Lfa 1 pe

Cells were washed with 1xPBS then stained for surface markers with fluorophore-conjugated antibodies in 1x PBS for 30 min at 4°C protected from light. The following antibodies were used for staining: CD3-APC-Cy7, CD4-PE-Cy7, CD8-Alexa-Fluor-700, CD69-Brilliant Violet-605, PD-1-PE, CD71-Per-CP-Cy7, LFA-1-PE, CD11a-FITC (all BioLegend). Cells were stained concurrently with 1 μL/mL Live/Dead dye (ThermoFisher Scientific). Cells were washed with 1x PBS then fixed with fix/perm buffer set (BD Biosciences) for 20 min at 4°C protected from light. Cells were stained for intracellular markers using fluorophore conjugated antibodies in permeabilization buffer (1% FCS, 0.1% saponin in 1x PBS). The following intracellular antibodies were used: GLUT-1-APC (Abcam), TNF-α-FITC and IFN-γ-V450 (Becton Dickinson). Samples were run on a BD Fortesssa. FlowJow v10 software was used for the analysis.

To assess degranulation, NK cells were co-incubated with opsonized target cell lines in the presence of GolgiPlug (1/1000 dilution, BD Biosciences), monensin (1/1000 dilution, Biolegend) and anti-CD107a PE mAb (clone H4A3, BD biosciences) or an isotype-matched control (PE-IgG1κ; clone MOPC-21, BD) for 4 h at 37°C. After incubation, cells were washed and stained with Zombie NIR viability dye (Biolegend), anti-CD56-AF488 mAb (clone HCD56, Biolegend), anti-CD107a-PE (clone H4A3) and anti-CD16-AF647 (clone; B.731, Biolegend) (42 (link)). To assess expression of other proteins, NK cells were co-incubated with opsonized target cell lines for 4 h, then washed and stained with Zombie NIR viability dye (Biolegend) and LFA-1-PE (Clone M24), NKp46-PercP-Cy5.5 (Clone 9E2) and NKp44-APC (Clone P44-8) or NKp30-AF647 (Clone P30-15) and NKG2D-PE (Clone 1D11, all from Biolegend). Isotype-matched control mAbs were also used accordingly (mouse IgG1 isotype control; clone MOPC-21, Biolegend). Finally, cells were fixed in 2% PFA/PBS and staining was assessed using a BD FACS LSRFortessa™ X-20 flow cytometer and analyzed by FlowJo V10 software (BD Biosciences). The gating strategy used for all flow cytometry assays is shown in Supplementary Figure 5.

Blood, bone marrow, BAL, and lung cells were counted using an automated cell counter (Nexcelcom Bioscience, Lawrence, MA). Cells were incubated with Fc block (anti-mouse CD16/CD32; eBioscience) in 100 μl of FACS buffer (PBS, 0.5 % EDTA) followed by the antibody panels below. Labeled cells were analyzed using BD FACSCanto RUO (BD Biosciences, San Jose, CA) and the FlowJo data analysis software (Ashland, OR). Experiments represent an average of n = 5/group with up to 3 replicates. Antibodies used included: anti-mouse CD62L-APC, ICAM1-FITC, Ly6G-FITC, Ly6G-PerCP/Cy5.5, LFA-1-PE, CD45-APC/Cy7, CD4-PE, CD8-PB, CXCR2-PerCP/Cy5.5, Gr1-PB, Ly6C-PB (BioLegend, San Diego, CA), CD11C-APC, CD11b-PECy7, CD3e PE/Cy7, CD3e-PerCP/Cy5.5, CD71-PE, F4/80-APC (eBioscience).