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

项目总结 Toll样受体9(TLR9)激动剂治疗增强人自然杀伤细胞(NK)的能力 直接杀死目标细胞(例如，恶性的；受感染的)细胞。TLR9激动剂的概念 治疗可提高人NK细胞介导抗体依赖细胞的能力 细胞毒性(ADCC)可以在文献中找到，但没有直接发表的数据 证明了这种增长是发生的。尽管如此，一些临床试验注册 表明他们的试验设计预测了TLR9激动剂治疗对NK细胞的强烈影响 功能。鉴于缺乏对TLR9激动剂影响的完整了解(S) 因此迫切需要确定TLR9激动剂对人NK细胞的影响。 人NK细胞介导ADCC的能力。因此，此应用程序的总体目标是 直接使用TLR9激动剂对人NK细胞介导的ADCC的影响 杀人作为一种积极的控制。我们的中心假设，这是根据我们的飞行员提出的 数据表明，TLR9激动剂导致NK细胞表面CD16的丢失--从而 降低这些细胞介导ADCC的能力。我们将通过完成以下操作来检验这一假设 具体目标如下：目标1：建立CD16从NK细胞和 当人的PBMC经历TLR9激动剂/-ADAM17抑制时，用于细胞因子的产生。 为此，我们将使用流式细胞术、ELISA和多重策略来跟踪CD16水平和 细胞因子贯穿我们的实验窗口。目的2：量化人类自然杀伤细胞的能力 当TLR9激动剂与ADAM17抑制剂联合应用时，介导杀伤作用。要做到这一点，我们将 使用我们的新NK-SADKA(自然杀伤细胞-同时ADCC和直接杀伤试验) 基于流式细胞术的方法。除了测量TLR9激动剂对 人类NK细胞，这一策略控制了潜在的人与人之间的反应变异 与这种药物的关系。除了我们新的杀伤试验，我们的研究是创新的，因为TLR9激动剂 利用的制造方式消除了与以下各项相关的混淆观察 用于产生其他TLR9激动剂的稳定方法。在此结束时 工作中，我们希望充分了解TLR9激动剂对人类NK细胞的影响 细胞介导ADCC的能力。我们生成的数据将与口译高度相关 数十项已完成的、正在进行的和计划中的临床试验的结果 TLR9激动剂治疗恶性肿瘤或感染的疗效。最后，我们的临床前研究 数据将提供将TLR9激动剂与ADAM17抑制相结合的潜力，以改善 ADCC介导的恶性或感染细胞的清除在未来的研究中。

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.