TLR agonists synergize with CD40L/IFN-γ to promote human dendritic cell synthesis of IL-12 (135.65)

Abstract CD40L provides a critical signal to license DC for the effective generation of T cell memory. To investigate additional signals that might synergize with CD40L, we tested TLR agonists to promote IL-12 production by DC matured with the combination of CD40L/IFN-γ. Human monocyte-derived immature DC established by standard 6 day culture in GM-CSF/IL-4 were activated with human CD40L/IFN-γ plus optimal concentrations of individual TLR agonists for an additional 48 h and the supernatants were assayed for IL-12 p70 by ELISA. TLR 1/2, 4, 5, 6, and 8 agonists each resulted in significant increases in IL-12 production as compared to CD40L/IFN-γ alone with mean fold increases of 2.3, 4.0, 2.1, 3.5, and 8.8 respectively. TLR agonists 3, 7, and 9 had no effect. To further assess the potential synergy of TLR agonists with CD40L/IFN-γ mDC, we studied the capacity of CMV pp65 NLV peptide-pulsed mDC to stimulate antigen-specific CD8+ T cells from normal CMV sero-positive donors. The antigen-specific CD8 response as measured by tetramer-fold increase was significantly higher at day 10 using TLR/CD40L/IFN-γ activated mDC compared to conventional CD40L/IFN-γ mDC. We conclude that multiple maturation signals are required to license DC for the optimal generation of T cell memory.