Prostaglandin e2

Monocyte-derived IL-15 DCs were prepared conforming to our previously described 48-hour culture protocol (18 (link), 20 (link)). Positively selected CD14⁺ monocytes (Miltenyi) are cultured in Roswell Park Memorial Institute medium (Life Technologies, Merelbeke, Belgium) with 2.5% heat-inactivated human AB serum (Life Technologies) at a final concentration of 1–1.2 × 10⁶ cells/mL. To generate mature IL-15 DCs, a 28-hour differentiation step using GM-CSF (800 IU/mL; Life Technologies) and IL-15 (200 ng/mL; Immunotools, Friesoythe, Germany) is followed by maturation induction with R848 (3 µg/mL; Alexis Biochemicals, San Diego, USA), interferon (IFN)-γ (250 ng/mL; Immunotools), tumor necrosis factor (TNF)-α (2.5 ng/mL; Life Technologies), and prostaglandin E2 (1 µg/mL; Pfizer, Puurs, Belgium) for 20 hours. To collect 48-hour wash-out supernatant, IL-15 DCs are harvested, thoroughly washed, and reseeded in fresh medium at a concentration of 1 × 10⁶ cells/mL in low-absorbing polypropylene tubes. After 48 hours, cell-free supernatant is collected and frozen at −20°C, until further use.

DC were generated as described previously [25 (link), 26 (link)] with minor adaptations specific for the IL-15 designer DC. Briefly, positively selected CD14⁺ monocytes were differentiated into immature DC in the presence of IL-4 (20 ng/mL; Life Technologies) and granulocyte-macrophage colony-stimulating factor (800 U/mL; Gentaur) in Roswell Park Memorial Institute 1640 (RPMI; Invitrogen) supplemented with 2.5% human AB serum (SanBio). After 5 days, 20 ng/mL tumor necrosis factor-α (Gentaur) and 2.5 μg/mL prostaglandin E2 (Pfizer, Puurs, Belgium) were added to induce maturation. Monocyte-derived DC (moDC) were harvested 40–44 hours later and electroporated by a time-constant (7 ms) pulse of 300 V using the Gene Pulser Xcell device (Bio-Rad) either without mRNA (mock EP DC), with 5 μg OSP-IL-15 mRNA (IL-15 EP DC), or with a combination of 5 μg OSP-IL-15 mRNA and 5 μg IL-15Rα mRNA (IL-15/IL-15Rα EP DC) in 200 μL Opti-MEM reduced serum medium without phenol red (Life Technologies). Immediately after electroporation, DC were resuspended in prewarmed Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen) + 10% fetal bovine serum (FBS) for further use.

IL-15 DCs were prepared as per our previously reported rapid DC culture protocol [11 (link), 12 , 14 (link)]. Briefly, monocytes were seeded in Roswell Park Memorial Institute medium (RPMI; Life Technologies) supplemented with 2.5% heat-inactivated human AB serum (hAB; Invitrogen, Merelbeke, Belgium) at a final concentration of 1.0–1.2 × 10⁶ cells/mL. Differentiation was induced with 800 IU/mL granulocyte macrophage colony-stimulating factor and 200 ng/mL IL-15 (Immunotools, Friesoythe, Germany). A TLR-activating maturation cocktail, comprising R848 (3 μg/mL; Alexis Biochemicals, San Diego, USA), tumor necrosis factor-α (2.5 ng/mL), interferon-γ (250 ng/mL; Immunotools) and prostaglandin E2 (1 μg/mL; Pfizer, Puurs, Belgium), was added after 24–48 hours of differentiation for 18–20 hours. All components were bought from Invitrogen, unless stated otherwise. Control 7-day IL-4 DCs from the same blood donors were prepared as previously described in detail [11 (link)]. All subsequent experiments were performed using the obtained activated IL-15 DCs and IL-4 DCs. For the collection of x-hour wash-out supernatant, mature DCs were harvested, washed thoroughly and resuspended in fresh medium, RPMI + 2.5% hAB, at a concentration of 1 × 10⁶ cells/mL. After x hours of culturing in low absorbing polypropylene tubes, cell-free supernatant was collected and frozen at −20°C until further use.