Interferon γ

The human myeloid leukemia cell line PLB-985 (certified mycoplasma negative, obtained from DSMZ, German collection of microorganisms and cell culture) was cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% GlutaMAX (Life Technologies, Carlsbad, CA) at 37°C with 5% CO₂. Cells were passaged twice weekly to maintain a cell density between 2 × 10⁵ and 1 × 10⁶ · mL⁻¹ and used until passage no. 10. For differentiation into neutrophil-like cells, cells were seeded at a density of 2 × 10⁵ · mL⁻¹ and cultured for 96 hr in RPMI-1640 medium with 10% FBS, 1% GlutaMAX and 1.25% DMSO (Pivot-Pajot et al., 2010 (link)). After 72 hr of incubation in the presence of DMSO, 2000 U · mL⁻¹ interferon-γ (ImmunoTools, Friesoythe, Germany) was added to the cell culture (Tlili et al., 2011 (link)). In a previous study, differentiation was checked by detecting the expression of the associated surface markers CD11b and CD64 (see Degrossoli et al., 2018 (link)). The viability of the cells was evaluated using trypan blue dye and was typically >90%.

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.

Bone marrow-derived macrophages were isolated from the long bones of hTNF-α tg mice and stimulated with 5 ng/ml M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) (d0) for 6 days with regular medium changes (Figure 1). On day 7, wells were subdivided into three groups and either stimulated with 5 ng/ml M-CSF for M0; 4 ng/ml GM-CSF (Miltenyi Biotec), 20 ng/ml interferon-γ (IFN-γ, ImmunoTools, Friesoythe, Germany), and 20 ng/ml lipopolysaccharide (LPS) for M1; and 5 ng/ml M-CSF and 20 ng/ml interleukin- (IL-) 4 (ImmunoTools) for M2 macrophages (Figure 1(a)) or stimulated with SN of FLS for 24 h, respectively (Figures 1(b) and 1(c)). Two hours after the addition of cytokines or SN, cells were irradiated (Figures 1(a) and 1(c)). Figure 1 summarizes these experimental set-ups. Cells were kept under standard conditions (37°C, 5% CO2; 95% humidity) in a medium containing Dulbecco's modified Eagle medium (DMEM, PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrom AG, Berlin, Germany), 5% horse serum (Sigma-Aldrich, Darmstadt, Germany), 1% penicillin/streptomycin (Gibco), and 50 μM 2-mercaptoethanol (Gibco).