Anti ifn γ bv 421

The ADCC assay for measuring intracellular NK cell IFN-γ and CD107a expression was conducted and analysed with the gating strategy as previously described^{23 (link)}. Briefly, 96-well plates were coated overnight at 4 °C with A/California/04/09 HA protein (1 μg/ml) and chimeric cH9/1 HA protein (1 μg/ml). The plates were then washed with PBS and incubated with heat-inactivated sera (prediluted 1:10) for 2 h at 37 °C. Plates were then washed again with PBS and incubated with 10⁵ CD16 176 v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking the expression of CD16 were added to an additional well per sample. Cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, the cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). The cells were acquired on BD Fortessa. Data analysis was performed using FlowJo version 10 (treeStar).

Procedure has been performed as previously described^{69 (link)}. Briefly, WT mice were infected with 10⁴ colony-forming units LmOVA. Mice were given 250 μg brefeldin A by i.p. injection, 6 h before being sacrificed. After 24 h, mice were euthanised, and the spleens were removed, fixed in 4% PFA and cryopreserved in OCT. Serial sections (30 µm in thickness) of frozen spleens were stained overnight at +4 °C in a humidified chamber with the following Abs: anti-B220 PB (1:300; 103230, Biolegend), anti-CD169 Alexa647 (1:300; 142407, Biolegend), anti-CD8 BV510 (1:100; 100751, Biolegend), anti-IFNγ BV421 (1:200; 505829, Biolegend), anti-I-A/I-E biotin (1:200; 107603, Biolegend), or anti-NKp46 (1:200; AF2225, R&D). For secondary detection, sections were then washed and incubated with Streptavidin-Cy3 (1:200; 016-160-084, Jackson Immunoresearch) and anti-goat IgG A647 (1:500; ab150131; Abcam) for 3 h at RT. All sections were analysed by confocal microscopy. Quantification of the interaction between DCs and IFNγ-producing cells was performed manually. DCs were randomly selected in the IFNγ-rich area. For each DC, the number of IFNγ-producing CD8 T cells and the number of IFNγ-producing NK cells was recorded and averaged.

After cell culture, whole blood samples (10x10⁵ cells/mL) were incubated with the following panel: antibodies from BioLegend, San Diego, CA, USA: anti-CD45-PerCP (Clone: HI30), anti-CD3-AF647 (Clone: UCHT1), anti-CD14-PECy7 (Clone: M5E2), anti-CD4-APC/Cy7 (Clone: OKT4), anti-CD8-PE/Dazzle594 or BV510 (Clone: SK1). After 15 min in the dark, blood was washed once with PBS (1 mL) by centrifugation at 900×g for 5 min at room temperature (RT); then, Fixation buffer was added (100 μL, Cat: 420801, BioLegend, San Diego, CA, USA), and the samples were incubated for 20 min. Then, samples were washed twice with 1 mL of Intracellular Staining Perm Wash buffer (Cat: 421002, BioLegend, San Diego, CA, USA); after the second wash, they were mixed with monoclonal antibodies against cytokines from BioLegend, San Diego, CA, USA: anti-TNFα-BV421 (Clone: MAb11), anti-IL-6-PE (MQ2-13A5), anti-IL-1β-FITC (Clone:JK1B-1), anti-IFNγ-BV421 (Clone:4S. B3), anti-IL-8a-PE (Clone: E8N1). For the exclusion of dead cells, a Zombie Aqua fixable viability kit (BioLegend, San Diego, CA, USA) was added and incubated for 30 min at RT. Last, the mixture was washed once with PBS. At least 30,000 events were acquired in a FACSAria IIu (BD, San Jose CA) flow cytometer. Analysis was performed using Infinicyt^™ Software 1.8.

The ADCC assay measuring intracellular NK cell IFNγ and CD107a expression was conducted as previously described with minor modifications.^{35 (link)} Briefly, 96-well plates were coated overnight at 4 °C with 1 μg/ml HA1 (A/California/06/2009(H1N1)) 6×His tagged (eEnzyme, USA) or chimeric cH6/1 in PBS. Plates were washed with PBS and incubated with heat-inactivated human sera for 2 h at 37 °C. After washing, 10⁵ CD16 176v NK-92 cells (mycoplasma-free, human NK cell line expressing high affinity 176V variant CD16 receptor) (kindly provided by Fox Chase Cancer Center, Philadelphia, PA, USA) were added per test well. As a negative control for each sample, NK-92 cells (lacking expression of CD16) were added to an additional well. The cells were incubated for 16 h at 37 °C with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA, 328610), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD). Cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen, Carlsbad, CA, USA), anti-CD3-PE CF594 (BD, Franklin Lakes, NJ, USA, 562280) and anti-CD56-APC (BD, 555518) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend, 502532). Cells were acquired on BD Fortessa (San Jose, CA, USA). Data analysis was done using FlowJo version 10 (treeStar, Ashland, OR, USA).