Background

Natural killer (NK) cells are key effectors for innate immunity that play a pivotal role in immune surveillance and cytotoxicity against cancer [1]. Recent works highlighted the potential of NK cells in cancer immunotherapy, owing to their directed cytotoxic capabilities and their roles in orchestrating broader immune responses to cancer cells [2]. With surface receptors recognizing other cells expressing low levels of major histocompatibility complex (MHC) class I molecules, NK cells can mount potent assaults on cancerous or infected cells and destroy those cells by secreting various cytokines [3]. NK group member 2D (NKG2D, also known as CD314) is one of the most common activating receptors on NK cells. By the binding of NKG2D receptors to the ligands on cancer cells, NK cells can be activated in immune recognition [4]. However, many cancer cells are enabled to aberrantly express the NKG2D ligands, thus avoiding recognition so as to inactivate and even destroy NK cells [5,6]. p53 protein acts as a powerful transcriptional factor that upregulates the stimulatory NKG2D ligands, including retinoic acid early transcript 1 (RAET1), histocompatibility antigen 60 (H60), and mouse UL16-binding protein-like transcript 1 (Mult1) of murine cancer cells, to activate NK cells against tumors [7,8]. Missense mutants of p53 proteins are frequently detected in cancer cells and exhibit gain-of-function to promote tumor progression [9]. Both loss of the function of wild-type p53 protein and gain-of-function of p53 mutants can alter NKG2D ligands, either by decreasing stimulatory ligands (such as Mult1, ULPB1) or by increasing inhibitory ligands (such as H60a, RAET1E2), and then inactivate NK cells [6,8,10,11]. In an effort to garner evidence on p53 and NK cell activation, we designed a method to quantitatively analyze cytotoxic NK cells using imaging flow cytometry (IFC)—a technology that combines multiparametric flow cytometry and fluorescence microscopy [8,12].

A wide variety of NK cell subsets exist in a variable tissue microenvironment, reflecting different maturation stages with functional capabilities. The NKG2D⁺ subset of NK cells is the major phenotype of NK cells and is highly cytotoxic to cancer cells [13–16]. It has been estimated that NKG2D⁺ splenic cells included only approximately 3% of γδ T cells, αβ CD8⁺ T cells, and macrophages [14–17]. IFC combines the high-event-rate nature of flow cytometry with the advantages of single-cell image acquisition associated with microscopy [18].

Tissue NK cells can be quantitatively analyzed using several methods, including traditional flow cytometry and advanced analysis techniques, such as multicolor flow cytometry or high-parameter cytometry, single-cell proteo-genomics, and molecular imaging. However, these often lack the detailed morphological information needed to distinguish true positive cells from nonspecific staining or background noise for precise quantification [19]. We developed IFC as a tool to measure the cytotoxic subtype of NK cells with the NKG2D marker through quantitative comparison with high-resolution confocal microscopy. We show that IFC can be used as a simple and direct measure of cytotoxic NK cells against tumors, as this technology possesses greater dimensionality than standard flow cytometry. This cell-imaging dimensionality significantly improves discrimination of true NKG2D⁺ NK cells from nonspecific staining or background noise using double-gating, including brightfield and fluorescence. IFC enables the analysis of spatial and structural details of cell populations with greater precision, which is critical for identifying cell features that conventional flow cytometry is unable to reach.

This protocol describes the steps to quantify cytotoxic NK cells (NKG2D⁺) of mice after injections of breast cancer cells. With minor modifications, such as replacing the conjugated CD8 antibody, we also used it to analyze CD8⁺ T cells [8]. This protocol can be applied to analyze cytotoxic NK cells with the NKG2D receptor in cell culture and animal tissues. Further analysis, such as multicolor IFC and cytokine assay, is required to precisely characterize the subsets and effects of NK cells in immunity [20].