F4 80 clone bm8.1

Lung ILC2 cell identification was performed as described previously (47 (link)). Lung tissues were digested in 8 mL RPMI 1640 containing liberase (50 μg/mL) and DNase I (1 μg/mL) for approximately 40 minutes at 37°C. Cell suspensions were filtered through 70 μm cell strainers and washed once with RPMI 1640. For ILC2 cell identification, total lung cell suspensions were blocked with 2.4G2 antibodies and stained with lineage cocktail mAbs: CD3ε (clone 145-2C11) (BioLegend, 100304), CD4 (clone GK1.5) (BioLegend, 100404), CD8α (clone 53-6.7) (Tonbo Biosciences, 30-0081-U500), CD11c (clone N418) (BioLegend, 117304), FceRIα (clone MAR-1) (BioLegend, 134304), NK1.1 (clone PK136) (BioLegend, 108704), CD19 (clone 6D5) (BioLegend, 115504), TER119 (clone TER-119) (BioLegend, 116204), CD5 (clone 53-7.3) (BioLegend, 100604), F4/80 (clone BM8.1) (Tonbo Biosciences, 30-4801-U500), Ly6G (clone RB6-8C5) (Tonbo Biosciences, 30-5931-U500), APC-conjugated streptavidin (BioLegend, 405207), PE-conjugated T1/ST2 (clone DIH9) (BioLegend, 145304), PerCP-Cy5.5-conjugated CD25 (clone PC61) (BioLegend, 102030), V450-conjugated Sca-1 (clone D7) (BD Biosciences, 560653), PE-Cy7-conjugated KLRG1 (clone 2F1/KLRG1) (BioLegend, 138416), APC-Cy7-conjugated CD45, and Fixable Viability Dye eFluor 506.

Tissues were embedded in OCT (Tissue-Tek) and stored at –80°C. Sections of 7 μm were cut using a cryostat and fixed in acetone at –20°C for 10 minutes. Sections were rehydrated with PBS + 1% BSA and incubated for 1 hour at room temperature with various combinations of primary antibodies PD-L1 (clone AF1019, R&D Systems, 1:500), CD8α (BD Biosciences, clone 53-6.7, 1:100), pan-cytokeratin–FITC (clone F3418, MilliporeSigma, 1:200), F4/80 (clone BM8.1, Tonbo, 1:100), and Ly6G (clone 1A8, BD, 1:100) diluted in PBS + 1% BSA. Slides were washed 3 times in PBS + 1% BSA and incubated with anti-goat AF647 (Jackson ImmunoResearch, 1:500) for 1 hour at room temperature in the dark. Following staining, slides were then washed 3 times with PBS + 1% BSA, washed 3 times with PBS, and mounted in DAPI Prolong Gold (Life Technologies). Images were acquired on a Leica DM6000 epifluorescence microscope at the University of Minnesota Center for Immunology using Leica LasX software and analyzed using Fiji2.0.