Flow Cytometric Analysis of Virus-Specific CD4 T Cells

As previously described [80 (link)], mice were injected intravenously with APC-CD45 antibody prior to sacrifice to mark CD4 T cells in the lung vasculature. Briefly, after labeling cells in vivo for 3 min with CD45-APC, lungs were harvested and processed into single-cell suspensions. A cocktail of antibodies, titrated to optimal concentration, was used to resolve surface markers on CD4 T cells and included CD19 (1D3, BD Horizon), CD4 (RAM4-5, BD Pharmigen), CD8a (53-6.7, Biolegend), TCRβ (H57-597, Biolegend), CD44 (IM7, Tonbo), CD62L (MEL-14, Biolegend), CD11a (2D7, BD Biosciences), CD69 (H1.2F3, Biolegend), and CD103 (2E7, Biolegend). For tetramer staining, PE-conjugated tetramer with the peptide indicated in the figure legends with the above-mentioned CD4 T cells markers were used to stain virus-specific CD4 T cells. Prior to staining, tetramer-specific CD4 T cells were enriched using negative MACS enrichment [62 (link)]. Data were acquired using a BD LSR-II instrument, configured with 488 (blue), 633 (red), 407 (violet), and 532 (green) nm lasers, as previously described [26 (link)]. Data were analyzed using Flowjo software (Flowjo, LLC.), version 10.