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

项目摘要 Toll样受体9（TLR 9）激动剂治疗增加人自然杀伤（NK）的能力 细胞直接杀死靶（例如，恶性的;感染的）细胞。TLR 9激动剂 治疗增加了人NK细胞介导抗体依赖性细胞增殖的能力， 细胞毒性（ADCC）可以在文献中找到，但没有直接发表的数据 证明了这种增长的发生。然而，一些临床试验注册 表明他们试验设计预期TLR 9激动剂治疗对NK细胞的强有力影响 功能协调发展的由于缺乏对TLR 9激动对肿瘤细胞增殖的影响的完全理解， 因此，迫切需要确定TLR 9激动对人NK细胞的作用。 人NK细胞介导ADCC的能力。因此，本申请的总体目标是 了解TLR 9激动对人NK细胞介导的ADCC的影响，同时使用直接 作为阳性对照。我们的中心假设是基于我们的飞行员 数据，是TLR 9激动导致CD 16从NK细胞表面的损失-从而 降低这些细胞介导ADCC的能力。我们将通过完成以下步骤来检验这一假设： 以下具体目标：目标1：建立CD 16从NK细胞脱落的时程， 对于当人PBMC经历TLR 9激动+/-ADAM 17抑制时的细胞因子产生。 为此，我们将使用流式细胞术、ELISA和多重策略来跟踪CD 16水平， 在我们的实验窗口中。目的2：量化人NK细胞的能力 当TLR 9激动与ADAM 17抑制组合时介导杀伤。为此，我们将 使用我们的新型NK-SADKA（自然杀伤细胞-同时ADCC和直接杀伤试验） 基于流式细胞术的方法。除了测量TLR 9激动对细胞凋亡的影响之外， 人NK细胞，该策略控制了应答中潜在的人与人之间的差异 这种药。除了我们新的杀伤试验，我们的研究是创新的，因为TLR 9激动剂 所使用的是以一种消除与以下因素相关的混淆观察结果的方式制造的 本发明还涉及用于产生其它TLR 9激动剂的稳定化方法。在这一结论 通过这项工作，我们希望能够全面了解TLR 9激动对人类NK细胞的影响， 细胞介导ADCC的能力。我们生成的数据将与解释高度相关 来自数十项已完成、正在进行和计划中的临床试验的结果， TLR 9激动剂作为恶性肿瘤或感染治疗的功效。最后，我们的临床前 数据将告知将TLR 9激动与ADAM 17抑制相结合以改善免疫应答的可能性。 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.