*2.2. Cell Culture*

Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation using Pancoll separating solution (PAN-Biotech, Aidenbach, Germany). Afterwards, PBMCs were washed twice and suspended to a final density of 1 × <sup>10</sup><sup>6</sup> PBMCs per mL in serum-free AIM-V culture medium (Invitrogen, Eggenstein, Germany). Activation of T lymphocytes among PBMCs was achieved by mitogen stimulation with 2 μg/mL phytohemagglutinin (PHA, life technologies/Gibco, London, UK).

JAK inhibitors tofacitinib, baricitinib, upadacitinib, and filgotinib and methotrexate (MTX) were all purchased from Selleckchem (Houston, TX, USA). These agents (stock solution: 10 mM in dimethyl sulfoxide (DMSO)) were either added simultaneously with PHA into cell culture plates or 48 h after PHA-stimulation, to investigate their impact on preactivated PBMCs. Cells treated with DMSO, diluted 1:1000, served as corresponding vehicle controls.
