The role of NFκB in the proliferation of CD4⁺CD80^{acq hi} T cells. (A) Naive CD4⁺ T cells were cocultured with PCC-pulsed P13.9 fibroblasts for 20 hours. After the separation from P13.9 APCs, NFκB activity of nuclear extracts from either naive CD4⁺ or CD4⁺CD80^{acq hi} T cells was investigated using EMSA. CD4⁺CD80^{acq hi} T cells were separated from PCC-pulsed P13.9 fibroblasts (0 hour) and further cultured at various time points in the absence of APCs and PCC peptide, and NFκB activity in these cells was examined. (B) CD4⁺CD80^{acq hi} T cells were cultured in the absence or presence of PCC peptide (1 μg/mL) without APCs, and NFκB activity in these cells was measured at 6 or 24 hours. (C) NFκB binding band in the cells mentioned in the discussion of Figure 4A was analyzed using competitive and supershift assays. In competitive assay (left), 50-fold of cold NFκB and AP1 oligmers were used to compete with labeled NFκB probe. In the supershift assay (right), a set of anti-NFκB antibodies was applied to detect NFκB subunits. (D) CD4⁺CD80^{acq hi} T cells were treated with 0.2 μM PDTC (an NFκB inhibitor) in the absence of APCs and PCC peptide for 6 hours. The effect of PDTC on 3H uptake of CD4⁺CD80^{acq hi} T cells was assessed. Data are expressed as average percentage inhibition of proliferation. This result is the average of 4 experiments. The CD4⁺CD80^{acq hi} T cells exposed to PDTC were further stained with propidium iodide. Viability of the CD4⁺CD80^{acq hi} T cells was determined by FACS analysis (data not shown). Error bars represent mean ± SD.