Transpresentation of interleukin-15 by IL-15/IL-15Rα mRNA-engineered human dendritic cells boosts antitumoral natural killer cell activity

Johan Van den Bergh, Yannick Willemen, Eva Lion, Heleen Van Acker, Hans De Reu, Sébastien Anguille, Herman Goossens, Zwi Berneman, Viggo Van Tendeloo, Evelien Smits, Johan Van den Bergh, Yannick Willemen, Eva Lion, Heleen Van Acker, Hans De Reu, Sébastien Anguille, Herman Goossens, Zwi Berneman, Viggo Van Tendeloo, Evelien Smits

Abstract

In cancer immunotherapy, the use of dendritic cell (DC)-based vaccination strategies can improve overall survival, but until now durable clinical responses remain scarce. To date, DC vaccines are designed primarily to induce effective T-cell responses, ignoring the antitumor activity potential of natural killer (NK) cells. Aiming to further improve current DC vaccination outcome, we engineered monocyte-derived DC to produce interleukin (IL)-15 and/or IL-15 receptor alpha (IL-15Rα) using mRNA electroporation. The addition of IL-15Rα to the protocol, enabling IL-15 transpresentation to neighboring NK cells, resulted in significantly better NK-cell activation compared to IL-15 alone. Next to upregulation of NK-cell membrane activation markers, IL-15 transpresentation resulted in increased NK-cell secretion of IFN-γ, granzyme B and perforin. Moreover, IL-15-transpresenting DC/NK cell cocultures from both healthy donors and acute myeloid leukemia (AML) patients in remission showed markedly enhanced cytotoxic activity against NK cell sensitive and resistant tumor cells. Blocking IL-15 transpresentation abrogated NK cell-mediated cytotoxicity against tumor cells, pointing to a pivotal role of IL-15 transpresentation by IL-15Rα to exert its NK cell-activating effects. In conclusion, we report an attractive approach to improve antitumoral NK-cell activity in DC-based vaccine strategies through the use of IL-15/IL-15Rα mRNA-engineered designer DC.

Keywords: IL-15 receptor α; Immune response; Immunity; Immunology and Microbiology Section; dendritic cells; interleukin-15 transpresentation; mRNA elektroporation; natural killer cells.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1. Interleukin-15 membrane expression and secretion…
Figure 1. Interleukin-15 membrane expression and secretion of IL-15 mRNA electroporated DC
A. Membrane-bound IL-15 expression was determined by flow cytometric staining of mock EP DC (dashed black line), IL-15 EP DC (grey triangles) and IL-15/IL-15Rα EP DC (black squares) 2h, 4h, 8h, 24h, 48h and 72h after electroporation. Expression levels (MFI) were transformed to relative levels compared to those of the corresponding mock EP DC, which were set to one. Data are shown as mean (± SEM) for 3 independent donors. B. IL-15 secretion was quantified using an ELISA on the same EP conditions (mock EP DC, IL-15 EP DC and IL-15/IL-15Rα EP DC) and the same time points after electroporation (2h, 4h, 8h, 24h, 48h and 72h) as shown in figure 1A. Data are shown as mean (± SEM) for 6 independent donors. Statistical comparison was performed between IL-15 EP DC and IL-15/IL-15Rα EP DC at each time point. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001, two-way ANOVA with Bonferroni posthoc test. Abbreviations: EP; electroporation, MFI; mean fluorescence intensity, SEM; standard error of the mean.
Figure 2. Phenotypic activation profile of DC-…
Figure 2. Phenotypic activation profile of DC- stimulated NK cells
NK cells were cultured alone (white bars) or in coculture with mock EP DC (light grey bars), IL-15 EP DC (dark grey bars) or IL-15/IL-15Rα EP DC (black bars) in a NK/DC-ratio of 5:1. After 48h, NK-cell phenotype was determined using flow cytometry. Data, shown as dMFI (as compared to isotype controls), are depicted as mean (± SEM) for 3 independent donors. As a positive control, NK cells were stimulated with recombinant human IL-15 (1 ng/mL) for 48h before measuring phenotypic NK-cell activation (striped bars). ns, not significant; *, p < 0.05; **, p < 0.01; ***,p < 0.001, repeated measures one-way ANOVA with Bonferroni posthoc test. Abbreviations: EP; electroporation, dMFI; delta mean fluorescence intensity, SEM; standard error of the mean.
Figure 3. Granzyme B, perforin and IFN-γ…
Figure 3. Granzyme B, perforin and IFN-γ production after NK/DC/tumor-cell coculture
Granzyme B A. and perforin B. secretion are shown for 48h NK/Daudi (ratio 5:1, white bars) and NK/DC/Daudi (ratio 5:1:1, mock EP DC light grey bars, IL-15 EP DC dark grey bars, IL-15/IL-15Rα EP DC black bars) cocultures. Data are shown as mean (± SEM) for 5 (granzyme B) and 8 (perforin) independent donors, respectively. C. Intracellular IFN-γ, measured after 24h NK/DC/Daudi coculture (ratio 5:1:1), is shown for 6 independent donors. As a positive control, NK cells were stimulated with IFN-α (1000U/mL) for 48h and K562 cells for 4h (striped bar) before measuring intracellular IFN-γ. ns, not significant; *,p < 0.05; **,p < 0.01; ***, p < 0.001, repeated measures one-way ANOVA with Bonferroni posthoc test. Abbreviation: SEM; standard error of the mean.
Figure 4. Cytotoxicity of NK/DC cocultures against…
Figure 4. Cytotoxicity of NK/DC cocultures against Daudi cells
The mean killing percentage (± SEM) of Daudi cells is shown for NK/Daudi (ratio 5:1, white bars), DC/Daudi (ratio 1:1, mock EP DC light grey bars, IL-15 EP DC dark grey bars, IL-15/IL-15Rα EP DC black bars) and NK/DC/Daudi (ratio 5:1:1, mock EP DC light grey bars, IL-15 EP DC dark grey bars, IL-15/IL-15Rα EP DC black bars) cocultures based on a 4h flow cytometric cytotoxicity assay following 44h NK-cell and/or DC cocultures. A. In some conditions, a transwell insert (pore size of 4 μM) was added before starting NK/DC cocultures. These data are shown as mean (± SEM) for 5-9 independent donors. B. 1h prior to the addition of NK cells, anti-IL-15 neutralizing IgG or corresponding IgG isotype control antibody (100 μg/4x105 DC) was added to DC. These data are shown as mean (± SEM) for 4-8 independent donors. **, p < 0.01; ***, p < 0.001, one-way ANOVA with Bonferroni posthoc test. Abbreviation: SEM; standard error of the mean.
Figure 5. Validation of the cytotoxic profile…
Figure 5. Validation of the cytotoxic profile of DC-activated NK cells against tumor cells for AML patients in remission
The killing percentage of A. Daudi, B. K562 and C. THP-1 is shown for NK/DC/tumor-cell (ratio 5:1:1) cocultures of three different AML patients in remission based on a 4h flow cytometric cytotoxicity assay following 44h NK/DC cocultures. AML 1 and AML 2 are already a few years in remission, while patient AML 3 was tested shortly after consolidation. *, p < 0.05, one-way ANOVA with Bonferroni posthoc test.

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