TCR-mediated activation of L-DP CD69^hi thymocyte-expressing CD40L transcripts leads to CD40L up-regulation and activation of thymic pDCs. (A) Reverse-transcribed polymerase chain reaction analysis of CD40L mRNA expression was performed using 2-fold serial dilutions of cDNA from primary S-DP CD69^lo, L-DP CD69^hi, and L-DP CD69^lo thymocyte populations isolated from the same thymus sample (top panel). cDNA obtained from phorbol myristate acetate plus ionomycin-stimulated PB CD4⁺ T lymphocytes was used as a positive control. (B) Flow cytometry analysis of CD69 versus CD40L expression on primary L-DP CD69^hi thymocytes before culture (left panel), after activation with anti-CD3 and anti-CD28 mAbs plus IL-2 for 24 hours (middle panel), or after culture in medium alone (right panel). NS indicates nonstimulated. Numbers in quadrants indicate percentages of positive cells. (C) Relative numbers of CD86-expressing pDCs after 24 hours of culture with IL-3 in the absence or presence of L-DP CD69^hi thymocytes either stimulated (DPCD40L⁺) or nonstimulated (NS) as in panel A. Data are mean ± SEM (n = 3). *P < .05. **P < .01.