Background:Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan. Methods:We performed 51Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues. Results:We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8+ T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues. Conclusion:Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.