Anti mouse ifn γ clone xmg1

Lungs were perfused with PBS containing EDTA (0.5 mM), minced, and digested in collagenase IV (5 mg/ml) and DNase I (23 (link)). Cells were filtered and washed with RBC lysis buffer (BD Biosciences, Franklin Lakes NJ) and kept on ice in media containing 10% serum. Dead cells were stained with Pac-Orange Live/Dead fixable dead staining dye (Invitrogen). Lung cells were then stained with fluorescent-labeled antibodies against various leukocyte surface markers (CD45, CD11b, CD11c, F4/80, CD103, MHCII, CD86, Clec9a, CD3ε, TCRβ, CD4, CD69, IFN-γ). Appropriate isotype-matched controls were used in all experiments. Antibodies were purchased from EBiosciences (San Diego, CA) or Biolegend (San Diego, CA). Cells were fixed and analyzed on a Canto2 (Becton-Dickinson, San Jose, CA) or FACSAria II (BD Biosciences) flow cytometer. Results were analyzed using FlowJo software (Tree Star, Ashland, OR). For analysis of intracellular IL-12 or IFN-γ, fresh aliquots of digested lung tissue were stimulated for 5 hours at 37°C, with Cell-stimulation Cocktail Buffer (40.5 μmol/L phorbol 12-myristate 13-acetate [PMA], 670 μmol/L ionomycin, 5.3 mmol/L brefeldin A, and 1 mmol/L monensin; eBioscience), fixed, permeabilized with Cell Permeabilization Buffer (eBioscience) and incubated with anti-mouse IL-12 clone C17.8 (eBioscience) or anti-mouse IFN-γ clone XMG1.2 (Biolegend).

FC receptors were blocked with anti-mouse CD16/32 (clone 93, Biolegend) before staining. Dead cells were identified using the Zombie Aqua Fixable Viability Kit (Biolegend). Cells were stained with the following antibody clones: anti-mouse CD8a (clone 53-6.7, Biolegend), anti-mouse/human CD44 (clone IM7, Biolegend), anti-mouse CD62L (clone MEL-14, eBioscience), anti-mouse/human Granzyme B (clone GB11, Biolegend), anti-mouse CTLA4 (clone UC1-4B9, Biolegend), anti-mouse CD25 (clone PC61.5, eBioscience), and anti-mouse IFN-γ (clone XMG1.2, Biolegend). Intracellular staining of Granzyme B, CTLA-4, and IFN-γ was performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). To count cells, 123count eBeads (eBioscience) were added to flow cytometry tubes immediately before flow cytometer acquisition. Data was acquired on a BD LSRFortessa and analyzed in FlowJo. Cells were gated for size, single cells, living cells, and CD8⁺ cells before examination of proliferation curves (Supplementary Fig. 8c). Cells were further gated on proliferation dye⁺ cells to exclude any CD8⁺ T cells in the APC fraction before quantification of division numbers and examination of surface and intracellular proteins. Statistical analyses of results from separate mice were performed using GraphPad Prism software.