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

Quantifying human natural killer cell-mediated antibody-dependent cellular cytotoxicity

Peripheral blood mononuclear cells (PBMCs) include cells that are essential to the functions of the immune system, both in the innate and adaptive immune responses. Natural killer (NK) cells are a primary cell type responsible for executing innate responses. NK cells can recognize and kill infected or malignant cells without the need for memory functions or antigen specificity (the topic of a separate presentation by fellow lab member, Angela Truong). However, NK cells can also destroy their targets using antibody-dependent cellular cytotoxicity, or ADCC, a combination of innate and adaptive immune techniques. ADCC requires an antigen-specific antibody to bind to the target cell. The other end of the antibody is bound to the NK cell to bridge the target and the effector cells. This bridging initiates a cytotoxic response that lyses the target cell. Our lab has developed a testing system, referred to as the Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA), that allows for the analysis of both ADCC and direct killing. The same donor samples are used for both the direct killing and ADCC portions of the assay to control for donor response variation. Within the ADCC portion of the assay (the focus of this presentation), aliquots of human lymphoma (e.g., Daudi) target cells are labeled with a fluorescent dye to distinguish them from the unlabeled human NK effector cells. Target and effector cells are combined (5:1 effector to target ratio) and co-incubated (2 hours) in the presence of an antibody known to facilitate ADCC (e.g., Rituximab which is an anti-CD20 antibody). Killing capacity is analyzed using a flow cytometer. Confirming that our ADCC arm of the NK-SADKA is functioning properly, the percent of NK cell-mediated ADCC killing of the target cells was found to be higher in samples with the ADCC-mediating antibody relative to samples without the antibody. Data-to-date will be presented. Our NK-SADKA, including the ADCC arm described herein, will have impacts on clinical trial interpretations and designs, particularly when those trials are using interventions (e.g., immunotherapies) intended to alter NK cell killing capacities

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