Alexa fluor 700 anti mouse cd4

Single lung-cell suspensions were incubated using a Zombie Aqua™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) at RT for 30 min to discriminate dead cells for analysis. Cells were washed with DPBS containing 1% FBS, blocked with anti-CD16/CD32 (clone 2.4G2, ATCC HB-197), and then stained with FITC anti-mouse CD19 (BD Biosciences), PE anti-mouse CD69 (BioLegend), PerCP-Cy5.5 anti-mouse CD3e (BD Biosciences), PE-Cy7 anti-mouse CD103 (BioLegend), APC anti-mouse CD44 (BioLegend), APC-Cy7 anti-mouse CD8a (BioLegend), Alexa Fluor 700 anti-mouse CD4 (BD Biosciences), or BV421 anti-mouse CD45.2 (BD Biosciences) antibodies for 30 min at 4 °C. For B-cell analysis, cells were stained with FITC anti-mouse IgA (Southern Biotech), PE anti-mouse CD19 (BioLegend), PerCP-Cy5.5 anti-mouse CD38 (BioLegend), PE-Cy7 anti-mouse CD3e (BD Biosciences), biotin anti-mouse CD45.2 (eBioscience), Alexa Fluor 700 anti-mouse IgD (BioLegend), Brilliant Violet™ 421 anti-mouse CD80 (BioLegend), or streptavidin APC-Cy7 (BD Biosciences) antibodies for 30 min at 4 °C. After staining, cells were washed with DPBS containing 1% FBS and analyzed on a FACS Fortessa (BD Biosciences) using FlowJo Software Version 10 (Tree Star, Ashland, OR, USA). The gating strategy is presented in Supplementary Fig. 1.