Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

Abstract

Immune therapy of cancer is among the most promising recent advances in medicine. Whether the immune system can keep cancer in check depends on, among other factors, the efficiency of immune cells to recognize and eliminate cancer cells. We describe a time-resolved single-cell assay that reports the quality, quantity, and kinetics of target cell death induced by single primary human natural killer (NK) cells. The assay reveals that single NK cells induce cancer cell death by apoptosis and necrosis but also by mixed forms. Inhibition of either one of the two major cytotoxic pathways, perforin/granzyme release or FasL/FasR interaction, unmasked the parallel activity of the other one. Ca influx through Orai channels is important for tuning killer cell function. We found that the apoptosis/necrosis ratio of cancer cell death by NK cells is controlled by the magnitude of Ca entry and furthermore by the relative concentrations of perforin and granzyme B. The possibility to change the apoptosis/necrosis ratio employed by NK cells offers an intriguing possibility to modulate the immunogenicity of the tumor microenvironment.

Rac1/ROCK-driven membrane dynamics promote Natural Killer cell cytotoxicity via necroptosis

“…We confirmed that chelating Ca 2+ by EGTA abrogated both granzyme-B-dependent apoptosis and necrosis induced by primary NK cells 15 in the cell line panel that we studied (data not shown). However, we did not observe high Ca 2+ concentration shifted NK cell killing to the necrosis mode, as reported by Backes et al [13]. Increasing Ca 2+ concentration up to 3.5 mM did not enhance necrotic killing of SMMC-7721 or U-2 OS cells.…”

“…Upon recognition of abnormal cell targets, NK cells can elicit direct target cell death by 4 three main mechanisms, i.e., lytic granule-mediated apoptosis, death ligand-mediated apoptosis and necrosis [11][12][13]. Lytic granule-mediated cytotoxicity is considered the principal cytotoxic pathway of NK cells.…”

“…In addition, NK cells express a variety of death ligands, such as Fas ligand (FasL) and TNF-related apoptosis inducing ligand (TRAIL), which engage cognate death receptors expressed on the target cell surface, triggering death by receptor-mediated extrinsic apoptosis [15,16]. Target cell death by NK cell-induced necrosis has only been reported in a few studies and its mechanistic basis is still largely unknown [13,17].…”

NK cells switch from granzyme B to death receptor–mediated cytotoxicity during serial killing

“…This could be due to an induction of pyroptosis mediated by activated caspase-3 as previously reported ( Rogers et al, 2017 ; Wang et al, 2017 ). Alternatively, this could be caused by the amount of perforin and GrzB released by the NK cells ( Backes et al, 2018 ). Although it was recently shown that the degranulation of as little as two to four granules is sufficient to cause cell death, it was also reported that NK cells release ∼10% of their total granules in a single killing event ( Gwalani and Orange, 2018 ).…”

Natural Killer Cells: Tumor Surveillance and Signaling

“…NK cells kill infected and transformed cells via a variety of mechanisms, including the delivery of lytic granules loaded with proteases and pore-forming proteins such as granzymes and perforin, release of cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ), upregulation of FASL and TNF-related apoptosis-inducing ligand (TRAIL) and by antibody-dependent cellular cytotoxicity (ADCC) [15][16][17][18][19]. There are multiple steps between NK cell: target cell engagement and cell killing, with receptor-ligand interactions thought to be the initiating step in the formation of an immunological synapse (IS) ( Figure 1A,B) [20,21].…”