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BACKGROUND. Respiratory syncytial virus (RSV) is an important cause of acute pulmonary disease and one of the last remaining major infections of childhood for which there is no vaccine. [CD4.sup.+] T cells play a key role in antiviral immunity, but they have been little studied in the human lung. METHODS. Healthy adult volunteers were inoculated i.n. with RSV A Memphis 37. [CD4.sup.+] T cells in blood and the lower airway were analyzed by flow cytometry and immunohistochemistry. Bronchial soluble mediators were measured using quantitative PCR and MesoScale Discovery. Epitope mapping was performed by IFN-[gamma] ELISpot screening, confirmed by in vitro MHC binding. RESULTS. Activated [CD4.sup.+] T cell frequencies in bronchoalveolar lavage correlated strongly with local C-X-C motif chemokine 10 levels. Thirty-nine epitopes were identified, predominantly toward the 3' end of the viral genome. Five novel MHC II tetramers were made using an immunodominant EFYQSTCSAVSKGYL (F-EFY) epitope restricted to HLA-DR4, -DR9, and -DR11 (combined allelic frequency: 15% in Europeans) and G-DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labeling revealed enrichment of resident memory [CD4.sup.+] T (Trm) cells in the lower airway; these Trm cells displayed progressive differentiation, downregulation of costimulatory molecules, and elevated CXCR3 expression as infection evolved. CONCLUSIONS. Human infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of Trm recognizing novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific [CD4.sup.+] cells, potentially accelerating the development of effective vaccines. TRIAL REGISTRATION. ClinicalTrials.gov NCT02755948. FUNDING. Medical Research Council, Wellcome Trust, National Institute for Health Research., Introduction T cells are a critical component of long-term adaptive immunity and have been shown to be essential for clearance of intracellular pathogens (1, 2), the development of high-affinity antibodies [...]