Cross-presentation of viral antigens is impaired upon TLR3 or TLR4 activation. Immature DCs or DCs matured with different TLR ligands were cocultivated for 24 hours with CMV-infected (+CMV and black columns) or uninfected (-CMV and gray columns) irradiated HLA-A2–negative fibroblasts and were subsequently used as stimulators in ELIspot experiments. Autologous HLA-A2–restricted pp65-peptide–specific CTLs were used as effectors. (A) Immature DCs cocultivated with CMV-infected fibroblasts (fibrobl+CMV) take up cellular material and subsequently process and cross-present these antigens on MHC class I molecules, whereas DCs cocultivated with uninfected fibroblasts (fibrobl-CMV) do not. Moreover, direct cocultivation of DCs with supernatant derived from CMV infection of fibroblasts (DC+CMV-sup) did not result in the presentation of viral antigens and subsequent stimulation of CTLs. (B) Cross-presentation of viral antigens on MHC class I molecules is strongly impaired by TLR3- or TLR4-activated DCs. Using combinations of TLR ligands such as Poly I:C/LPS and Pam₃Cys/R848 did not further enhance the suppressive effect of the corresponding TLR3 or TLR4 single stimuli, respectively. The data shown are representative of at least 3 independent experiments.