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

University Of Nebraska Omaha, Omaha NE

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Abstract

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.