Effect of anti-CD40 on the capacity of mature DCs to generate a specific memory T-cell response. (A-B) Purified CD4⁺ T cells were cocultured for 5 days with tetanus toxoid and tuberculin-loaded autologous immature (A) or LPS-matured (B) DCs (see “Materials and methods”), in the presence of anti-CD40 mAb or an isotype control. Proliferation was assayed by measuring thymidine incorporation. Specific proliferation is expressed as Δ cpm after subtracting T-cell proliferation in cocultures without antigen. The results are the means ± SEM of 3 independent experiments. (C) Immature DCs were activated for 24 hours with 5 μg/mL of the indicated antibody and stained with anti-CD1a-PECy5 and anti-CD83-FITC before flow cytometry. Numbers in the quadrants indicate the percentages of double-positive cells. These results are representative of 5 independent experiments. (D) Supernatants of immature DCs activated for 24 hours with 5 μg/mL anti-CD40 were tested by enzyme-linked immunosorbent assay (ELISA) for their IL-12p40 content. The results are the means ± SEM of 2 independent experiments. (E) Antigen-loaded DC/CD4 T cells were cocultured in the presence of anti-CD40 mAb or an isotype control as in panels A and B. Six hours after the beginning of coculture, cells were stained with anti-CD40-PE, anti-CD3-PECy5, and annexin V FITC. For DC apoptosis analysis, cells were gated according to their forward/side scatter (FS/SS) properties, and annexin V staining was analyzed in the CD40⁺CD3^- population. For T-cell apoptosis, annexin V staining was analyzed in the CD3⁺CD40^- population. Results are expressed as Δ apoptosis = (percentage of annexin V⁺ cells in coculture with anti-CD40) - (percentage of annexin V⁺ in coculture with the isotype control).