Ficoll

Clinical grade CS1 lentiviral vector was constructed consisting of a CS1-specific scFv linked to an intracellular 4-1BB co-stimulatory and CD3ζcytoplasmic domain by a modified IgG4 hinge region, (i.e., deleted CH2 region for enhanced persistence). A truncated human EGFR (huEGFRt) was used as a transduction marker and was separated from the codon optimized CS1:4-1BB: z sequence by a T2A ribosomal skip sequence. Leukapheresis products from healthy human donors were obtained and PBMCs separated by density gradient centrifugation using Ficoll (Amersham Biosciences). Subsequently, T naïve/memory (Tn/mem) cells were isolated by CD62L + microbeads from the resulting negative fraction, following depletion of CD14 + and CD25 + cells (AutoMACS, Miltenyi Biotech). Following selection, Tn/mem cells were activated with CD3/CD28 microbeads, transduced with CS1 lentivirus and expanded as previously described (6). CAR T cells were characterized for CAR percentage based on EGFR expression and banked in liquid nitrogen for animal experiments. All healthy donor samples were obtained under approved COH IRB protocols (IRB 09025).

Peripheral blood mononuclear cells (PBMCs) were isolated within 24 h of blood collection using the Ficoll (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation method. The isolated PBMCs were then suspended in a freeze medium containing 10% DMSO (Sigma-Aldrich, St. Louis, MI, USA) and 90% heat-inactivated foetal calf serum (GIBCO, Life Technologies, USA) or 90% human AB serum (Sera Laboratories International, Sussex, UK) and frozen at a speed of 1 °C/min to −80 °C and subsequently stored in liquid nitrogen. Upon thawing, each vial of the frozen PBMCs was thawed rapidly in a 37 °C water bath and re-suspended gently in AIM-V media (Life Technologies, Grand Island, NY, USA) supplemented with 5% normal human serum (HS, Sigma-Aldrich, St. Louis, MI, USA) (complete AIM-V media).

Human immortalized gastric epithelium GES-1 and human monocyte THP-1 cell lines were routinely cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, at 37°C in a humidified incubator under 5% CO2. For the induction of THP-1-derived M0 macrophages, 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) was added to THP-1 cells in RPMI-1640 for 24 h. Cultures were checked for mycoplasma on a regular basis. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll (Amersham, Uppsala, Sweden) gradient density centrifugation [28] . The cells were suspended in 2 mL of IMDM media supplemented with Ultraglutamine and then placed in a T25 flask at a density of 1*10^6 cells per square centimeter. The cells were left to adhere for a duration of 60 min at a temperature of 37°C, after which the cells were subjected to two washes with PBS in order to eliminate any cells that were not adhered to the surface. Cells were subsequently cultured in IMDM containing L-glutamine, 10% FCS, 1% penicillin-streptomycin, and 50 ng/ml GM-CSF (Sigma-Aldrich).

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll (Amersham Biosciences, Piscataway, NJ, USA). Following isolation of the PBMC layer, two washing steps were performed with phosphate-buffered saline (PBS, Biomed Lublin, Poland). Viable cells were cryopreserved in fetal bovine serum (FBS, Gibco, Thermo Fisher, Carlsbad, CA, USA) with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and stored in liquid nitrogen until flow cytometric analyses.

CD14⁺ monocytes were purified from blood of human donors (approved by the local ethical committee, 2007-0019) by centrifugation though Ficoll (Amersham, GE Healthcare, Little Chalfont, United Kingdom) and subsequent immunomagnetic isolation with MagCellect Streptavidin Ferrofluid (R&D Systems, Minneapolis, MN, USA). A more detailed experimental description has been published previously [27 (link)]. Cells were cultured at 37 °C in 5 % CO₂, supplied with αMEM (Invitrogen) containing 10 % fetal calf serum (FCS) (Biological Industries, Kibbutz Beit-Haemek, Israel) and 25 ng/ml macrophage colony-stimulating factor (M-CSF, R&D Systems). After a 2-day culture period, the medium with FCS and M-CSF was refreshed, and 25 ng/ml human receptor activator of nuclear factor kappa-B ligand (RANKL, R&D Systems) was added. The cells were subsequently seeded in either 8-well Lab-Tek chamber slides (Nunc, Roskilde, Denmark) or 96-well plates (Greiner, Frickenhausen, Germany) depending on the assay. Here we define preOCs as mononucleated CD14⁺ cells, and OCs are defined as multinucleated cells differentiated from preOCs with M-CSF and RANKL, as described above.