NP _311–325 :I-A ^b –specific and Der p 1 _117–127 :I-A ^b –specific CD4 ⁺ T cells are not crossreactive or activated by heterologous responses. (A) In mice administered respiratory HDM (top) or PR8 (bottom) as illustrated in the experimental timeline shown, lung-draining mediastinal lymph nodes and lung tissues were isolated and stained with Der p 1_117–127:I-A^b and NP_311–325:I-A^b tetramers and analyzed by flow cytometry. Representative flow plots are shown. Lung samples were pregated on cells that are negative for the intravenous Thy1.2 label to demarcate cells located in the lung parenchyma. (B) Representative flow cytometry plots of CD4⁺ T cells isolated from the mediastinal lymph node or lung 43 d after infection with PR8 (top) or following PR8 and respiratory HDM (bottom) as illustrated in the experimental timeline shown. Lung samples were pregated on cells negative for the intravenous Thy1.2 label to demarcate cells located in the lung parenchyma. (C) Summary data of the total number of NP-specific or Der p 1–specific tetramer-positive cells in the mediastinal lymph node or lungs of mice from each condition as represented in B. Data are pooled from three to six mice per group from three independent experiments and were analyzed by unpaired t test. Graphs show mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. IAV, influenza A virus. o.p., oropharyngeal. pfu, plaque-forming units.