Mechanisms of CD4⁺ and CD8⁺ T-cell deletion by imDCs. TEA CD4⁺ (A-B) or 2C CD8⁺ (C-D) cells stained with calcein AM green were incubated for 5 hours in the absence (white) or presence of imDCs from CB6/F1 (H-2^bd, black), irrelevant FVB (H-2q, gray) donors, or with mDCs from CB6/F1 at the indicated ratios. At the end of the culture, the total number of stained TEA CD4⁺ T cells or 2C CD8⁺ cells was evaluated by FACS. The activity of imDCs from different donors preincubated with the NO inhibitor L-NAME (100 ng/mL) for 20 minutes at 37°C, on TEA CD4⁺ (B) or 2C CD8⁺ (D) cells was assessed by live-cell determination as described in panels A and C. The effect of various imDC pretreatments (E-F) and the effect of supernatants (Sup) from culture with cognate (black) or noncognate (gray) imDCs on 2C CD8⁺ cell deletion by imDCs. The results shown represent the average ± SD (n ≥ 3). *P < .01. NS indicates not significant.