Isolation and Activation of Immune Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of HDs by centrifugation on Ficoll-Paque Plus (GE Healthcare Lifesciences) gradients^{38 (link)}. CD4⁺ T, CD8⁺ T, NK and B cells were isolated from PBMCs using negative selection-based cell isolation kits from Miltenyi, #130-096-495, #136-096-533, Stem Cell Technologies, #19055, and Biolegend #480061, respectively, following manufacturers’ protocol. Isolated cells were cultured in RPMI medium supplemented with 10% (v/v) exosome-depleted and heat-inactivated FBS at 37 °C in the atmosphere of 5% CO₂ in air^{16 (link)}. T cells were activated with CD3/CD28 T cell activator (25 µl/mL, Stem Cell) and IL-2 (150 IU/mL, Peprotech) at 1 × 10⁶ cells/mL in RPMI medium for 12 h. NK cells were activated by 48 h incubation with IL-2 (100 IU/mL, Peprotech) and IL-15 (150 IU/mL, Peprotech) at 1 × 10⁶ cells/mL. B cells were activated by 12 h incubation with IL-4 (100 IU/mL, R & D System) and CD40L (0.5 ug/mL, Cell Signaling Technology, #3583 S) at 1 × 10⁶ cells/mL.