Interleukin-15 Dendritic Cells Harness NK Cell Cytotoxic Effector Function in a Contact- and IL-15-Dependent Manner

Abstract

The contribution of natural killer (NK) cells to the treatment efficacy of dendritic cell (DC)-based cancer vaccines is being increasingly recognized. Much current efforts to optimize this form of immunotherapy are therefore geared towards harnessing the NK cell-stimulatory ability of DCs. In this study, we investigated whether generation of human monocyte-derived DCs with interleukin (IL)-15 followed by activation with a Toll-like receptor stimulus endows these DCs, commonly referred to as "IL-15 DCs", with the capacity to stimulate NK cells. In a head-to-head comparison with "IL-4 DCs" used routinely for clinical studies, IL-15 DCs were found to induce a more activated, cytotoxic effector phenotype in NK cells, in particular in the CD56bright NK cell subset. With the exception of GM-CSF, no significant enhancement of cytokine/chemokine secretion was observed following co-culture of NK cells with IL-15 DCs. IL-15 DCs, but not IL-4 DCs, promoted NK cell tumoricidal activity towards both NK-sensitive and NK-resistant targets. This effect was found to require cell-to-cell contact and to be mediated by DC surface-bound IL-15. This study shows that DCs can express a membrane-bound form of IL-15 through which they enhance NK cell cytotoxic function. The observed lack of membrane-bound IL-15 on "gold-standard" IL-4 DCs and their consequent inability to effectively promote NK cell cytotoxicity may have important implications for the future design of DC-based cancer vaccine studies.

Fig 1. IL-15 DCs induce an activated cytotoxic effector phenotype in NK cells and enlarge the proportion of the CD56^bright NK cell subset.

(A) Bar graphs showing the MFI (± SEM) of CD56, CD69, CD25, HLA-DR, NKG2D, NKp30 and NKp46 on unstimulated NK cells ((-), white bars) and NK cells stimulated for 48 hr with autologous IL-15 DCs (grey bars) or IL-4 DCs (dashed bars). Expression levels of the indicated markers are shown for the total CD56⁺ NK cell population, as well as for the CD56^bright and CD56^dim NK cell subpopulations. Data are from one experiment with 6 different donors (*, P<0.05; **, P< 0.01; ***, P<0.001; ns, not significant). (B) Bar graphs showing the relative distribution of CD56^bright and CD56^dim subsets in unstimulated NK cells ((-)) and in NK cells cultured for 48 hr with IL-15 DCs or IL-4 DCs. Data are expressed as mean (± SEM) percentages of 6 donors from 1 experiment (*, P<0.05).

Fig 2. With the exception of GM-CSF, exposure of NK cells to IL-15 DCs does not trigger cytokine or chemokine secretion.

(A) Bar graphs showing the IFN-γ secretion level (pg/mL), as determined by ELISA, in 48-hr culture supernatants collected from NK cell monocultures, IL-15 DC/IL-4 DC monocultures, and NK+IL-15 DC/IL-4 DC co-cultures. Where indicated, the effect of an additional 4-hr stimulation with the K562 tumor cell line was examined. Values represent means (± SEM) of triplicate cultures from 6 individual donors (ns, not significant). (B) Cytokine/chemokine levels (pg/mL; log scale) in 48-hr IL-15 DC-stimulated NK cell culture supernatants, as measured by ECLIA. Observed concentrations (grey bars) were compared with the expected concentrations (white bars) calculated as the sum of the DC and NK cell monoculture concentrations. Values represent means (± SEM) of duplicate measurements of 3 separate donors (*, P<0.05; Wilcoxon matched pairs).

Fig 3. IL-15 DCs but not IL-4 DCs promote NK cell-mediated tumor cell killing.

Unstimulated NK cells ((-), white bars) and NK cells exposed for 48 hr to IL-15 DCs (grey bars) or IL-4 DCs (dashed bar) were analyzed by flow cytometry for cytotoxicity against K562 and Daudi tumor cells. Target cell killing was determined by Annexin-V/PI staining after 4 hr incubation at an E:T ratio of 5:1. Results are expressed as mean (± SEM) percentage killing, which was calculated using the formula specified in “Methods”. Data are from two independent experiments involving 10 different donors (***, P<0.001; ns, not significant).

Fig 4. IL-15 DCs enhance NK cell cytotoxicity by a cell contact-dependent mechanism involving membrane-bound IL-15.

(A) NK cell cytotoxicity was determined after 48-hr co-culture with IL-15 DCs in the absence (grey bars) or presence (TW, dark grey bars) of a Transwell system. NK cells cultured in medium alone for 48 hr ((-), white bars) were used as controls to determine baseline cytotoxicity. K562 and Daudi target cells were added at an E:T ratio of 5:1 and assessed for viability after 4 hr by Annexin-V/PI staining and flow cytometry. Results are expressed as mean (± SEM) percentage killing, which was calculated using the formula specified in “Methods”. Data are from two independent experiments involving 10 different donors (*, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant). (B) Flow cytometric analysis of membrane-bound IL-15 expression on monocytes and mature IL-15 DCs at different time points of culture: at harvest of the DCs (IL-15 DCs), at 48 hr post-harvest (48-hr IL-15 DCs), at the start of IL-15 DC-NK cell co-culture (IL-15 DCs + NK) and after 48 hr of co-culture (48-hr IL-15 DCs + NK). One representative sample out of 3 different donors is shown. (C) NK cells were cultured in the absence (left panel) or presence of autologous IL-15 DCs (right panel) in medium without neutralizing Ab ((-), white bars) or in medium containing either neutralizing anti-IL-15 mAb (αIL15, dark grey bars) or its corresponding isotype control Ab (IgG₁, light grey bars). After 16 hr, Daudi tumor cells were added at an E:T ratio of 5:1 for an additional 4 hr after which target cell killing was determined by Annexin-V/PI staining and flow cytometry, as described above. Bars represent mean (± SEM) percentage killing of three individual donors from one experiment (*, P<0.05; ns, not significant).