Recombinant human il 2

Peripheral blood mononuclear cells (PBMC) were isolated from blood donor derived buffy coats through gradient centrifugation (Lymphoprep, Axis shields diagnostics, Dundee, Scotland). Naive CD45RA + CD4⁺T-cells were subsequently isolated with magnetic bead separation using the “Naive CD4⁺T-cell Isolation Kit II, human” (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then activated and polarized toward T_H1 using Dynabeads™. Human T-Activator CD3/CD28 (1 bead/cell) (Dynal AS, Lillestøm, Norway), 5 ng/μl recombinant human IL-12p70, 10 ng/μl recombinant human IL-2 and 5 μg/μl anti-IL-4 antibodies (clone MAB204) (all three from, Bio-Techne, Minneapolis, USA), in RPMI 1640 media (Gibco, Paisley, United Kingdom). A portion of T-cells used for RNA and protein isolation was obtained at baseline and after 0.5 h (only RNA), 1 h, 2 h, 6 h, 24 h and 5 days (only protein; point not used in the analysis). The cells were washed twice in PBS, snap frozen in a dry ice ethanol bath and stored at −80°C until use. During the protein and RNA extractions, multiple samples were pooled from twelve different individuals to reach the necessary amount of material for the subsequent analysis steps.

PBMCs or purified NK cells were stimulated through various means. For the co-culture assay, PBMCs or NK cells were cultured with B-cell targets at 1–2 × 10⁶/mL for 24 hours. Alternatively PBMCs or NK cells were cultured with anti-CD3 (OKT3, 0.5 μg/mL, in house) and anti-CD28 (CD28.2, 1 μg/mL, eBioscience), recombinant human IL-2 (25 ng/mL, Biotechne), recombinant human IL-15 (10 ng/mL, Biotechne) or SEB (Sigma) (1 ng/mL) for 24 hours.

The pre-incubated cells were activated and differentiated towards T_H1 using Dynabeads™ Human T-Activator CD3/CD28 (1 bead/cell) (Dynal AS, Lillestøm, Norway), 5 µg/ml recombinant human IL-12p70, 10 µg/ml recombinant human IL-2 and 5 mg/ml anti-IL-4 antibodies (clone MAB204; all from, Bio-Techne, Minneapolis, USA). P4 was added together with the activation and differentiation mixture to the cells pre-incubated with P4 cells. The cells were incubated at 37°C, with 5% CO₂ in RPMI 1640 media containing L-glutamine, 10% FBS and 1% Penicillin/Streptomycin (all from Gibco^®, ThermoFisher Scientific). Cells were either cultured in 6-well (for RNA-seq) or 96-well plates (for ATAC-seq) in biological triplicates at a density of 1 million cells/ml. Cells were subsequently harvested at 0.5, 1, 2, 6 and 24 hrs and processed for RNA-seq and ATAC-seq. An overview of the study design is shown in Figure 1.