Dissecting the Differential Impacts of Toll-like Receptor 9 Agonism on the Capacity of Human Natural Killer Cells to Mediate Target Cell Killing

项目概要 Toll 样受体 9 (TLR9) 激动剂治疗可增强人类自然杀伤 (NK) 的能力 细胞直接杀死目标（例如，恶性的；感染的）细胞。 TLR9激动剂的概念 治疗增加人类 NK 细胞介导抗体依赖性细胞的能力 细胞毒性（ADCC）可以在文献中找到，但没有直接公开的数据 证明这种增加的发生。尽管如此，多项临床试验注册 表明他们的试验设计预期 TLR9 激动剂治疗对 NK 细胞产生强大影响 功能。鉴于缺乏对 TLR9 激动剂的影响的完整了解 人类 NK 细胞，迫切需要确定 TLR9 激动剂对 人类 NK 细胞介导 ADCC 的能力。因此，该应用程序的总体目标是 了解 TLR9 激动剂对人类 NK 细胞介导的 ADCC 的影响，同时使用直接 杀死作为阳性对照。我们的中心假设是根据我们的试点制定的 数据显示，TLR9 激动作用导致 NK 细胞表面 CD16 丢失，从而 降低这些细胞介导 ADCC 的能力。我们将通过完成来检验这个假设 具体目标如下： 目标 1：建立 NK 细胞中 CD16 脱落的时间过程， 当人类 PBMC 经历 TLR9 激动/- ADAM17 抑制时产生细胞因子。 为此，我们将使用流式细胞术、ELISA 和多重策略来追踪 CD16 水平和 细胞因子贯穿我们的实验窗口。目标 2：量化人类 NK 细胞的能力 当 TLR9 激动与 ADAM17 抑制相结合时介导杀伤。为此，我们将 使用我们的新型 NK-SADKA（自然杀伤细胞 – 同时 ADCC 和直接杀伤测定） 基于流式细胞术的方法。除了测量 TLR9 激动剂对 人类 NK 细胞，该策略控制潜在的人与人反应差异 对这种药。除了我们新的杀伤测定之外，我们的研究还具有创新性，因为 TLR9 激动剂 使用的制造方式消除了与相关的混淆观察结果 用于生成其他 TLR9 激动剂的稳定方法。在此结论中 通过这项工作，我们希望能够充分了解 TLR9 激动剂对人类 NK 的影响 细胞介导 ADCC 的能力。我们生成的数据将与解释高度相关 数十项已完成、正在进行和计划中的临床试验的结果评估 TLR9 激动剂治疗恶性肿瘤或感染的功效。最后，我们的临床前 数据将揭示将 TLR9 激动剂与 ADAM17 抑制相结合的潜力，以改善 在未来的研究中 ADCC 介导的恶性或感染细胞的清除。

PROJECT SUMMARY Toll-like receptor 9 (TLR9) agonist treatment increases the ability of human natural killer (NK) cells to directly kill target (e.g., malignant; infected) cells. The concept that TLR9 agonist treatment increases the capacity of human NK cells to mediate antibody dependent cellular cytotoxicity (ADCC) can be found in the literature, yet there are no published data directly demonstrating that this increase occurs. Nevertheless, several clinical trials registrations indicate that their trial design anticipates robust impacts of TLR9 agonist therapy on NK cell functions. Given the lack of a complete understanding of the impact(s) of TLR9 agonism on human NK cells, there is an urgent need to determine the effects of TLR9 agonism on the capacity of human NK cells to mediate ADCC. Thus, the overall objective of this application is to understand the impacts of TLR9 agonism on human NK cell-mediated ADCC while using direct killing as a positive control. Our central hypothesis, which was formulated based on our pilot data, is that TLR9 agonism causes a loss of CD16 from the surface of NK cells – thereby reducing the capacity of these cells to mediate ADCC. We will test this hypothesis by completing the following specific aims: Aim 1: Establish a time course for CD16 shedding from NK cells and for cytokine production when human PBMCs experience TLR9 agonism +/- ADAM17 inhibition. To do this, we will use flow cytometry, ELISA, and multiplex strategies to track CD16 levels and cytokines throughout our experimental window. Aim 2: Quantify the capacity of human NK cells to mediate killing when TLR9 agonism is combined with ADAM17 inhibition. To do this, we will use our novel NK-SADKA (Natural Killer cell – Simultaneous ADCC and Direct Killing Assay) flow cytometry-based approach. In addition to measuring the impact of TLR9 agonism on human NK cells, this strategy controls for potential human-to-human variations in the response to this drug. Beyond our new killing assay, our study is innovative because the TLR9 agonist utilized is manufactured in a way that eliminates confounding observations associated with stabilization methods used in the generation of other TLR9 agonists. At the conclusion of this work, we expect to have a full understanding of the impacts TLR9 agonism has on human NK cells’ ability to mediate ADCC. The data we generate will be highly relevant for interpreting outcomes from the dozens of completed, ongoing, and planned clinical trials evaluating the efficacy of TLR9 agonists as treatments for malignancies or infections. Finally, our preclinical data will inform the potential for combining TLR9 agonism with ADAM17 inhibition for improved ADCC-mediated clearance of malignant or infected cells in future studies.