A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells

Abstract

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.

Keywords: IL-15; NF-κB; adoptive cellular immunotherapy; dendritic cell; natural killer cell.

Figure 1

Composition and expression of the chimeric IL15 constructs. (A) Originally, the protein sequence of the complete IL-15 transcript variant 1 was fused to that of the IL-15Rα via a flexible SG-linker (SG₃(SG₄)₃SG₃S). This construct is referred to as chIL15old. To improve the construct, the first 48 AA of the IL-15 transcript variant 1 that constitute the signal peptide and the pro-peptide were replaced with the signal sequence of CD25. The signal sequence of the IL-15Rα was removed so that the linker was directly fused to the first sushi-domain. This altered construct is referred to as chIL15. (B) To measure the expression of the chIL15old and the chIL15 construct on the surface of DCs, cytokine matured DCs were electroporated with mRNA encoding the chIL15old (red line) or chIL15 (blue line), or as a negative control were mock electroporated (grey histogram). The cells were stained with an IL-15Rα-specific antibody and analyzed by flow cytometry. A histogram of DCs electroporated with chIL15old, chIL15, or without RNA (mock) and stained for IL-15Rα 24 h after electroporation is shown (n = 3 for chIL15, n = 1 for chIL15old). (C) The expression kinetics of chIL15 was determined 2, 4, 6, and 24 h after electroporation. The specific mean fluorescence intensity (MFI) was calculated by subtracting the isotype control MFI from the MFI measured with the IL-15Rα-specific antibody (different symbols represent the three individual experiments). The interaction p-value between the mock and the chIL15 condition was calculated by two-way ANOVA, † p ≤ 0.05.

Figure 2

Transfection of DCs with caIKKβ combined with chIL15 leads to an improved NK-cell activation. DCs matured with the standard cytokine cocktail were electroporated with RNA encoding caIKKβ and chIL15 either alone or in combination. As a control, DCs were mock electroporated. Transfected DCs were co-cultured with autologous peripheral blood mononuclear cells (PBMCs) 2–4 h after electroporation at a ratio of 1:10 (final concentrations 2 × 10⁵ DCs/mL and 2 × 10⁶ PBMCs/mL). As controls, transfected DCs and PBMCs were cultured alone. Cells were harvested and supernatant was sampled after 48 h of co-culture. (A) The expression of surface markers CD69, CD25, and CD54 was determined via flow cytometry on cells negative for CD3 and positive for CD56 (using the gating strategy shown in Supplemental Figure S3). A representative histogram for each activation marker out of three independent experiments is shown. (B) The depicted values show the fold upregulation of each surface marker, calculated relative to the mean fluorescence intensity (MFI) of PBMCs cultured in absence of DCs. The average fold induction of three different donors with SD is shown. p values were calculated with a one-way ANOVA and subsequent Tukey test, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, numbers indicate p values of 0.05 < p ≤ 0.1. (C) The secretion of IFNγ was measured in the supernatant by Cytometric Bead Array. Average cytokine concentrations with SD are shown from three independent donors. p values were calculated with a one-way ANOVA and subsequent Tukey test, *** p ≤ 0.001, ** p ≤ 0.01, numbers indicate p values of 0.05 < p ≤ 0.1. (D) The cytolytic capacity of stimulated cells was determined in a ⁵¹chromium release assay. The K562 cell line was used as a target at the indicated target ratios. Average values with SD of three independent donors, each analyzed in triplicates, are shown. p values were calculated with a one-way ANOVA and subsequent Tukey test, ** p ≤ 0.01, * p ≤ 0.05, numbers indicate p values of 0.05 < p ≤ 0.1. The respective brackets refer to the mock-transfected co-cultures.