Cd20 bv605

NK cells were enriched from PBMCs by the depletion of unwanted cells using biotinylated antibodies and MoJo Streptavidin Nanobeads (BioLegend); more details are available in SI Appendix. The enriched NK cells were stained with CD3-BV605 (clone: UCHT-1, BioLegend), CD19-BV605 (clone: SJ25C1, BioLegend), CD20-BV605 (clone: 2H7, BioLegend), HLA-DR-BV510 (clone: L243, BioLegend), CD56-BV421 (clone: 5.1H11, BioLegend) and NKp46-BV421 (clone: 9E2, BioLegend) and sorted as CD56⁺NKp46⁺CD3⁻CD19⁻CD20⁻HLA-DR⁻ cells. Sorted XCR1⁻ and XCR1⁺ cDC1 and NK cells were cocultured in a 1:5 ratio and either stimulated with a cocktail of pIC (2.5 µg/mL), R848 (2.5 µg/mL), and CpG (2.5 µg/mL) or cultured in DC-medium alone. For some experiments, DCs and NK cells were separated in Transwell plates (96 well, 0.4 µm insert). NK cells were cultured in the well, while DCs were added to insert. In order to analyze the influence of secreted cytokines on NK cells, NK cells were cultured alone and supernatants of stimulated DCs were added corresponding to a 1:5 DC:NK cell ratio. After 18 h, cells were harvested and analyzed by flow cytometry for activation of NK cells (CD69). The supernatants were stored at −80 °C until analysis with LEGENDplex Human Interferon Panel (BioLegend) for secretion of IFNγ.