Anti cd4 antibody

Live cells were stained with anti-CD4 antibody (eBioscience) and Fixable Viability Dye eFluor 780 (eBioscience) on ice, followed by fixation and permeabilization using the Foxp3 Transcription Factor Staining Kit (eBioscience). Intracellular staining was performed at room temperature using anti-IL-2 and anti-IFN-γ (eBioscience), followed by analysis with a BD LSRFortessa X-20 flow cytometer. FACS data for Fig. 6c, d are provided in Source Data file.

For flow cytometric analysis of single-cell suspensions, one part of the sample was used for staining of regulatory T cells with the Mouse Regulatory T Cell Staining Kit (eBioscience, San Diego, CA). Remaining lung cells were subjected to density gradient centrifugation using Pancoll (PAN Biotech, Aidenbach, Germany) to isolate mononuclear cells. Isolated cells were cultured over night in anti-CD3/anti-CD28-coated plates to perform intracellular cytokine staining. On the following day, cultivated cells were restimulated with leukocyte activation cocktail with Golgi Plug (BD Pharmingen) for 4 h. Afterwards, cells were stained with anti-CD4 antibody (eBioscience), fixed in 2% formaldehyde, permeabilized in 0.3% saponin buffer and stained with antibodies against IL-17A, IFNγ (both from eBioscience) and IL-4 (Biozol) to distinguish between different T helper cell subpopulations. Multicolor analysis of stained cells was conducted with a BD FACSCanto II flow cytometer (BD Biosciences, Heidelberg, Germany) and BD FACSDiva software.