Interleukin-2 stimulation increases NK cell cytotoxicity

Interleukin-2 regulates NK cell activity and can increase their cytotoxicity (Henney et al., 1981;

Lehmann et al., 2001). To investigate if and how IL-2 stimulation affects NKiToxMel, non-stimulated and IL-2 non-stimulated (0.05 µg/mL) NK cells from the same donor were used in cytotoxic assays. In Figure 11, the results depicting NK cell cytotoxicity against three different melanoma cell lines, as well as the control K562, are summarised. In all cell lines, the non-stimulated NK cells showed significantly lower cytotoxicity than the IL-2 non-stimulated NK cells (see Figure 11 A). This was not only visible at the initial phase of the experiment (Figure 11 B),

the first hour (Figure 11 B) more than half (~56 %) of the 1205Lu were eliminated. At the end of the assay, all 1205Lu were killed by stimulated NK cells (see Figure 11 C). Non-stimulated NK cells only killed ~10 % after 60 min and reached a killing plateau of ~34 % after 220 min.

WM3734 had an intermediate susceptibility to NK cells. Figure 11 C shows that IL-2 stimulated NK cells killed ~42 % of WM3734 cells, whereas their susceptibility to non-stimulated NK cells is reduced to ± 12 %. WM88 are the least susceptible melanoma cells among those that were tested, as non-stimulated NK cells were not able to kill WM88 to any detectable amount. Even after stimulation, NK cells only marginally killed this melanoma cell line (12 %).

Figure 11. Interleukin-2 stimulation increases cytotoxicity against cancer cells. NK cell-mediated killing of the melanoma cell lines 1205Lu, WM3734 and WM88 as well as the control cell line K562 was determined in 4 h a cytotoxic assay (A). NK cells from four different donors either IL-2 stimulated (1-2 days) or non-stimulated were used as effector cells. For further statistical analysis the killing after 60 min (B) as well as the killing after 240 min (C) were used. Bars and graphs indicate means ± SEM. Statistical significance of paired, two-tailed Student's t‐test is indicated with * for p<0.05 and ** for p <0.01.

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As previously shown (Cappello, 2015), the difference between non-stimulated and IL-2 stimulated NK cells are due to alterations in the NK cell receptor profile and differences in the content of lytic granules before and after stimulation. The representative histograms (Figure 12 A) depict altered expression levels of the NK cell receptors CD16, NKp30, NKG2D, the adhesion molecule CD56 as well as perforin and granzyme B. The median fluorescence intensity (MFI) was used to compare the expression profile of non-stimulated and IL-2 stimulated NK cells (see Figure 12 B). To exclude fluorescence signals, which were caused by non-specific antibody binding, the MFI of the corresponding isotype controls were subtracted. In comparison to the other analysed parameters, the median fluorescence intensities (MFIs) of the activating receptors NKp30 and NKG2D were low (Net MFI of 50 and 187, respectively) in non-stimulated NK cells. After IL-2 stimulation, the MFI increased to 154 for NKp30 and 908 for NKG2D. The detected amount of CD56 expression on the NK cell surface increased almost fourfold (MFI of 882 to MFI of 3215) after stimulation. The CD16 surface expression showed only a small increase from MFI of 3600 to 4800 after IL-2 stimulation. In addition to the higher expression of activating NK cell receptors, the NK cells tended to have a higher content of intracellular granzyme B and perforin (from 2822 to 5642 and from 6264 to 9120, respectively).

These findings are also in agreement with observations from other groups (Balsamo et al., 2013) and partially explain the lower cytotoxicity of non-stimulated NK cells to melanoma cells.

Figure 12. Interleukin-2 stimulation alters expression profile of NK cells. The expression levels of CD16, NKp30, NKG2D, CD56, perforin and granzyme B were analysed with flow cytometry. (A) Histograms show the cell distribution according to the emitted fluorescence of the indicated fluorochrome conjugated antibodies (see Table 3). The following colour code was used: light grey: non-stained samples;

dark grey: corresponding isotype controls; blue: non-stimulated NK cell samples; red: IL-2 stimulated NK cells (B) The bar graphs show the net median fluorescence intensity (MFI) (minus MFI of corresponding isotype control). Bars indicate SEM of 3 NK cell donors. Data were analysed with Flow Jo software Version 10. Data were previously shown in (Cappello, 2015)

CD16 APC-Cy7 CD65 PE-Cy7 NKG2D PerCP-Cy5.5

Perforin FITC Granzyme B Alexa 647 NKp30 PE

A

Granzyme B CD16 Perforin NKp30 CD56

Net median fluorescenceintensity

The NK cells were used on different days after IL-2 stimulation. Therefore, it was important to estimate the dependence of NK cell cytotoxicity towards melanoma in relation to the duration of IL-2 stimulation. As depicted in Figure 13 A, the duration of IL-2 stimulation influenced the NK cell cytotoxicity towards cancer cells only marginally. Figure 13 B shows that NK cells after 1-3 days of IL-2 stimulation had significant higher cytotoxicity (~73 % and ~54 % after 60 min) towards the cell lines K562 and 1205Lu. WM3734 cells and WM88 cells were best eliminated by NK cells stimulated for 4-7 days with IL-2 (compare Figure 13 C). Nevertheless, these minor alterations in NK cell cytotoxicity in the course of IL-2 stimulation can be neglected as the general susceptibility of melanoma cells to NK cells is unchanged.

Figure 13. NK cell cytotoxicity stays nearly unchanged in the course of Interleukin-2 stimulation.

The susceptibility of the melanoma cell lines to NK cells that were stimulated with IL-2 over multiple days: K562 1-3 d (n=84), 4-7 d (n=39), 8-11 d (n=33); 1205Lu 1-3 d (n=15), 4-7 d (n=2), 8-11 d (n =5);

WM3734 1-3 d (n=50), 4-7 d (n=21), 8-11 d (n=8); WM88 1-3 d (n=5), 4-7 d (n=8). (A) Kinetics of 4 h cytotoxic assay. For further statistical analysis the killing after 60 min (B) as well as the killing after 240 min (C) were used. Bars and graphs indicate means ± SEM. Statistical significance of unpaired, two-tailed Student'st‐test is indicated with * for p<0.05.

Altogether, the data presented here indicate that IL-2 stimulation increased the NK cell