Apcef780

Antibodies for flow cytometry included the following clones and conjugates: anti B220 (clone RA3-6B2, BV421, Biolegend, and APCCy7, eBiosciences); anti CD93 (clone AA4.1, APC, Invitrogen); anti CD23 (clone B3B4, PECy7, eBiosciences); anti IgM (clone B76, AF488, homemade); anti Fas (clone Jo2, PE, BD Pharmingen); anti IL-4Rα (clone mIL4R-M1, PE, BD Biosciences); anti CD3 (clone 17A2, PB, Biolegend); anti CD4 (clone RM4.5, APC, BD Biosciences and BV510, Biolegend); anti CD8β.2 (clone 53–5.8, FITC, BD Biosciences); anti Fas-L (clone mFL3, PE, eBiosciences); anti TCR-β (clone H57-597, PB, Biolegend and PercpCy5.5, eBiosciences); anti CD24 (clone M1/69, BV711, BD Biosciences); anti CD8α (clone 53–6.7, APCeF780, eBiosciences); anti CD122 (clone 5H4, PE, eBiosciences); anti CD44 (clone IM7, BV605, BD Biosciences); anti CD49d (clone R1-2, APC, Biolegend); anti CXCR3 (clone CXCR3-173, PECy7, Biolegend); anti T-bet (clone 4B10, BV421, Biolegend); anti Eomes (clone 1219A, AF488, R&D Systems).

Cells were stained with fluorochrome- or biotin-labeled antibodies for 30 min on ice. The following antibodies conjugated to FITC, PE, PerCp-Cy5.5, PerCP-ef710, PeCy7, APC, APC-ef780, Pacific Blue, or Brilliant Violet 421 were purchased from eBiosciences or BD Biosciences: CD3ε, CD4, CD8α, CD19, Ter119, CD122, NK1.1, NKp46, DX5, CD69, CD11b, CD25, Klrg1, IL7Rα, Sca1, cKit, CD43, B220, and Gr1. Propidium iodide was used to exclude dead cells. Cells were acquired on a FACS Canto, LSRII, or Fortessa or sorted with a FACS ARIAII and analyzed with FLOWjo. Through this study mNK cells were identified using a lineage cocktail containing CD19, CD3, CD4, CD8, and Ter119 antibodies and propidium iodide to exclude dead cells. NK cells were either Lin-CD122+NK1.1⁺ or Lin-CD122+NKp46/NK1.1+ and DX5⁺. NKP were defined as Lin-CD122⁺NK1.1^-.

After 6 days (immature-) or 8 days (mature-) moDC were incubated on ice (while shaking) for 30 min in ice cold FACS buffer (PBS (Lonza, BE17,516F) containing 0.5% BSA fraction V (Roche, 10735086002, Basel, Switzerland), 2.0 mM EDTA (Merck, 108418, Kenalworth, NJ, USA) and 0.05 NaN₃) to facilitate the detachment of DC from the surface. Surface marker expression was analysed by using fluorochrome-conjugated antibodies directed against CD14 (FITC; BD Biosciences, 555397), CD86 (V450; BD Biosciences, 560357), CD83 (FITC; BD Biosciences, 556910), HLA-DR (APCef780; eBiosciences, 47-9956-42, San Diego, CA, USA), CD80 (PE-Cy5; BD Biosciences, 559370), PD-L1 (PE-Cy7; BD Biosciences, 558017), CD1a (PerCP/Cy5-5; Biolegend, 300130, San Diego, CA, USA). 10 µg/mL of human Fc Block (BD Bioscience, 554220) was added to the antibody mixture to block non-specific binding. Compensation beads (eBiosciences, 01-2222-41) stained with single antibodies were run for every experiment. Cells were washed with 200 µL FACS buffer and stained by incubating the antibody mixture for 30 min in the dark at 4 °C. Before measuring DRAQ7 (Abcam; ab109202, Cambridge, UK) was added and incubated for 10 min in the dark to stain nonviable cells. Cells were resuspended in 100 µL FACS buffer and acquired on a BD FACS Canto II (BD Biosciences) and analysed using the FlowJo software V10.