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. 2013 Jul;173(1):131-9.
doi: 10.1111/cei.12095.

Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody-based anti-tumour immunotherapy

Q Zhou  1 , A Gil-Krzewska, G Peruzzi, F Borrego
Affiliations

Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody-based anti-tumour immunotherapy

Q Zhou et al. Clin Exp Immunol. 2013 Jul.

Abstract

Activation of human natural killer (NK) cells is associated with the cleavage of CD16 from the cell surface, a process mediated by matrix metalloproteinases (MMPs). In this report, we examined whether inhibition of MMPs would lead to improved NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) function. Using an in-vitro ADCC assay, we tested the anti-tumour function of NK cells with three different therapeutic monoclonal antibodies (mAbs) in the presence of MMPs inhibitor GM6001 or its control. Loss of CD16 was observed when NK cells were co-cultured with tumour targets in the presence of specific anti-tumour antibodies, and was found particularly on the majority of degranulating NK responding cells. Treatment with MMPs inhibitors not only prevented CD16 down-regulation, but improved the quality of the responding cells significantly, as shown by an increase in the percentage of polyfunctional NK cells that are capable of both producing cytokines and degranulation. Furthermore, MMPs inhibition resulted in augmented and sustained CD16-mediated signalling, as shown by increased tyrosine phosphorylation of CD3ζ and other downstream signalling intermediates, which may account for the improved NK cell function. Collectively, our results provide a foundation for combining MMPs inhibitors and therapeutic mAbs in new clinical trials for cancer treatment.

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Figures

Figure 1
Figure 1
Matrix metalloproteinases (MMPs) inhibition preserved CD16 cell surface expression on natural killer (NK)-responding cells. Freshly isolated NK cells were incubated at a 1:1 ratio with the tumour cell lines SKBr3, Raji or A431, in the absence or presence of therapeutic monoclonal antibodies (mAbs) trastuzumab (anti-HER2), rituximab (anti-CD20) or cetuximab [anti-epidermal growth factor receptor (EGFR)], at 10 μg/ml for 4–6 h in the presence of the MMPs inhibitor GM6001 or control (50 μM). CD16 and CD107a/b expression were measured by flow cytometric analysis. Cells were gated electronically on CD3−CD56dim NK cells. (a) Representative dot-plots of CD16 expression on NK cells cultured with SKBr3 tumour cells in the presence or absence of trastuzumab and treated with GM6001 or its control (CTRL) are shown. (b) Bar graphs showing the percentage of NK cells expressing CD16 are shown. Bars represent the average ± standard error of the mean. Results are from four independent experiments with NK cells from four donors. (c) Representative dot-plots of CD16 and CD107a/b expression on NK cells cultured with tumour cell lines in the presence of therapeutic mAbs and treated with GM6001 or its control are shown. *P < 0·05 compared to CTRL.
Figure 2
Figure 2
Matrix metalloproteinases (MMPs) inhibition improved natural killer (NK) cell polyfunctionality. (a) Representative dot plots of interferon (IFN)-γ- and tumour necrosis factor (TNF)-α-producing NK cells cultured with SKBr3 tumour cells in the absence or presence of trastuzumab, and treated with the MMPs inhibitor GM6001 or its control (CTRL) are shown. (b) Bar graph representation of the percentage of IFN-γ single-producing, TNF-α single-producing and IFN-γ and TNF-α double-producing NK cells cultured with SKBr3 tumour cells in the absence or presence of trastuzumab and with GM6001 or its control. Bars represent the average ± standard error of the mean (s.e.m.). Results are from four independent experiments with NK cells from four donors. (c) Bar graph representation of the mean fluorescence intensity (MFI) of IFN-γ and TNF-α expression by NK cells cultured with SKBr3 tumour cells in the presence of trastuzumab with GM6001 or its control. Data were normalized according to the controls. Results are from three independent experiments with NK cells from three donors. (d) Bar graph representation of the possible combinations of three effector functions (degranulation as shown by the expression of CD107a/b, IFN-γ and TNF-α production) on the x-axis and the percentage of distinct functional populations within the CD56dim NK cells on the y-axis is shown. Bars represent the average ± s.e.m. Results are from three independent experiments with NK cells from three donors. *P < 0·05 compared to CTRL.
Figure 3
Figure 3
Cross-linking of CD16 in the presence of matrix metalloproteinases (MMPs) inhibition led to increased and sustained CD16 mediated signalling through phosphorylation of CD3ζ. (a) Freshly isolated natural killer (NK) cells were incubated with mouse anti-human CD16 (clone 3G8) at 20 μg/ml for 30 min on ice, washed with phosphate-buffered saline (PBS) to remove unbound antibodies and then followed by 4 h incubation at 37°C with goat anti-mouse secondary immunoglobulin (IgG) at 20 μg/ml in the presence of the MMPs inhibitor GM6001 or its control (CTRL). Then, flow cytometric analyses were performed. Cells were gated electronically on CD3−CD56dim NK cells and representative dot-plots of the CD16 expression from two experiments is shown with NK cells from two donors. (b) Cells were treated as in (a). Representative dot-plots of interferon (IFN)-γ- and tumour necrosis factor (TNF)-α-producing NK cells from two experiments are shown with NK cells from two donors. (c) Cells were stimulated as in (a) and (b), and harvested at different time-points (0, 5, 30, 60, 120 and 240 min) and lysed. Total cell lysates were resolved on 4–20% Mini-protean® precast gels, transferred to a polyvinylidene difluoride (PVDF) membrane, followed by immunoblotting with appropriate antibodies. A representative image of Western blot is shown. (d) Graph representations of the quantification of phospho-CD3ζ from two independent experiments with NK cells from two donors are shown. Data were analysed using ImageJ software and normalized according to total CD3ζ.
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