HLA-DP2–PLXNA4/Be tetramer staining of ex vivo BAL cells from CBD patients. (A) The binding kinetics of soluble AV22 TCR to biotinylated HLA-DP2–PLXNA4 with and without Be are shown. Four concentrations of soluble AV22 TCR were injected through the flow cells before and after loading with 200 µM BeSO₄, and the surface plasmon resonance signal was obtained. Data shown are representative of three independent experiments. (B) Staining of the AV22 T cell hybridoma with HLA-DP2–mimotope-2, HLA-DP2–mimotope-2/Be, and HLA-DP2–PLXNA4/Be tetramers individually (top) and costaining with Be-saturated mimotope-2 (BV421 labeled) and PLXNA4 (PE labeled) tetramers (bottom). The fluorescence intensity of cells was evaluated after staining with 20 µg/ml of tetramers for 2 h at 37°C. Representative results from three independent experiments are shown. (C) Density plots showing control HLA-DP2–mimotope-2 without Be (left) and HLA-DP2–PLXNA4/Be (right) tetramer staining of CD4⁺ T cells from ex vivo BAL cells of a HLA-DP2⁺ CBD patient. Results are representative of four HLA-DP2⁺ CBD subjects stained. (D) Summary of the frequency of tetramer staining of ex vivo BAL CD4⁺ T cells from four HLA-DP2⁺ CBD patients individually stained with the HLA-DP2–mimotope-2/Be and HLA-DP2–PLXNA4/Be tetramers. (E) Density plots of ex vivo BAL cells from four DP2⁺ CBD patients costained with HLA-DP2–PLXNA4/Be and HLA-DP2–mimotope-2/Be tetramers are shown.