Fig 2: Antibody titration and validationAll antibodies were titrated separately; here are a few examples to illustrate. We started with the manufacturer recommended dilution and made at least 6-point 1:2 dilution series plus unstained control.(A) WC1-IgG2a-FITC: recommended dilution 1:10 corresponds to 1 μg/mL.(B) CD45RO-IgG3-PE, the manufacturer does not provide the concentration corresponding to the recommended dilution 1:10.(C) CD25-IgG3: recommended dilution 1:100 corresponds to 10 μg/mL.(D) CD8-IgG1: recommended dilution 1:40 corresponds to 25 μg/mL.(E) IgG2a-APC-Cy7: recommended dilution 1:250 corresponds to 1 μg/mL.(F) IgG1-PE-Cy7: recommended dilution 1:40 corresponds to 5 μg/mL. Highlighted are the working dilutions that were defined for further use in the study.

Fig 3: FCM gating strategy for components of the pulmonary T cell compartment(A–C) Example of gating strategy for multiparameter FCM analysis to identify ovine γδ T cells (A), CD4+ and CD8+ T cells (B), and Tregs (C) among cell suspension isolated from BAL, lung tissue and LN cell suspensions. The neonatal lambs were infected with RSV A2 and euthanized at day 3 post-infection. (A) Phenotype of ovine γδ T cells. Gating strategy following multiparameter FCM staining uses Abs against CD4, CD8, CD25, WC1, IFN-γ and IL-17. Cells showing low forward scatter and side scatter profiles were gated. Then, the dead cell population (positive for Live/Dead) was excluded, followed by a FSC-H/FSC-A contour plot (to exclude doublets from the analysis). Then, we defined among this population the cells negative for both CD4 and CD8 markers. Among these cells, γδ T cells were defined as the WC1+ cells. Maturation was evaluated based on CD25 expression, whereas activation was evaluated on the basis of IFN-γ and/or IL-17 production. (B) Phenotype of ovine T cells. Gating strategy following multicolor FCM staining uses Abs against CD4, CD8, CD25, CD45RO, IFN-γ and IL-4. Cells showing low forward scatter and side scatter profiles were gated. Then, the dead cell population (positive for Live/Dead) was excluded, followed by a FSC-H/FSC-A contour plot (to exclude doublets from the analysis). Then, we defined among this population either the cells positive for CD4 and negative for CD8 markers (CD4+ T cells), either cells positive for CD8 and negative for CD4 markers (CD8+ T cells). Herein, example is given for the CD4+ T cell subset, where maturation was evaluated based on CD25 expression, whereas activation was evaluated based on IL-4 or IFN-γ production. (C) Phenotype of ovine Tregs. Gating strategy following multicolor FCM staining uses Abs against CD4, CD25, FoxP3 and TGF-β. Cells showing low forward scatter and side scatter profiles were gated. Then, the dead cell population (positive for Live/Dead) was excluded, followed by a FSC-H/FSC-A contour plot (to exclude doublets from the analysis). Next, we defined among this population the cells positive for CD4 marker (CD4+ T cells). Among this cell subset, Tregs were defined as the FoxP3+CD25+ cells. Activation was evaluated based on TGF-β production.