HDM-exposed NP-specific CD4 ⁺ T _RM maintain decreased T-bet expression after heterosubtypic infection and are associated with improved disease severity. (A) Schematic of the experimental timeline for data in B–H. (B) The total number of CD4⁺ cells per 20 μl peripheral blood of mice at experimental day 48 prior to FTY720 administration and on day 55 prior to sacrifice. Data are pooled from 17–23 mice per time point from three independent experiments. (C) Representative flow cytometry plots of cells isolated from the lung at experimental day 55. Box gate denotes the Thy1.2⁻ NP-specific CD4⁺ cells in E. (D) Summary data of the number of NP-specific CD4⁺ cells in the lung at experimental day 55 as represented in C. Data are pooled from 6–12 mice per group from three independent experiments. (E) Representative flow cytometry plots of NP-specific CD4⁺ T cells isolated from the lung at experimental day 55. (F) Summary data of the percentage of T-bet⁺ NP-specific cells, the geometric mean fluorescence intensity of T-bet in NP-specific cells, and the percentage of ST2⁺ NP-specific cells as represented in E. Data are pooled from 10–12 mice per group from three independent experiments. (G) Graph summarizing morbidity following X31 infection as a percent of starting (experimental day 50) weight. Data are pooled from 2–12 mice per group from five independent experiments. (H) Quantification of viral load in whole lung tissue at experimental day 55 as measured by qRT-PCR. The dotted line indicates limit of detection. Data are pooled from 2–12 mice per group from five independent experiments. *, P < 0.05; ^## or **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Graphs show mean ± SD. Data in B, D, F, and G were analyzed by unpaired t test. Data in H were analyzed by Mann–Whitney test. pfu, plaque-forming units. IAV, influenza A virus. o.p., oropharyngeal. gMFI, geometric mean fluorescence intensity.