Cd69 apc cy7

Cryopreserved PBMC from 3 HUU at birth were thawed and counted as described above. Cells were stimulated for 24h in complete RPMI+10% FBS with TLR cocktail in duplicate. BFA (5ug/mL) was added to one replicate at the beginning of the stimulation, or for the last 4 hours of culture to the second replicate. Cells were washed with PBS, stained with Zombie Yellow Fixable Viability dye and surface stained with HLA-DR Alexa Fluor 488, PD-L1 BV421, CD19 PerCP-Cy5.5, CD69 APC-Cy7 (Biolegend), CD56 PE, CD16 PE-CF594, CD14 PerCP-Cy5.5, CD20 PerCP-Cy5.5 and CD3 Alexa Fluor700 (BD Biosciences). Cells were washed and treated with BD lysing solution and BD permeabilization buffer 2 (BD Biosciences) then stained for IL-8 PE-Cy7 (Biolegend) and IFNγ APC (eBiosciences). Cells were washed and fixed with PBS+1% paraformaldehyde before acquisition on the cytometer (Beckman Coulter Gallios).

Single‐cell suspensions were stained with a mixture of 5 to 6 fluorophores including and 7‐aminoactinomycin D (7‐AAD) for viability and acquired on the BD Verse flow cytometer with data analyzed using FlowJo software (BD Biosciences). Human B‐cell subsets were identified using the following antibodies: CD19‐APC, CD27‐BV510, CD38‐APC‐Cy7, CD24‐BV421, and IL‐10‐PE‐Cy7 (all from BioLegend). Mouse B‐cell subsets were identified using the following antibodies: B220‐PE, CD23‐APC, CD21‐PE‐Cy7, IgM‐BV421, CD138‐APC, CD86‐PE‐Cy7, CD69‐APC‐Cy7, and IL‐10‐APC‐Cy7 (all from BioLegend). Intracellular IL‐10 expression was detected as previously reported.³³ Immunophenotype of human and murine B‐cell subsets analyzed are listed in Supplementary Table 1.

We used A549 cells, a transformed human cell line, in these studies as a proxy for lung alveolar epithelial cells. We maintain large stocks of A549 cells at low passage and verify that they are free from mycoplasma contamination routinely. A549 cells were seeded in 24-well plates and incubated for 24 h at 37 °C with 5% CO₂. Confluent monolayers of A549 cells were infected with 0.5 MOI of pH1N1 and cells were incubated at 37 °C with 5% CO₂ in the presence of 1 µg/mL of TPCK-trypsin. Mock-infected cells received infection media instead of virus. Mature BMdEos suspended in growth media supplemented with 10 ng/mL of IL-5 and 5 ng/mL of rmGM-CSF were added at 1:1 ratio to A549 cells directly or indirectly by using Transwell^® permeable supports (0.4 µM permeable membrane) (Castor, Kennebunk, ME, USA). Plates were incubated for 3 days at 37 °C and 5% CO₂ and cells were individually stained for flow cytometry. A549 cells were stained with anti-human HLA-A2 (PE-Cy7; Biolegend), CD40 (BV421; Biolegend), CD69 (A700; Biolegend), and influenza A PB1 (PE). Eosinophils were stained with anti-mouse MHC-I (PE-Cy7; Biolegend), CD80 (BV605; Biolegend), Siglec-F (PE-CF594, BD Biosciences), CD69 (APC-Cy7; Biolegend), and influenza A PB-1 (PE). Staining and acquisition were performed individually on each cell type with a BD LSR Fortessa and analyzed using FlowJo v10.5.2 (Treestar) software.

Antibodies used for flow cytometry were purchased from BioLegend (San Diego, CA) and include: anti‐CD4‐Alexa Fluor 700 (RPA‐T4), CD69‐APC/Cy7 (FN50), and CD25‐PE (M‐A251).