Expression and immunostimulatory function of OX40L on monocyte-derived DCs. (A, top row) OX40L expression was determined by fluorescence-activated cell sorting (FACS) on immature DCs (gray histograms) or 24 hours after maturation with cytokine cocktail/PGE₂ (iDCs ± CC/PGE₂), with 3 μg/mL soluble trimeric CD40 ligand (iDCs ± sCD40L), or with cytokine cocktail plus sCD40L (iDCs ± CC/PGE₂/sCD40L). (A, bottom row) OX40L expression was determined on immature DCs (gray histograms), immature DCs transfected with prostate-specific antigen (PSA) control mRNA (iDCs ± PSA RNA), cocktail-matured and control RNA–transfected DCs (iDCs ± CC/PGE₂/PSA RNA), or OX40L-transfected and cocktail-matured DCs (iDCs ± CC/PGE₂/OX40L mRNA). (B) Allogeneic mixed lymphocyte reactions (MLRs). Allogeneic CD4⁺ T cells were stimulated with immature DCs (iDC) or cocktail-matured DCs (iDC + CC/PGE₂) at the indicated responder-to-stimulator ratios. DCs were transfected with PSA mRNA (□), green fluorescent protein (GFP) mRNA (▴), or OX40L mRNA (▪). Proliferation was assessed by incorporation of tritiated thymidine. Results are presented as the mean stimulatory index with standard deviation (SD) calculated from triplicate wells. (C) DCs were transfected with increasing amounts of OX40L mRNA, matured with cytokine cocktail, and used as stimulators in an allogeneic MLR at a stimulator-to-responder ratio of 1: 10. (D) Allogeneic MLR in the presence of increasing concentrations of OX40 agonist (recombinant human OX40L protein). Allogeneic CD4⁺ T cells were incubated with OX40L RNA–transfected and cocktail-matured DCs (+ OX40L mRNA) or control (PSA) RNA–transfected and cocktail-matured DCs (–OX40L mRNA) at a stimulator-to-responder ratio of 1: 10. Increasing amounts of OX40L protein (range, 0.01-0.5 μg/mL) plus anti-FLAG antibody (1 μg/mL) were added to reactions. No stimulation of CD4⁺ T cells could be observed in the absence of DCs (Control). (E) Allogeneic MLR in the presence of OX40L-neutralizing antibody. Immature or cocktail-matured DCs were transfected with control (PSA) mRNA or OX40L mRNA and used as stimulators for allogeneic CD4⁺ T cells (stimulator-to-responder ratio, 1:10). Anti-OX40L antibody (5 μg/mL) was added as indicated. As a control, an anti-CD8 antibody (5 μg/mL) was used in these assays. Results are presented as the mean stimulatory index with SD calculated from triplicate wells.