Flow cytometry

Lungs, MLNs, and peripheral blood were collected from immunized and control animals. Lung lobes were separated and cut into small pieces in phosphate-buffered saline (PBS) containing 0.5% fetal bovine serum (FBS) and 0.05 M Hepes buffer with collagenase IV (120 U/ml; Worthington Bio, NJ) and deoxyribonuclease I (40 U/ml; bioWORLD, OH). Single-cell suspensions were made using the gentleMACS Lung Dissociation Protocol (52). MLNs were passed through a 70-μm cell strainer to obtain a single-cell suspension of lymphocytes. Red blood cells were lysed using ACK lysis buffer (Gibco, MD). Single-cell suspensions were washed with PBS and resuspended in flow cytometry staining buffer (PBS + 2% FBS + 0.1% sodium azide). Cells were stained with fluorescently labeled antibodies, and tetramers and samples were analyzed on LSRFortessa or FACS Canto (Becton Dickinson, NJ) flow cytometry instruments. Data were analyzed using FlowJo (TreeStar). Antibodies used were the following: anti-CD8α (53-6.7), anti-CD69 (H1.2F3), anti-CD103 (2E7), anti–TNF-α (MPX-XT22), anti–interferon-γ (XMG1.2), anti-CD11a (H155-78), anti-CD49a (Hα31/8), anti-CD11c (N418), anti-CD11b (M1/70), anti-CD24 (M1/69), anti-CD64 (X54-5/7.1), anti-SiglecF (1RNM44N), and anti-CD45 (30-F11). CD103⁺ and CD11b⁺ DCs in the lung were identified by using a previously established gating scheme (fig. S8) (35).