Therapeutic aim 5 medium

Human DCs were ex vivo generated from adherent monocytes isolated from PBMC of healthy donors and patients with stage IV malignant melanoma. PBMC were cultured in serum-free therapeutic AIM-V medium (GIBCO, Carlsbad, CA, USA) at a concentration of 13 × 10⁶ cells/mL in six well plates (BD Biosciences, Hershey, PA, USA) at 37 °C and 5% CO₂ for 2 h. Thereafter, non-adherent cells were removed, and the adherents (monocytes) were maintained and incubated for 22 additional hours in the presence of 500 U/mL recombinant human IL-4 (rhIL-4) and 800 U/mL of rhGM-CSF (US Biological, Swampscott, MA, USA) in order to induce DC-lineage differentiation as previously described [18 (link)]. The obtained cytokine-activated monocytes (AM), which showed an immature DC-like phenotype, were then stimulated for 24 additional hours with 10 ng/mL of HP (Abcam, Cambridge, UK), the lysate TRIMEL (100 µg/mL) as positive control or 10 ng/mL of insulin (INS; Novus Biologicals, Centennial, CO, USA) as negative protein control. In addition, all the in vitro experiments were performed using unstimulated iDCs (AM) as internal control.

Human melanoma cell lines (Mel1, Mel2 and Mel3), established from three tumor infiltrated lymph nodes from patients with metastatic HLA-A2+ stage IV melanoma and positive for several melanoma associated antigens, were cultured in RPMI-1640 medium (Invitrogen, Waltham, MA, USA) containing 10% FBS (Invitrogen, Waltham, MA, USA) until 90–95% confluence. Before use, all the cell lines were tested by PCR techniques, to check the absence of potentially infecting virus or mycoplasma. The presence of contaminating bacteria was also ruled out by periodical culture testing in agar. The allogeneic cell lysate TRIMEL was prepared as previously described [18 (link),21 (link)]. Briefly, the three different melanoma cell lines were mixed in equal proportions (1 × 10⁷ cells for each cell line), resuspended in the therapeutic AIM–V medium (GIBCO, Carlsbad, CA, USA) at a concentration of 4 × 10⁶ cells/mL, HS-treated by incubating the cells one hour at 42 °C, then two hours at 37 °C and finally lysed by performing three freeze-thaw cycles using liquid nitrogen. The obtained cell lysates were sonicated, snap frozen, and stored at −80 °C, until further use [5 (link)]. Three independently produced batches of the lysate TRIMEL were prepared.