(A) Cells recovered from a suction blister induced over a MT site 21 days following the induction were stimulated in vitro with PPD every 14 days (indicated by arrows). Peripheral blood CD4⁺ T cells from the same donor were cultured in parallel. (B) Dot plot shows IFN-γ production by the cell line in response to PPD stimulation. The percentage of cells expressing IFN-γ is indicated. (C) PPD-specific CD4⁺ T cell line was stimulated with immobilized anti-CD3 mAb. The cells were then washed and stimulated with PPD and antigen-presenting cells for 3 days, and proliferation was measured by [³H]-thymidine incorporation. The mean of triplicate wells ± SEM is shown (representative of 3 separate experiments). PPD-specific CD4⁺ T cells that were not anergized were used as control. (D) Anergized PPD-specific cells described in C were mixed with an equal number of autologous non-anergized PPD-specific cells and stimulated with PPD and autologous antigen-presenting cells. Proliferation was measured on day 3 and is expressed as mean ± SEM. Responders, non-anergized PPD cell lines cultured alone; control, responders with non-anergized PPD-specific cells. Results are representative of 2 separate experiments. (E) Foxp3 expression was measured by real-time quantitative RT-PCR using 18s rRNA as an internal control. Relative quantity values are plotted in a log-scale bar chart using the triplicate values to estimate SD. Cells recovered from the blister are indicated as “blister cells ex vivo.” Blister cells expanded in vitro prior to anergy induction were used as a control.