Transcriptional responses of antigen-specific CD8 T cells after secondary influenza virus challenge

To evaluate the transcriptional responses of effector and memory virus-specific CD8 T cell subsets after primary and secondary influenza infection, we sorted subsets of FluNP 366-374/Db+ CD8+ T cells from the BAL, lung, and spleen at the peak of primary x31 influenza infection (day 10), resting memory (day 30), or 3 days after secondary PR8 influenza challenge (Day 30+3). Cells from the circulation were excluded from BAL and lung based on intravital labeling with anti-CD45. Memory cells from the lung were further separated in TRM and TEM subsets based on expression of CD69 at the day 30 and day 30+3 timepoints. Memory cells from the BAL were separated into long-term airway resident versus recently-recruited cells based on CD11a expression at the day 30 and days 30+3 timepoints. The results show that virus-specific BAL and lung TRM cells, but not lung TEM cells or spleen TEM cells, rapidly alter their transcriptional program and increase expression of effector molecules within 3 days of a secondary influenza challenge. Transcriptional profiling of FluNP 366-374/Db-specific CD8 T cells at the peak of the initial effector T cell response (day 10), resting memory (day 30), or after secondary heterosubtypic influenza challenge (day 30+3). Five minutes prior to euthanasia, mice were administered anti-CD45-PECF594 intravenously to label cells in the vasculature. Cells were isolated from the BAL, lung, and slpeen and sorted on the markers listed below. 5 mice were pooled for each timepoint to obtain sufficient cells for RNA sequencing analysis and there were 3 independent replicates.