以及选择性的CDK5 我们将确定CDK5抑制的NK细胞是否可以减轻各种表型 转化生长因子-β治疗引起的变化。我们将测量NK细胞表达的任何变化 激活/抑制受体、溶解颗粒细胞毒酶和细胞因子释放。我们还将确定如何 转化生长因子β诱导NK细胞p35表达，探讨CDK5/p35活性的分子机制 调节NK细胞毒性。P35基因敲除的NK细胞的整个转录组测序将用于揭示 CDK5信号通路下游的差异表达通路。我们也希望探索治疗的方法 抑制CDK5/p35在加强NK细胞免疫治疗中的作用。用体外细胞毒试验检测人外周血中 我们将确定p35基因敲除是否能使NK细胞抵抗转化生长因子-β诱导的 抑制细胞毒性。同样，使用已建立的患者来源的异种移植小鼠模型，我们将测试 P35基因敲除的NK细胞能否增强NK细胞对患者来源B细胞的过继治疗 急性淋巴细胞白血病(B-ALL)，已知通过升高转化生长因子-β导致NK功能障碍 分泌物。这个项目的发现将促进我们对信号通路的基本理解， 调节NK细胞毒性和调节NK功能障碍，可能导致改善基于NK细胞的癌症 免疫疗法。

PROJECT SUMMARY/ABSTRACT Natural killer (NK) cells are cytotoxic lymphocytes with important immune functions in killing virally infected cells and cancer cells. NK cells have been explored for cancer immunotherapy and have advantages over T cell- based therapies. However, their cytotoxicity and tumor immunosurveillance functions are often dysfunctional in cancer patients, in large part due to elevated levels of TGF-β, a potent immunosuppressive cytokine. Cyclin- dependent kinase 5 (Cdk5) is a Cdk family proline-directed serine/threonine kinase. Unlike other Cdk members, its kinase activity is primarily dependent on binding of the coactivator p35 or p39, and it has unclear cell cycle roles. Cdk5 was thought to primarily function in neuronal cells, but recent research has discovered new roles for Cdk5 and p35 in other cell types, including cancer and immune cells. For the first time, we have discovered that Cdk5 and p35 protein are both expressed in NK cells and appear to play an important role in regulating NK cell cytotoxicity. Additionally, TGF-β appears to induce p35 expression in NK cells in a dose-dependent manner. Based on our preliminary data, we hypothesize that Cdk5/p35 kinase activity negatively regulates NK cell cytotoxicity and is a key mediator of TGF-β-induced NK cell dysfunction, and we also hypothesize that Cdk5/p35 inhibition can be utilized to enhance NK cell immunotherapy. First, we will explore how the Cdk5/p35 and TGF- β signaling pathways overlap in NK cells. Using genetic tools to knock down p35, as well as the selective Cdk5 inhibitor roscovitine, we will determine whether Cdk5-inhibited NK cells can mitigate the various phenotypic changes caused by TGF-β treatment. We will measure any changes in the expression of NK cell activating/inhibitory receptors, lytic granule cytotoxic enzymes, and cytokine release. We will also determine how TGF-β induces p35 expression in NK cells, then investigate the molecular mechanism of how Cdk5/p35 activity regulates NK cytotoxicity. Whole transcriptome sequencing of p35 knockdown NK cells will be used to reveal differentially expressed pathways downstream of Cdk5 kinase signaling. We also wish to explore the therapeutic potential of Cdk5/p35 inhibition in enhancing NK cell immunotherapy. Using in vitro cytotoxicity assays against cancer cell lines, we will determine whether p35 knockdown will enable NK cells to resist TGF-β-induced suppression of cytotoxicity. Similarly, using established patient-derived xenograft mouse models, we will test whether p35 knockdown NK cells are able to enhance NK cell adoptive therapy against patient-derived B cell acute lymphoblastic leukemia (B-ALL), which is known to cause NK dysfunction through elevated TGF-β secretion. Discoveries from this project would advance our basic understanding of the signaling pathways that regulate NK cytotoxicity and mediate NK dysfunction, potentially leading to improved NK cell-based cancer immunotherapies.