Cd16 cd32

P1 and P21 male and female murine lungs were isolated as described above. Cells were blocked for 30 min with CD16/CD32 (Tonbo Biosciences). For intracellular analyses, cells were stimulated with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (750 ng/mL, Sigma-Aldrich), and GolgiStop (BD Biosciences) for 5 hr, and then surface stained with fluorochrome-conjugated antibodies for 30 min: CD3 (clone: 145–2 C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), and CD8a (clone: 53–6.7, Biolegend). Cells were then permeabilized with FOXP3 Fixation/Permeabilization Kit (BD Biosciences) as indicated by the manufacturer, and stained for TBET (clone: 4B10, Biolegend), GATA3 (clone: L50-823, BD Biosciences), FOXP3 (clone: FJK-16s, eBioscience), RORγt (clone: Q31-378, BD Biosciences), IFNγ (clone: XMG1.2, Biolegend), IL-4 (clone: 11B11, BD Biosciences), IL-10 (clone: JES5-16E3, Biolegend), and IL-17 (clone: TC11-18H10, Miltenyi Biotec) for 30 min. Cells were read using an LSRII flow cytometer using FACSDiva software. Flow data was analyzed using FlowJo (Tree Star Inc). Flow cytometry analysis for this project was done on instruments in the Stanford Shared FACS Facility.

Fluorescently labeled antibodies to cell surface antigens were diluted in PBS containing 1% FCS, 0.05% sodium azide, and 0.5 μg/mL CD16/CD32 (Tonbo; 2.4G2). Cells were incubated for 30 minutes at 4°C, washed, and resuspended in FACS buffer. The following antibodies were used for multi-parameter FACS analysis: CD3 (Tonbo; 17A2), CD44 (Tonbo; IM7), GITR (eBioscience; DTA-1); CD4 (RM4-5, RM4-4), CD8 (53-6.7), CD69 (H1.2F3), B220 (RA3-682), CD103 (2E7), Nrp-1(3E12), ICOS (C398.4A), and CTLA-4 (UC10-4B9) were all purchased from BioLegend, unless specified otherwise. To remove innate cells in our analysis of tissue-resident CD4⁺ T cells and tetramer analysis, antibodies directed against CD11c (N418), CD11b (M1/70), IA/IE (M5.114.15.2), HLA-DP (purified from hybridoma, B7.21), and Ly6G (1A8) were purchased from eBioscience. For HLA-DP2-CCL4/Be tetramer staining, lung cells were stained as previously described (13 (link), 17 (link)). Intracellular staining for FoxP3 was performed according to the manufacturer’s protocol (Thermo Fisher Scientific). Data were obtained using a BD FACS Canto and Celesta Flow Cytometers and analyzed using FlowJo software (v9.9.6; Treestar, Inc.).