CD40L^G219R protein expression. (A) CD40L expression on Con A–expanded T-cell blasts from a healthy control (CD40L) and an individual expressing the CD40L^G219R mutant (CD40L^G219R). Cells were stimulated or not (No stim.) with phorbol 12-myristate 13-acetate and ionomycin (PMA/Iono) for 6 hours and analyzed by flow cytometry with anti-CD40L TRAP-1 or anti-CD40L 24.31 antibodies. Gray histograms correspond to staining with control immunoglobulin. (B) CD40L expression on wild-type 3T3 cells (3T3) and 3T3 expressing green fluorescent protein (GFP)-CD40L (CD40L) or GFP-CD40L^G219R (CD40L^G219R). After infection with retroviral plasmids that contained CD40L^G219R or wild-type CD40L with a GFP reporter gene, cells were analyzed by flow cytometry for their GFP content (GFP) or for their expression of CD40L with TRAP-1 or 24.31 mAbs. Shaded histograms as in (A). (C) CD40L expression on ConA–, PHA-expanded T-cell blasts and 3T3 cells expressing CD40L or CD40L^G219R was detected with CD40-Ig (histograms on right). Histograms on left correspond to staining with control immunoglobulin. The profile of cells expressing CD40L^G219R (blue line) was overlaid on the profile of cells expressing CD40L (red line). Max indicates maximum.