Identification and mutation of the CD40L cleavage site leads to retention of ligand at the cell membrane. (A) Amino acid alignment of FasL and CD40L. Alignment identified a region in the extracellular domain of CD40L (EMQK) with significant similarity to the cleavage site of FasL (EKQI). CD40L mutant (ncCD40L) is not cleaved into soluble CD40L. EJ cells were infected with 100 MOI of either RAdMock (RAdM) or RAdCD40L (RAdL) or RAdncCD40L (RAdncL) or left without infection. (B) Samples were collected from the culture media 24 and 72 hours post-infection for sCD40L ELISA assay. Results represent a mean of triplet samples +/- SD. Two-tailed t-test RAdL vs. RAdncL: 24 h, p = 0.0067; 72 h, p < 0.0001. (C) CD40L expression was examined by western blot 72 hours after infection. β-actin was used as a loading control. (D) Adenovirus-delivered CD40L is expressed on the cell membrane. EJ cells were infected with 100 MOI of either RAdCD40L (wild-type) or RAdncCD40L (mutated cleavage resistant) for 28 hours. Harvested cells were incubated with mouse anti-CD40L-APC conjugated antibody or isotype control and then analysed by flow cytometry.