IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells

Abstract

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.

Keywords: IL-12; IL-15; IL-18; NK cells; cancer immunotherapy; cytokine-preactivated NK cells; memory-like; mitochondria.

Figure 1

IL-12/15/18-induced cell death in human NK cells and mitochondrial superoxide levels. (A) Viability of IL-12/15/18-stimulated NK cells (activated NK) measured by flow cytometry immediately after cytokine stimulation (day 0; n=10) or after culturing them for seven days with IL-15 or IL-2 (day 7; n=8). Live cells (L/D-Apotracker-), early apoptotic cells (L/D-Apotracker+) and late apoptotic (L/D+Apotracker+) are represented with white, light red and dark red colors, respectively. Statistically significant differences (p<0.05) between control and activated NK cells in live, early apoptotic and late apoptotic cells are represented with *, # and &, respectively. (B) Mitochondrial superoxide levels upon IL-12/15/18 stimulation measured by flow cytometry using the fluorescent probe MitoSOX normalized to mitochondrial mass and measured with MitoTracker Deep Red (MTDR) (n=6). Significant differences were determined with Wilcoxon matched-pairs signed rank test (*p<0.05).

Figure 2

IL-12/15/18-stimulated human NK cells and mitochondrial dynamics. (A) Representative electron microscopy micrographs (top) and morphometric analyses of mitochondrial length and cristae density (bottom). Each dot represents a mitochondria using NK cells from 3 different experiments. Scale bar: 500 nm. (B) Representative Western Blots of total cell lysates from control and activated NK cells stimulated as indicated (top) and densitometric analyses of OPA1 levels normalized to the mitochondrial protein TOM40 (n=4). Vinculin was used as a loading control. (C) Representative Western Blots of total cell lysates from control and CIML NK cells treated as indicated (left) and autophagic flux determined by the densitometric quantification of LC3-II levels in experiments as in the left panel (right; n=3). Autophagic flux was calculated by subtracting the LC3-II expression in cells exposed to vehicle control (distilled water) from the LC3-II expression in cells exposed to chloroquine. (D) Mitophagic activity determined by flow cytometry in NK cells transfected with mKeima (n=3). Bars represent mean. a.u., arbitrary units; CQ, chloroquine. Significant differences were determined with Wilcoxon matched-pairs signed rank test, or Mann-Whitney test for panel (A) (**p<0.01, ****p<0.0001).