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. Ca2+ 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 Ca2+ 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.
Highlights
Immune therapy of cancer is among the most promising recent advances in medicine
We found that the apoptosis/necrosis ratio of cancer cell death by natural killer (NK) cells is controlled by the magnitude of Ca2؉ entry and by the relative concentrations of perforin and granzyme B
The assay developed here allows quantification of target cell death induced through contact with primary human natural killer cells regarding kinetics, modes, and mechanisms on a single-cell level
Summary
Jurkat E6-1 and K562 cells are well-characterized targets for NK cytotoxicity. The FRET-based apoptosis reporter Casper3-GR [20] was stably transfected into both cell lines to create stable, monoclonal target cell lines named Jurkat pCasper and K562 pCasper to analyze NK cell–mediated cytotoxicity on a single-cell level. With 1 mM extracellular Ca2ϩ present, Jurkat pCasper target cells were killed by NK cells as shown before in AIMV medium, which contains 800 M free Ca2ϩ [29] with a balanced ratio of apoptosis and primary necrosis, analyzed during 80 min (Fig. 6a). In cases where FasR-deficient K562 pCasper cells were used as targets, NK cells killed the target mostly by primary necrosis with very little apoptosis in the presence of 800 M free Ca2ϩ in AIMV medium (Fig. 6c), induced by the perforin/granzyme mechanism. Consistent with target cell necrosis, fluorescence in the GFP and FRET decreased in parallel, whereas the donor ratio remained relatively unchanged (Fig. 7e) These examples show that the single-cell apoptosis/necrosis assay can be transferred into a 3D matrix– based environment. NK vs spheroid MCF-7 pCasper b 0 min 50 μm Apoptosis c 0 min 3 μm 5 min 3 μm 20 min Necrosis
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