Recombinant cytokines

Peripheral blood mononuclear cells (PMBCs) were obtained from anonymous donors as previously described [23 (link)]. The CD8 T cells were isolated by negative selection using an enrichment kit for CD8 T cells (Stem Cell Technologies). These isolations yielded cells that were consistently >98% positive for CD8 T cells (data not shown). CD8 T cells were activated for 5 days with magnetic Dynabeads (Invitrogen) bound with anti-CD3 (OKT3, BioLegend) and anti-CD28 (CD28.2, BioLegend) antibodies in the presence of 100 U/mL IL-2. Following activation and expansion, the stimulatory anti-CD3/anti-CD28 beads and IL-2 were removed, and the cells were rested for 24 h in complete RPMI (RPMI 1640 supplemented with 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mM l-glutamine (Gibco)) before further experimentation. Activated CD8 T cells were split into different flasks and exposed to different doses of recombinant cytokines (R&D Systems) or exposed to IL-12 for different times. Media was not changed in between the times. Cells were then washed three times in complete RPMI to remove the recombinant cytokines.

Cells were removed from spleens of naïve mice and a single cell suspension was made by homogenization. Following lysis of red blood cells, CD4⁺ T cells were isolated using a negative selection kit (Miltenyi Biotec, San Diego, CA, USA). Cells were cultured in complete medium in 96-well plates that had been coated with 2.5 μg/ml of α-CD3 for 2 hours at 37°C. For primary skewing, T cells were activated with 3 μg/ml α-CD3 for 48 hours or 5 days to drive Th0 differentiation. In addition to soluble α-CD28, the following skewing conditions were used: Th1 cells – rIL-12 (100 ng/ml) and α-IL-4 (10 μg/ml), Th2 cells – rIL-4 (100 ng/ml) and α-IL-12 (10 μg/ml) and α-IFN-γ (10 μg/ml), and Th17 cells – rIL-6 (100 ng/ml), rTGF-β (20 ng/ml), α-IL4 (10 μg/ml), α-IL-12 (10 μg/ml) and α-IFN- γ (10 μg/ml). For secondary skewing, cells were rested in complete medium for three days, then restimulated with plate-bound α-CD3 and soluble α-CD28 for 48 hours. For skewing of Jurkat cells, the cells were expanded in RPMI with 10% FCS and penicillin/streptomycin. Following transfection (see following section), cells were cultured on plate-bound α-CD3 in the presence of soluble α-CD28, α-IL-12 and recombinant human IL-4. All antibodies were purchased from eBiosciences, and all recombinant cytokines were purchased from R&D Systems (Minneapolis, MN, USA).

Peripheral blood mononuclear cells (PBMCs) were obtained from 50 ml of buffy coats (New York Blood Center) by Ficoll gradient centrifugation. Human monocytes were isolated by CD14-positive selection (STEM-CELL) from PBMCs. CD14⁺ monocytes were cocultured with GBM6 cells at 1:1 ratio for in vitro proliferation or invasion assays. CD14⁺ monocytes (300,000 to 500,000/ml) were plated in RPMI 1640 plus 10% of FBS and in vitro differentiated to macrophages by adding macrophage-CSF (25 ng/ml). Interferon-γ (50 ng/ml), IL-4 (40 ng/ml), or IL-10 (50 ng/ml) polarizing cytokines were added on day 3, and M0, M1, M2a, and M2c monocyte-derived macrophages were harvested on day 7. Macrophage polarized phenotype was confirmed using anti-human CD11b, CD64, CD23, CD80, CD163, and CD14 (BioLegend). Recombinant cytokines were purchased from R&D Systems.