Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T-cells recognising peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection.
To investigate the kinetics, phenotypes and function of influenza virus-specific CD8+ resident-memory T-cells (Trm) in the lower airway and infer the molecular pathways associated with their response to infection in vivo.
Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A(H1N1)2009. Blood, upper and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed using self-reported diaries and nasal viral load by qPCR. T-cell responses were analysed by three-colour FluoroSpot, flow cytometry with MHC I-peptide tetramers and RNAseq, with candidate markers confirmed using immunohistochemistry of endobronchial biopsies.
Following challenge, 57% of participants became infected. Pre-existing influenza-specific CD8+ T-cells in blood correlated strongly with reduced viral load, which peaked at day 3. Influenza-specific CD8+ T-cells in BAL were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between pre-infection CD8+ T-cells in BAL and blood by RNAseq revealed 3928 differentially expressed genes, including all major Trm cell markers. However, gene-set enrichment analysis of BAL CD8+ T-cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (Cxcl1, Cxcl10 and Cxcl16) that were also expressed by CD8+ cells in bronchial tissues.
CD8+ Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical trial registration available at www.clinicaltrials.gov, ID: NCT02755948.

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