Cd44 im7

The anti-mouse monoclonal antibodies Eomes (Dan11mag), PLZF (Mags.21F7), CD27 (LG.7F9), CD122 (TM-b1) and T-Bet (eBio4B10) were purchased from eBioscience; RORγt (Q31-378), CD8a (53-6.7), and CD44 (IM7) were from BD Pharmingen; B220 (RA3-6B2), Ly9 (Ly9.ab3) and CD4 (GK1.5) were purchased from BioLegend; CD3 (145-2C11) was from Tonbo Bioscience, and PBS57-loaded mCD1d tetramer was kindly provided by the NIH Tetramer Core Facility. Data were acquired with either FACSCanto II or LSRII Fortessa flow cytometers (BD Biosciences).

Bronchoalveolar lavage (BAL) was collected using HBSS + 30μM EDTA; the cellular component was isolated and resuspended in HBSS for enumeration. The superior/upper right lobe (URL) was harvested and processed into a single cell suspension, as previously described^{26 (link)}. Each BAL and URL sample represents a single adult animal; infant BAL samples (2-4 infant mice) were pooled to acquire sufficient cell numbers for flow cytometry. To maintain consistency with the BAL, the same infant URL samples were pooled prior to enumeration. Cells (0.5 – 1 × 10⁶) were surface stained with antibodies against TCR_β-H57-597, CD62L-MEL-14, and CD19-6D5 (Biolegend, San Diego, CA), CD4-GK1.5, CD8a-53-6.7, CD44-IM7 (BD Biosciences, San Jose, CA). Cells were fixed and permeabilized for transcription factor staining using the BD Pharmingen Transcription Factor Buffer Set, according to manufacturer recommendations (BD Biosciences). Intracellular staining of GATA3-16E10A23 and Tbet-4B10 (Biolegend) was performed following overnight incubation in BD Fix/Perm solution. Where indicated, BAL samples were incubated with RSV F-protein MHC I pentamer (H-2kd KYKNAVTEL, ProImmune, Sarasota, FL). Samples were run on a BD LSRFortessa (BD Biosciences) managed by the University of Pittsburgh United Flow Core and analyzed using FlowJo V10 software (FLOWJO, Ashland, OR).

Tetramer-enriched samples were stained for surface markers for 30 minutes on ice using antibodies to: CD4 (GK1.5, BD), CD8 (53–6.7, BD), CD90.1 (HIS51), CD90.2 (53–2.1, 30-H12), CD3e (145-2C11, BD), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7, BD), CD45.1 (A20, Biolegend), CD45.2 (104), B220 (RA3–6B2), and/or PD1 (J43). Cellular viability was confirmed using GhostDye Red 780 (Tonbo Biosciences). For transcription factor expression analysis, stained cells were fixed and permeablized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained overnight at 4°C with antibodies to T-bet (4B10, Biolegend), Foxp3 (FJK-16s), RORγt (Q31-378, BD), and Bcl-6 (K112-91, BD). For thymic dendritic cell and thymic epithelial cell analyses cells, following enrichment cells were stained with antibodies to CD11c (N418), CD19 (6D5, Biolegend), I-A/I-E (M5/114.15.2, Biolegend), CD8 (53–6.7, BD), CD90.2 (53–2.1, 30-H12), CD11b (M1/70), and CD45.2 (104, BD). Antibodies were purchased from eBioscience unless otherwise indicated. Cells were analyzed by flow cytometry on a Fortessa (BD). Data were analyzed using FlowJo software (TreeStar).