Expression data from NP366-374/Db and PA224-233/Db CD103 positive and negative T-cells

To investigate the role of TCR-pMHC interaction in regulating lung tissue-resident CD8 T cell differentiation, polyclonal responses were compared against NP366-374/Db and PA224-233/Db, two immunodominant epitopes that arise during influenza A infection. To characterize the molecular programs associated with the lung tissue-resident CD8 T cell subpopulations, we measured by microarray the global gene transcriptional profile of NP366-374/Db CD103+ and NP366-374/Db CD103-, PA224-233/Db CD103+, PA224-233/Db CD103- lung resident T cells. For each microarray dataset, lung tissue-resident CD8 T cells from 9 female B6 mice were sorted and pooled. Previously, influenza A/PR8/8/34 (H1N1)-infected mice were re-challenged with influenza A/HK/X31 (H3N2) virus 35 days after primary infection. Five days post re-challenge, lungs were harvested after in vivo CD8alpha staining was performed. After cell suspension from the collagen-digested lungs was prepared, non-CD8 T cells were removed using mouse CD8+ T Cell Isolation Kit (Miltenyi Biotec, Inc.). The untouched CD8 T cell-enriched fraction was stained with H-2Db Influenza NP366-374 Tetramer, H-2Db Influenza PA224-233 Tetramer, followed by additional staining with anti-CD8beta and anti-CD103 Abs. Then 4-way sorting was performed using a FACsAria IIu (BD Biosciences) to obtain NP366-374/Db CD103+, NP366-374/Db CD103-, PA224-233/Db CD103+ and PA224-233/Db CD103- CD8 T cells.