Evaluation of NF-kB Nuclear Translocation in Natural Killer Cells by Imaging Multispectral Flow Cytometry as a Marker of Anticancer Immune Activation

Innate immunity has a potent antitumor function and occurs as the first line of defense in cancer immunosurveillance. Natural killer (NK) cells are one of the most important effectors of innate immunity and exert their cytotoxic activity as a result of signals mediated by activating and inhibitory receptors able to recognize ligands on cancer cells. Of note, NK cells play a pivotal role in the efficacy of anticancer therapies, as those employing monoclonal antibodies, by mediating antibody-dependent cell cytotoxicity. Additionally, some types of chemotherapeutics, such as taxanes, can modulate NK cell function. Thus, evaluating the activation state of NK cells is crucially useful for both monitoring the response to therapy and optimizing therapeutic strategies for improving patient’s outcome. NK cell function is usually assessed by flow cytometry assays evaluating markers like perforin and granzyme or cytotoxic activity, which, however, represent late end point measures of NK cell activation. In this context, NF-kB represents an important mediator of pro-inflammatory gene expression in several immune cells, including NK cells. Upon activation, the NF-kB p65 subunit translocates from the cytoplasm to the nucleus and promotes the transcription of various genes, as those encoding for perforin, granzyme, and interferon-gamma. Therefore, assessing the nuclear translocation of this transcription factor represents a valuable strategy to study the triggering of immune effectors. In this chapter, we describe an imaging multispectral flow cytometry assay able to evaluate the nuclear translocation of the NF-kB p65 in NK cells as a marker of anticancer immune activation.