Tcrβ h57 597

Splenocytes were isolated as previously described(23 ). PBS-perfused kidneys and lungs were minced and digested with collagenase IV (100 μg/ml) for 30 minutes at 37 °C to prepare single cells.
Isolated cells were stained with the following antibodies specific for: TCRβ (H57-597, BioLegend), CD3ε (145-2C11, eBiosciences), CD4(GK1.5, BioLegend), CD8α (53-6.7, eBiosciences), B220 (RA3-6B2, BioLegend), CD25(PC61, BioLegend), I-A/I-E (M5/114.15.2, BioLegend), Nkp46 (29A1.4, BioLegend) for 30min at 4 °C. For intracellular staining, cells were stimulated with 20 ng/ml of phorbol-myristate acetate (PMA, Sigma) and 1 mg/ml of ionomycin(Sigma) for 4 hours, washed and stained with TCRβ, CD4, CD8, I-A/I-E. BD cytofix/cytoperm plus with Golgi stop staining kits (BD Biosciences) were used according to the manufacturer’s protocol. Antibodies of IFN-γ (XMG1.2, BioLegend) and IL-17A (TC11-18H10.1, BioLegend) were used for detecting intracellular cytokines.

Fluorochromes used for surface staining included PE, APC, Alexa Fluor 647, PeCy7, PerCP, and eFluor450. Conjugated anti-mouse CD4 (GK1.5), CD8 (53-6.7), CD25 (PC61, eBio 3C7), CD44 ((IM7), c-Kit (2B8), CD28 ((E18), CD5 (53-7.3), γδTCR (GL3) and TCRβ (H57-597) antibodies were from Biolegend (San Diego, CA, USA) eBioscience (San Diego, CA, USA) and BD-Pharmingen (San Diego, CA, USA). Lineage depletion for CD25/44 staining was accomplished by staining cells with a cocktail of biotinylated mAb (anti-mouse CD3, CD4, CD8, B220, Mac-1, Gr-1, Ter119 and NK1.1; (Biolegend, eBioscience and BD-Pharmingen) and gating out Streptavidin (SA-) PeCy7 or SA-Alexa Fluor 594 positive cells (Invitrogen, Carlsbad, CA, USA).

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).