Human ab serum

PBMCs were isolated from the blood of patients and HSC donors on the Ficoll-Paque Plus gradient (GE Healthcare, Uppsala, Sweden). The fresh cells were washed with PBS containing 2% human serum albumin. They were subsequently stimulated for 1 h with a mixture of 15 amino acid long overlapping peptides (PepMix^TM) covering BKPyV VP1 and BKPyV LTAG (JPT Peptide Technologies GmbH, Berlin, Germany). The PepMixes were used at concentrations of 0.1 µg per 15 million PBMC. The stimulated cells were resuspended in 15 mL of culture medium (CTL) composed of 50% RPMI 1640 with HEPES and glutamine, 45% Click’s medium (both from FUJIFILM Irvine Scientific, Santa Anna, CA, USA), 5% human AB-serum (Capricorn Scientific, Ebsdorfergrund, Germany), 1% penicillin-streptomycin-glutamine (GIBCO, Dublin, Ireland) and supplemented with IL-4 and IL-7 (CellGenics, Freiburg, Germany) at a concentration of 16.6 ng and 10 ng/mL, respectively. They were then cultured in 45 cm² culture flasks (Corning, Corning, NY, USA) under 5% CO₂ at 37 °C. On day 5 of culture, 10 mL of exhausted medium were replaced with the fresh CTL containing 25 ng/mL of IL-4 and 15 ng/mL of IL-7. The culture was harvested on the 12th day, and the frequency of the BKPyV-specific T-cells among expanded T-cells was measured by ELISPOT-IFNγ or by flow cytometry with intracellular cytokine staining (IC FACS).

CxCa and OPSCC tumors were obtained and handled as described previously [5 (link), 20 (link)]. In brief, tumor material was cut into small pieces and the pieces were incubated for 60 min at 37 °C in Iscove’s Modified Dulbecco’s Medium (IMDM, Lonza, Verviers, Belgium) with 10% human AB serum (Capricorn Scientific, Esdorfergrund, Germany) and supplemented with high dose of antibiotics (50 µg/ml Gentamycin (Gibco/Thermo Fisher Scientific (TFS), Bleiswijk, the Netherlands), 25 µg/ml Fungizone (Invitrogen/TFS), 100 IU/ml penicillin (pen; Gibco/TFS) and 100 µg/ml streptomycin (strep; Gibco/TFS)). Next, the tumor pieces were put in culture in IMDM supplemented with 10% human AB serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamin (Lonza, Breda, Netherlands; hereafter referred to as IMDM complete) and 1000 IU/ml human recombinant IL-2 (Aldesleukin, Novartis, Arnhem, the Netherlands). Cultures were replenished every 2–3 days with fresh IMDM complete and IL-2 to a final concentration of 1000 IU/ml and when sufficient T cells were obtained after 2–4 weeks the cells were harvested, cryopreserved and stored in liquid nitrogen until use.