FIG. 4.

Characterization of the BRI4PA clones by tetramer staining and proliferation to PA 112-127. (A) BRI4PA clones were generated by single-cell sorting the tetramer-positive PA 112-127 population, followed by 12 to 14 days of mitogen stimulation and 12 to 14 days of antigen-specific stimulation. The clones were tetramer stained with DRB1*0404 mortalin10A, as a negative control, and the DRB1*0404 PA112-127 specific tetramer; subsequently, samples were stained with anti-CD4 monoclonal antibody and analyzed by flow cytometry. (B) BRI4PA clones were exposed to PA 112-127 in the presence of human DRB1*1302, DRB1*0404, and DRB1*0407 antigen-presenting cells (APC); [³H]thymidine incorporation after 72 h is shown. (C) BRI4PA.18 cells were labeled with CFSE prior to specific stimulation with 10 μg/ml PA 112-127 and irradiated DRB1*0407 PBMC. Six days after stimulation, the sample was tetramer stained with the same tetramers as described for panel A. The percentages reported for flow cytometry are based on all cells in the live lymphocyte gate. On the histogram plot, the black line is the CFSE profile of the culture containing PA 112-127, and the gray line is the profile of a culture without peptide.