Cd28 apc cd28.2

BAL cells were stained using fluorochrome-conjugated mouse monoclonal antibodies directed to human CD11b-PE (clone D12), CD45-PerCP (TU166), CD20–PE-Cy7 (L27), CD16-AF647 (3G3), CD3–APC-Cy7 (SP34-2),CD8-AmCyan (SK1), CD95-PE (DX-2), CD69-PerCP (FN50), CCR4–PE-Cy7 (1G1), CD28-APC (CD28.2), and CD4-V450 (L200) (All from BD Biosciences), as well as HLA-DR–Pacific Blue (L243; BioLegend). Fluorescence was measured on a FACS Canto II and analyzed using BD FACS Diva software (both BD Biosciences) and FlowJo software (Tree Star Inc.) (Supplemental Methods, Supplemental Figure 4). Extrapolated cell number was determined for each of the analyzed cell types present in BAL fluid as follows: “number of BAL cells/mL” × “frequency of the population.”

We performed flow cytometry analysis using the following antibodies: CD3-Percp-Cy5.5 (HIT3a, BD Bioscience), CD8-APC (RPA-T8, BD Bioscience), CD27-PE (M-T271, BD Bioscience), CD28-APC (CD28.2, BD Bioscience), CD45RO-PE (UCHL1, BD Bioscience), CD62L-FITC (DRGE-56, BD Bioscience), αvβ3-FITC (LM6090, EMD Millipore,), αvβ5-FITC (P1F6, EMD Millipore), and NRP-1-PE (AD5-17F6, Miltenyi Biotec GmbH). All samples tested were suspended in FACS buffer and stained with indicated antibodies for 30 min in 4 °C in darks, and then, washed twice, and resuspended in FACS buffer before analysis. For IFN-γ detection, BD™ Cytometric Bead Array (CBA) Human IFN-γ kit (BD Bioscience, USA) was used. Samples were analyzed with BD Accuri C6 (BD Bioscience).