Facs lysing solution 1x

One hundred microliters of whole blood were stained with anti-CD3-PECy7 (BioLegend, San Diego, CA, USA; clone HIT3a), anti-CD8-PECy5 (BioLegend; clone SK1), anti-PD-L1-PE (BioLegend; clone 29E.2A3), anti-CD14-PECy7 (BD Bioscience, San Jose, CA, USA; clone M5E2), anti-CD41a-FITC (Immunotools, Friesoythe, Germany; clone HIP8), and anti-CD62P-APC (Immunotools; clone HI62P). The cells were then incubated for 15 min in the dark before adding 2 mL of BD FACS lysing solution 1X (BD Bioscience) for 10 min. Finally, the cells were washed with 2 mL of PBS 1X and resuspended in 400 µL of PBS 1X before acquisition by flow cytometry (MACSQuant Analyzer 10 flow cytometer; Miltenyi Biotec, Bergisch Galdbach, Germany). Negative gates for each marker were defined by fluorescence minus one (FMO) controls.

Lung immune cells were obtained and analyzed one day after the last challenge (6 to 7 mice per group). Lungs were fragmented and transferred to a conical tube containing digestion solution. Samples were incubated at 37°C under agitation for 1 h. After incubation, cells were dispersed through a 40 µm nylon filter by using a 10 ml syringe. After red blood cells lysis, using a BD FACS Lysing Solution 1X, cells were washed with complete RPMI-1640, passed through a 40 µm nylon filter and resuspended. Total cell counts were determined with Kova slide®. Cytology was determined by flow cytometry as in BAL but on 3.10⁶ cells. Lung cells (8.10⁶ cells/ml) were stimulated for 5 h at 37°C 5% CO2 with phorbol myristate-Acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml) in presence of monensin (2 ng/ml) for Th17 assessment or brefeldin A (1 ng/ml) for Th1/Th2 and Treg assessment. T cell cytokine production was determined using flow cytometry after cells staining with the following antibodies: fixable viability 450, anti-CD16-CD32, extracellular corresponding marking mix, intracellular labeling mix after fixation and permeabilization (Table 1A).

Quantification of blood immune cell phenotypes of peripheral venous EDTA whole blood was performed using the BD Trucount™ System (Multitest™ 6-Color TBNK: CD3-FITC, CD16-PE, CD56-PE, CD45-PerCP-Cy5.5, CD4-Pe-Cy7, CD19-APC, CD8-APC-Cy7 (Trucount) with tubes; BD Biosciences cat #337,166) according to the manufacturer’s instructions. In brief, 20 μL of BD Multitest 6-Color TBNK antibody solution was mixed with 50 μL of whole blood in Trucount tubes. The solution was vortexed and incubated for 15 min at RT in the dark. Next, 450 μL of BD FACS Lysing Solution (1x), which was previously diluted 1:10 from the 10 × stock solution in distilled aqua, was added to the sample, vortexed, and incubated again for 15 min at RT in the dark to allow lysis of the erythrocytes. Samples were immediately measured on the LSR-II flow cytometer by recording 200,000 cell events and analyzed using FACSDiva software (version 8.0.1). The gating strategy is depicted in suppl Fig. 1.