Golgiplug

PBMCs and dMCs were thawed and rested overnight in low dose IL-15 as described above. For stimulation by PMA/Ionomycin, 1 × 10⁶ mononuclear cells were treated with eBioscience™ cell stimulation cocktail (Invitrogen) for 4 h in 96-well U bottom plates at 37 °C, 5% C0₂. Golgi Plug (BD Biosciences) and Golgi Stop (BD Biosciences) were added for the final 3 h of stimulation. For K562 stimulations, mononuclear cells were co-cultured with K562 target cells at a ratio of 10:1 for 6 h in 96-well U bottom plates at 37 °C, 5% CO₂. Golgi Plug (BD Biosciences) and Golgi Stop (BD Biosciences) were added for the final 5 h of stimulation. Cells were then stained and acquired as outlined above. To analyse polyfunctional NK responses to stimulation, Lin− CD56+ subsets were manually gated on the tSNE plot to show dNK1 (c10–c13), dNK2 (c9), dNK3 (c5, c8), and pbNK (c1, c2) subsets. Boolean gating arrays were created using FlowJo to determine the frequency of cells within these subsets that stained for one, two, or three readouts. Readouts were defined as: CD107a and/or IFNγ and/or at least one of the cytokines MIP1α, MIP1β, GM-CSF, XCL1 (Cyto). Non-responding cells that expressed none of the functional readouts were excluded.

Four million of the freshly purified human monocytes/naïve CD8 T cells were electroporated either with or without 1.5 μm of hsa-miR-125b miRCURY™ LNA inhibitor (EXIQON, Woburn, MA, USA) or Scramble-miR inhibitor Control (Negative control A, EXIQON). The electroporation was carried out using a P3 Primary Cell 96-well Nucleofector™ Kit (Lonza) following the manufacturer's instructions. Next day (16 h after transfection), monocytes were stimulated for 3 h with LPS (100 ng/mL) plus GolgiPlug™ (1 μL/mL; BD Biosciences, San Jose, CA, USA), and naïve CD8 T cells were stimulated by anti-CD3/CD28 antibodies for at 3 h in the presence of GolgiPlug. The levels of CCL4 in electroporated cells were determined by intracellular staining with anti-CCL4 (R&D systems) as described below.

To determine intracellular cytokine production by isolated murine lung ILC2s, sorted cells in complete ILC2 medium (see above) were stimulated in 96-well round-bottom plates (15,000 cells/well) for 48 h with medium only; recombinant murine IL-2, IL-7, IL-9, and IL-33 (10 ng/ml; R&D Systems); or combinations thereof. GolgiPlug (BD Biosciences) was added for the last 6 h of culture. To analyze cytokine production following in vivo stimulation, lung cell suspensions (1 × 10⁶ cells/well in a 96-well plate) in complete ILC2 medium were stimulated with Cell Stimulation Cocktail (eBioscience) according to the manufacturer’s instructions in the presence of GolgiPlug (BD Biosciences). Cells were stained with respective surface antibodies and viability dye as described above, and intracellular cytokine staining was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s protocol. Stained cells were acquired on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) and analyzed using FlowJo X (BD Biosciences). All antibodies used for flow cytometry analyses are listed in Table 1.