Synergia PRR-fecta: Harnessing pathogen-recognition receptor synergy to produce high levels of IL-12p70 & prime dendritic cells for use in cancer immunotherapy

Abstract

Progress in the production of consistent, viable anti-cancer vaccines has been hampered by the lack of a fitting adjuvant capable of eliciting a targeted, cytotoxic response against tumours. The cytokine interleukin 12p70 (IL-12p70) as secreted by dendritic cells (DCs) forms a key part in the formation of type I T helper cells and effector cytotoxic T lymphocytes (CTLs). IL-12p70 is a heterodimeric protein consisting of a p35 and p40 subunit, both units are secreted following recognition of pathogen- and damage-associated molecular patterns (PAMPs/DAMPs) by various pattern-recognition receptors (PRRs) located on the cell surface and in the cytosol, most notably the family of Toll-like receptors (TLRs). Previous work has shown the ability of several TLRs to act in synergy, leading to the production of high amounts of IL-12p70 in DCs, such combinations may prove to be effective as adjuvants. Here we assayed a broad range of ligand combinations against TLRs and other PRRs in human monocyte-derived DCs (MoDCs), using ELISA to measure cytokine levels, and flow cytometry to characterize the expression of surface molecules on MoDCs following stimulation. We have identified 8 combinations capable of cueing the secretion of high levels of IL-12p70, of which 3 were chosen for further characterisation, with their effects on IFN-β and IL-10 also determined: 1. pIC (a TLR3 agonist) + R848 (a TLR7 & TLR8 agonist); 2. Pam3 (a TLR2:1 agonist) + pIC with LyoVec™ (a RIG-I & MDA5 agonist); and 3. Zymosan-δ (a Dectin-1 agonist) + R848. These combinations were also noted to stimulate MoDCs and upregulated expression of HLA-DR, CD80 and CD86. Based on common signalling, and past work on the synergistic production of IL-12p70, we propose a model for the transcription factors required. Combinations of PRR ligands may offer new routes for further expanding the arsenal of DC-based immunotherapies against cancer.