High-parameter flow cytometry analysis of immune cells

High-parameter flow cytometry was performed on BAL cells and PBMCs at preinfection, at pretreatment, during treatment, at posttreatment, and at necropsy as previously described (17 (link), 34 (link), 66 (link), 74 (link), 77 (link)). The single cells prepared from lung, BAL, PBMCs, and other tissues were stained with surface and intracellular markers to study T cell phenotypes. Tissues obtained at necropsy were digested using Liberase and DNase (both Sigma-Aldrich), filtered, and subjected to RBC lysis (ACK Lysis Buffer, Gibco). The cells were then counted and used for staining for flow cytometry. The cells were first stained with extracellular/surface antibodies: CD3, CD4, CD8, CD45, CD28, and CD95 for 25 minutes at room temperature, followed by the Fixable Viability Stain 575V (BD Biosciences). The cells were then fixed and permeabilized using Fixation/Permeabilization Kit (BD Biosciences) for 30 minutes at 4°C. Subsequently, the cells were stained with intracellular antibody (Ki67) to study the T cell proliferation. Cells were then washed and acquired on a BD FACSSymphony flow cytometer. Analysis was performed using FlowJo (v10.5.3) using previously published gating strategies (Supplemental Figure 4) (30 (link), 32 (link)–34 (link)). The details of antibodies used in flow cytometry experiments are provided (Supplemental Table 3).