A research-driven approach to the identification of novel natural killer cell deficiencies affecting cytotoxic function

Abstract

Natural killer cell deficiencies (NKDs) are an emerging phenotypic subtype of primary immune deficiency. NK cells provide a defense against virally infected cells using a variety of cytotoxic mechanisms, and patients who have defective NK cell development or function can present with atypical, recurrent, or severe herpesviral infections. The current pipeline for investigating NKDs involves the acquisition and clinical assessment of patients with a suspected NKD followed by subsequent in silico, in vitro, and in vivo laboratory research. Evaluation involves initially quantifying NK cells and measuring NK cell cytotoxicity and expression of certain NK cell receptors involved in NK cell development and function. Subsequent studies using genomic methods to identify the potential causative variant are conducted along with variant impact testing to make genotype-phenotype connections. Identification of novel genes contributing to the NKD phenotype can also be facilitated by applying the expanding knowledge of NK cell biology. In this review, we discuss how NKDs that affect NK cell cytotoxicity can be approached in the clinic and laboratory for the discovery of novel gene variants.

Graphical abstract

Figure 1.

The 2 classes of NKDs and the critical steps of development and cytotoxic function. The focus of this review is on the functional arm.

Figure 2.

General approaches to studying the mutational impact and genotype-phenotype causality in candidate genes for NKDs. HTS, high-throughput screening; iPSC, induced pluripotent stem cell; RNAseq, RNA sequencing; WES, whole exome sequencing; WGS, whole genome sequencing.

Figure 3.

Examples of the NK cell IS during cytotoxicity. (A) Coincubation and subsequent fixed-cell imaging of NK cells (NK-92) conjugated to a K562 erythroleukemic target cell using spinning disk confocal microscopy. The image shows the formation of a lytic synapse between the NK and target cells where polymerized actin (white) is relatively enriched at the lytic immune synapse. The lytic granules (green; labeled using perforin as a marker for lytic granule content) are converged around the MTOC (red), with some having polarized toward the target cell (cyan; indicated with a membrane dye) as shown by the MTOC near the immune synapse. Image was cropped to focus on a single conjugate. (B) Structured illumination–total internal reflection fluorescence (SI-TIRF) microscopy of the actin meshwork at the IS of an NK cell (YTS) that was plated and fixed on an activating glass surface and then subsequently stained for F-actin using phalloidin. The activating surface is composed of anti-CD18 for adhesion and anti-CD28 for activation, thereby creating a representative target cell on a glass surface, enabling an en face assessment of actin architecture during IS formation. (C) Time lapse SI-TIRF microscopy of a YTS cell on an activated surface. YTS cells were placed on an activating glass surface and fixed at certain time points (2, 5, 10, and 20 minutes) and stained for F-actin using phalloidin to examine the evolution of the IS. The cell initially touches down upon the glass surface and initially extends out lamellipodia and filopodia as it spreads. By 20 minutes, a mature IS is formed as shown by the intricate dense actin mesh. Scale bars, 10 μm.