Hgm csf

About 4x10⁶ THP-1 cells were seeded in T75 flasks and differentiated towards immature dendritic cells (iDC) over a 5-day culture in 20 mL serum-supplemented RPMI 1640 in the presence of 100 ng/mL hIL4 (R&D Systems, 204-IL-020/CF) and 100 ng/mL hGM-CSF (Sigma-Aldrich, #GF304). To allow full maturation towards mature dendritic cells (mDC), iDC were collected via centrifugation, resuspended in serum free RPMI 1640 media supplemented with 200ng/mL hIL4, 100ng/mL hGM-CSF, 20ng/mL hTNFα (Sigma-Aldrich, #GF314), and 200ng/mL ionomycin (Tocris Bioscience, 2092/1), and plated at density 10,000/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Cells were kept in culture for 1 day to allow differentiation towards mDC, before beginning the transfection with MessengerMax-formulated mRNA to induce expression of the E6-E7 fusion protein in the vaccine. The day following the transfection, human antigen specific E7_11-20 T cells (Charles River Laboratories, #ASTC-1099) were added to the 96-well plate at density 20,000 cells/well. ASTC and mDC cells were kept in coculture for an overnight. The plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions and read under the CTL Immunospot Analyzer (ImmunoSpot^®). CD209 monoclonal antibody eB-h209 (eBioscience™) was used to characterize DC differentiation via Flow Cytometry.

TF-1-CN5a.1 (product CRL-2512™, American Type Culture Collection, ATCC^®, Manassas, VA, USA) cells were obtained from LGC Standards (Teddington, UK). The cells were maintained in complete growth medium of Roswell Park Memorial Institute (RPMI) 1640 medium, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate (RPMI-1640, ATCC modification; Gibco^TM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco^TM, Thermo Fisher Scientific), 2 ng/mL human granulocyte macrophage colony-stimulating factor (hGM-CSF) (Sigma-Aldrich, St. Louis, MO, USA), 0.4 mg/mL G-418 (Calbiochem^TM, Merck), and 100 U/mL penicillin – 100 µg/mL streptomycin (Gibco^TM, Thermo Fisher Scientific). The cells were maintained as stationary suspension cultures in non-treated Nunc™ EasyFlask™ 75 cm² flasks (Thermo Fisher Scientific) in a fully humidified 5% CO₂ atmosphere at 37 °C. Cell number and viability were determined using trypan blue.