After total lung leukocyte preparation was performed, nonspecific Fc binding was blocked with a CD16/32 antibody. Subsequently, primary antibodies were added to cell samples and incubated for 30 minutes in the dark at 4°C. Primary antibodies used were anti-CD45, CD11b, CD4, IL-17A, MHCII (I-Ab), CD103, SiglecF (BD Bioscience, San Jose, CA), CD11c, IFNγ (eBioscience San Diego, CA), CD64 (Biolegend, San Diego, CA). For intracellular cytokine staining, cells were first incubated with PMA (10 ng / mL) plus ionomycin (10 μM), with the addition of Golgi-stop reagent (BD Bioscience, San Jose, CA) for 4 h at 37°C. Staining was then performed as above.
T-Distributed Stochastic Nearest-neighbor Embedding (tSNE) was implemented as a plugin in Flowjo v 10.4. Briefly, flow cytometry data for individual samples was concatenated into a single FCS file and a pre-gating strategy was implemented to rid the dataset of cell debris and doublets. Cleaned data was further gated as being Thy1.2+, IL-17+ and the tSNE algorithm was run to separate groups based on the indicated surface markers using the following parameters: perplexity 35, learning rate 1500 for 3000 iterations.
T-Distributed Stochastic Nearest-neighbor Embedding (tSNE) was implemented as a plugin in Flowjo v 10.4. Briefly, flow cytometry data for individual samples was concatenated into a single FCS file and a pre-gating strategy was implemented to rid the dataset of cell debris and doublets. Cleaned data was further gated as being Thy1.2+, IL-17+ and the tSNE algorithm was run to separate groups based on the indicated surface markers using the following parameters: perplexity 35, learning rate 1500 for 3000 iterations.