Figure 4. HLA-DP2–mimotope-2/Be tetramer staining of Be-specific T cell lines and ex vivo BAL cells from CBD patients. (A) Approximately 2,000 resonance units (RU) of biotinylated HLA-DP2–mimotope-2 and control HLA-DR52c–WIR complex were immobilized in flow cells of a BIAcore streptavidin biosensor chip. Various 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 T cell hybridomas (Be-specific AV22, insulin B:9-23–specific 8-1.1, and dengue virus–specific DV-13) with HLA-DP2–mimotope-2 (DP2-Mim2) and IAg7-insulin (IAg7-Ins) tetramers prepared into complexes either in the presence or absence of BeSO4. Cells were stained with 20 µg/ml of tetramers, and fluorescence intensity was evaluated by flow cytometry. Representative results from three independent experiments are shown. All hybridomas expressed high levels of TCR (not depicted). (C) Detection of HLA-DP2–mimotope-2/Be tetramer–binding T cells from parental T cell line derived from CBD patient 1332. BAL T cells after one and two cycles of stimulation with BeSO4 and after Vβ5.1 sorting were stained with the HLA-DP2–mimotope-2/Be (top) and IAg7-insulin (bottom) tetramers, and the frequency of CD4+ T cells stained with each tetramer is shown in each density plot. Representative staining results from two independent experiments are shown. (D) HLA-DP2–mimotope-2/Be tetramer staining of ex vivo BAL cells from CBD patients who are HLA-DP2+ (8722) and HLA-DP2− (1089). Density plots show tetramer staining of CBD patients, excluding cells staining with CD8, CD14, and CD19, and gating for CD3 and CD4 expression. Results are representative of ex vivo BAL cells from eight HLA-DP2+ and four HLA-DP2− CBD subjects. (E) Frequency (mean) of CD4+ T cells staining with the HLA-DP2–mimotope-2/Be tetramer is shown for ex vivo BAL cells (n = 12) and BAL T cell lines (n = 7) derived from DP2+ and DP2− CBD patients. Frequency was determined by subtracting nonspecific staining observed with the control tetramer from staining with the HLA-DP2 tetramer. (F) Flow cytometric analysis of dual intracellular cytokine and tetramer staining of ex vivo BAL cells from a DP2+ CBD patient is shown. Density plots show IFN-γ (left) and IL-2 (right) expression relative to HLA-DP2–mimotope-2/Be tetramer staining. Data are representative of the eight HLA-DP2+ CBD subjects studied. (G and H) For ex vivo BAL cells, the mean frequency of IFN-γ– and IL-2–expressing CD4+ T cells that bind the HLA-DP2–mimotope-2/Be tetramer (G) and the frequency of tetramer-positive cells that secrete these cytokines (H) are shown. (E, G, and H) Mean values for each group are indicated with horizontal bars.
Figure 4.

HLA-DP2–mimotope-2/Be tetramer staining of Be-specific T cell lines and ex vivo BAL cells from CBD patients. (A) Approximately 2,000 resonance units (RU) of biotinylated HLA-DP2–mimotope-2 and control HLA-DR52c–WIR complex were immobilized in flow cells of a BIAcore streptavidin biosensor chip. Various 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 T cell hybridomas (Be-specific AV22, insulin B:9-23–specific 8-1.1, and dengue virus–specific DV-13) with HLA-DP2–mimotope-2 (DP2-Mim2) and IAg7-insulin (IAg7-Ins) tetramers prepared into complexes either in the presence or absence of BeSO4. Cells were stained with 20 µg/ml of tetramers, and fluorescence intensity was evaluated by flow cytometry. Representative results from three independent experiments are shown. All hybridomas expressed high levels of TCR (not depicted). (C) Detection of HLA-DP2–mimotope-2/Be tetramer–binding T cells from parental T cell line derived from CBD patient 1332. BAL T cells after one and two cycles of stimulation with BeSO4 and after Vβ5.1 sorting were stained with the HLA-DP2–mimotope-2/Be (top) and IAg7-insulin (bottom) tetramers, and the frequency of CD4+ T cells stained with each tetramer is shown in each density plot. Representative staining results from two independent experiments are shown. (D) HLA-DP2–mimotope-2/Be tetramer staining of ex vivo BAL cells from CBD patients who are HLA-DP2+ (8722) and HLA-DP2− (1089). Density plots show tetramer staining of CBD patients, excluding cells staining with CD8, CD14, and CD19, and gating for CD3 and CD4 expression. Results are representative of ex vivo BAL cells from eight HLA-DP2+ and four HLA-DP2− CBD subjects. (E) Frequency (mean) of CD4+ T cells staining with the HLA-DP2–mimotope-2/Be tetramer is shown for ex vivo BAL cells (n = 12) and BAL T cell lines (n = 7) derived from DP2+ and DP2− CBD patients. Frequency was determined by subtracting nonspecific staining observed with the control tetramer from staining with the HLA-DP2 tetramer. (F) Flow cytometric analysis of dual intracellular cytokine and tetramer staining of ex vivo BAL cells from a DP2+ CBD patient is shown. Density plots show IFN-γ (left) and IL-2 (right) expression relative to HLA-DP2–mimotope-2/Be tetramer staining. Data are representative of the eight HLA-DP2+ CBD subjects studied. (G and H) For ex vivo BAL cells, the mean frequency of IFN-γ– and IL-2–expressing CD4+ T cells that bind the HLA-DP2–mimotope-2/Be tetramer (G) and the frequency of tetramer-positive cells that secrete these cytokines (H) are shown. (E, G, and H) Mean values for each group are indicated with horizontal bars.

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