Human ab serum

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood on Ficoll-Paque gradient (GE Healthcare Life Sciences, USA). After washing in RPMI 1640 medium (Lonza, Switzerland) the cells were counted centrifuged and resuspended in human AB serum (Lonza, Switzerland) containing 10% DMSO for cryoprotection. Cells were aliquoted in cryovials and stored in a –80°C mechanical freezer. Thawing was carried out on the day of fluorescent cell labeling as quickly as possible in 37°C water bath and DMSO (Sigma-Aldrich, Hungary) was washed out twice in RPMI 1640 medium.

A harmonised IVS protocol was designed based upon features of the “best-performing” protocol(s) from Phase I, defined as one which delivered high antigen-specific T cells whilst maintaining low background and low inter-assay variation. The harmonised IVS protocol was then tested by 6 centres in Phase II. In brief, PBMCs were thawed, washed and resuspended in X-Vivo 15 medium (Lonza) supplemented with 1 % l-glutamine (200 mM; PAA), 1 % Penicillin/Streptamycin (10,000U/mL and 10,000mcg/mL; PAA) and 10 % human AB serum (Lonza). Cells were counted and volume adjusted to 4 × 10⁶ viable cells/mL. One mL of cell suspension was added to each well of a 24-well, flat-bottomed plate, along with 1 mL of X-Vivo 15 working medium plus antigenic peptides (Peptide Synthetics Peptide Protein Research Ltd.) and recombinant IL-2 (R&D Systems Europe Ltd.) to give a final concentration of 1 μg/mL and 20 IU/mL, respectively, in a final volume of 2 mL. Plates were incubated at 37 °C with 5 % CO₂. On days 4, 6, 8 and 11, 1 mL of culture medium was removed from each well and replaced with 1 mL of X-Vivo 15 working medium plus recombinant IL-2 to a final concentration of 20 IU/mL. On day 13, the cells were harvested, washed twice and resuspended into X-Vivo 15 working medium. Cells were counted and volume adjusted to 1 × 10⁶ viable cells/mL for subsequent ELISPOT assay and multimer staining.

RPMI-1640, penicillin/streptomycin (P/S), 10X HEPES and 10X HBSS from Life Technologies (Carlsbad, CA). Human AB serum from Lonza (Basel, Switzerland). Fetal Bovine Serum from Lonza for MDM culture and from Gemini (West Sacramento, CA) for HEK293 culture. BSA, Acetylcholine, Probenecid, Dopamine, SKF81297, Sulpiride and SCH23390 from Sigma-Aldrich (St. Louis, MO). SKF38393 and Flupenthixol dihydrochloride from Tocris Biosciences (Minneapolis, MN). Quinpirole from Tocris or Sigma-Aldrich. All DR agonists and antagonists were resuspended in distilled H₂O. TAK779 was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH [41] (link). Macrophage colony stimulating factor (M-CSF) was from Peprotech (Rocky Hill, NJ), and was resuspended at 100 µM in distilled H₂O.

Peripheral blood mononuclear cells (PBMCs) obtained from 4 patients and 4 controls were challenged with diluent (D; PBS) or allergen extracts (A) from birch pollen (ALK Abelló, 100 μg/mL) at a density of 10⁶ cells/mL for 7 days in RPMI 1640 supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 5% human AB serum (Lonza, Switzerland), 5 μM β-mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA), and 50 μg/mL gentamicin (Sigma- Aldrich, St. Louis, Missouri, USA) [7 (link)].