Monocyte cell contact increases CD40L mRNA in CD4⁺ T cells. Purified CD4⁺ T cells were cultured alone or with U937 cells and stimulated for 6 hours as indicated at the bottom of the figure. (A) Flow cytometric analysis of CD40L surface expression. (B) RNA was isolated from an aliquot of the same cells shown in panel A. Equal amounts of total RNA from each sample were loaded per lane; and after electrophoresis, the gel was stained for 28S and 18S rRNA (top panel). The gel image was acquired on a Biorad Fluor-S MultiImager with Quantify One Version 4.2 software. Northern blot of the same gel probed for CD40L mRNA (bottom panel). The position of CD40L mRNA is indicated. (C) Results of quantitative RT-PCR for CD40L mRNA performed on an aliquot of the same RNAs used in the Northern blot. Data are mean ± SE for 3 replicates of each sample, expressed as the number of CD40L mRNA copies per nanogram of rRNA. Representative results from one of 4 donors are shown. (D) PBMCs were plated in the presence (Act D) or absence (Control) of a transcriptional inhibitor and stimulated with anti-TCR mAb. Control samples were harvested and stained for CD40L at the indicated times (solid line). Act D was added to the PBMC cultures at 0, 1, 2, or 3 hours, and the Act D–treated samples were harvested and stained for CD40L at 6 hours (hatched columns). CD40L expression is plotted relative to the 6-hour control (100%). Representative results from one of 4 donors are shown.