Cytokine cocktail

Whole blood was obtained at the time of diagnosis from patients with gliomas (i.e., prior to surgery, radiation and chemotherapy), diluted 1:1.5 with RPMI 1640 l-glutamine (2 mM) with antibiotics (penicillin, 100 IU/mL, and streptomycin, 100 µg/mL) (Life Technologies, Carlsbad, USA) and incubated (i) without cytokines (RPMI only), (ii) with a IL-7 (10 ng/ml)/IL-2 (500 IU/ml) cytokine cocktail or (iii) with a IL-2 (1000 IU/ml)/IL-15 (10 ng/ml)/IL-21 (10 ng/ml) cytokine cocktail (Prospec, Ness Ziona, Israel). The diluted blood was co-incubated in pre-coated plates with a panel of different TAA and viral antigens (Supplementary Table 1) for 7 days at 37 °C and 5% CO₂ as described previously [23 (link), 24 (link)]. To gauge the basal IFN-γ production to NY-ESO-1, survivin as well as commonly recognized target antigens, blood was incubated with medium (negative control) and non-tumor-related antigens, i.e., the Epstein–Barr virus nuclear antigen 3 (EBNA-3). Positive controls were phytohemagglutinin (PHA, Sigma-Aldrich), OKT3 (anti-CD3 monoAb, Biolegend, CA, USA, 30 ng/ml) and SEA + SEB (staphylococcal enterotoxin A and B). IFN-γ production was then quantified in the cell culture supernatant by ELISA (Mabtech, Stockholm, Sweden).