Development and implementation of natural killer cell simultaneous ADCC and direct killing assay

Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing

Functional NK Cell-Mediated ADCC Killing Assay and Protocol in B-Cell Lymphoma

In the Denton Immunobiology Lab, we use human natural killer (NK) cells to kill cancer cells. Specifically, my project is using NK cells to kill lymphoma cells in an indirect manner termed ADCC (antibody dependent cellular cytotoxicity). To measure how much killing occurs in our experiments we utilize a flow cytometry-based assay. I participated in the establishment of the baseline parameters of our flow cytometry-based “killing assay” protocol and will discuss some of these in my poster. For my FUSE project, I proposed to evaluate the impact of anti-PD1 immunotherapy on NK cell killing capacity. My lab colleagues and I preformed many experiments jointly because of the limited number of human cells available and the overlap in control conditions that we tested. In my poster, I will present our data-to-date regarding the impact on NK cell killing of lymphoma cell plus/minus anti-PD1 immunotherapy

Establishing staining parameters for successfully discriminating target cells from effector cells using flow cytometry

Establishing staining parameters for successfully discriminating target cells from effector cells using flow cytometry Arriana Blackmon1, Anna R. Mahr1, Maia Bennett1, and Paul W. Denton, PhD1 1 Department of Biology, University of Nebraska at Omaha, Omaha, NE Background: The Denton Immunobiology laboratory has an overall research goal of improving human immune functions - with a particular emphasis on natural killer (NK) cells. As their name implies, NK cells kill target cells (e.g., infected or malignant cells). To study human NK cell killing activity ex vivo, our laboratory has developed a specialized technique we call the Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA). This name is given because we can use this strategy to test NK cell killing following two distinct mechanisms of target cell recognition (direct or via antibody “bridging”). Our assay requires that we be able to clearly distinguish targets from effectors (the NK cells) when we assess killing efficacy. Developing and optimizing that method is the focus of this abstract. We assess killing using a flow cytometer. Therefore, we sought a fluorescent strategy to separate targets from effectors. A cost effective and efficient method for causing cells to fluoresce is staining with CFDA-SE. When this molecule is taken into cells, the molecule is converted to CFSE a protein dye that fluoresces green when excited by blue light. We determined to apply this dye to our target cells such that we can focus our analyses on “green” cells to determine their viability within the NK-SADKA. For this to work, we needed to determine parameters (e.g., concentration) for how to treat target cells with CFDA-SE to achieve optimal fluorescence (e.g., ~ 1 log higher than background). We tested staining characteristics for two different target cells (i.e., lymphoma cells as ADCC targets and leukemia cells as direct killing targets). Our data support both cell types being labeled with 1.2 μM CFDA-SE to achieve optimum fluorescence. This finding allows us to implement our NK-SADKA in many context, including assessing the impact of interventions (e.g., immunotherapies) on the ability of human NK cells to mediate ADCC as well as direct killing of target cells

Development and implementation of natural killer cell simultaneous ADCC and direct killing assay

Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing