Transcriptome profiling of influenza-specific memory CD8 T cells following transfer into the airways or in vitro culture

To examine differences in the transcriptome of resident memory CD8 T cells in the lung, influenza nucleoprotein-specific CD8 T cells from the airways , lung interstitium, lung vasculature, and spleen were FACS sorted and RNA isolated for RNA-seq. In some experiments, FACS sorted cells were transferred into the airways, peritoneum, or in vitro culture before subsequent isolation and RNA isolation. C57Bl/6 mice were infected with influenza x31 and rested for 35 days to establish T cell memory. Five minutes prior to euthanasia mice were injected with anti-CD45.2-PE to discriminate between tissue resident and circulating cells, and cells were isolated from the airways by bronchoalveolar lavage, from the lung parechyma by enzymatic digestion, and from the spleen by mechanical disruption. Cells were sorted on CD8+ CD44Hi FluNP 366-374 Db tetramer+ populations, and cells from the airways and parenchyma were further sorted on intravital label negative (i.e. tissue resident) cells. Lung vasculature cells were lung cells that stained positive for the intravital label. This cohort was used as a replicate cohort. In some experiments, FACS sorted cells from the airways or spleen were transferred into the airways or peritoneum of congenic (CD45.1+) mice, or placed in an in vitro culture, before subsequent isolation and RNA isolation 48 hours post-transfer.