Crosstalk between TLR8 and RIG-I-like receptors enhances antiviral immune responses.

Background: Toll-like receptor (TLR) agonists have been investigated due to their potential dual effects as latency reverting agents and immune modulatory compounds in people living with HIV (PLWH). Here, we investigated whether co-stimulation of TLR7/8 agonists with RIG-I-like receptor (RLR) agonists enhances antiviral immunity. Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (DCs) were incubated with TLR and RLR-agonists for 24 h and innate and adaptive immune responses were determined (maturation markers, cytokines in supernatant, ISG expression). Results: Both TLR7 and TLR8 agonists induced pro-inflammatory cytokines in DCs as well as PBMCs. TLR8 agonists were more potent in inducing cytokine responses and had a stronger effect on DC-induced immunity. Notably, while all compounds induced IL-12p70, co-stimulation with TLR8 agonists and RLR agonist polyI: C induced significantly higher levels of IL-12p70 in PBMCs. Moreover, crosstalk between TLR8 and RLR agonists induced a strong type I Interferon (IFN) response as different antiviral IFN-stimulated genes were upregulated by the combination compared to the agonists alone. Conclusion: Our data strongly suggest that TLR crosstalk with RLRs leads to strong antiviral immunity as shown by induction of IL-12 and type I IFN responses in contrast to TLRs alone. Thus, co-stimulation of TLRs and RLRs might be a powerful strategy to induce reactivation of latent reservoir as well as antiviral immunity that eliminates the reactivated cells.

Dendritic Cells Require PINK1-Mediated Phosphorylation of BCKDE1α to Promote Fatty Acid Oxidation for Immune Function.

Dendritic cell (DCs) activation by Toll-like receptor (TLR) agonist induces robust metabolic rewiring toward glycolysis. Recent findings in the field identified mechanistic details governing these metabolic adaptations. However, it is unknown whether a switch to glycolysis from oxidative phosphorylation (OXPHOS) is a general characteristic of DCs upon pathogen encounter. Here we show that engagement of different TLR triggers differential metabolic adaptations in DCs. We demonstrate that LPS-mediated TLR4 stimulation induces glycolysis in DCs. Conversely, activation of TLR7/8 with protamine-RNA complex, pRNA, leads to an increase in OXPHOS. Mechanistically, we found that pRNA stimulation phosphorylates BCKDE1α in a PINK1-dependent manner. pRNA stimulation increased branched-chain amino acid levels and increased fatty acid oxidation. Increased FAO and OXPHOS are required for DC activation. PINK1 deficient DCs switch to glycolysis to maintain ATP levels and viability. Moreover, pharmacological induction of PINK1 kinase activity primed immunosuppressive DC for immunostimulatory function. Our findings provide novel insight into differential metabolic adaptations and reveal the important role of branched-chain amino acid in regulating immune response in DC.

Evaluation of novel synthetic TLR7/8 agonists as vaccine adjuvants.

Small-molecule adjuvants that boost and direct adaptive immunity provide a powerful means to increase the effectiveness of vaccines. Through rational design several novel imidazoquinoline and oxoadenine TLR7/8 agonists, each with unique molecular modifications, were synthesized and assessed for their ability to augment adaptive immunity. All agonists bound human TLR7 and TLR8 and induced maturation of both human mDCs and pDCs. All agonists prompted production of type I interferon and/or proinflammatory cytokines, albeit with varying potencies. In most in vitro assays, the oxoadenine class of agonists proved more potent than the imidazoquinolines. Therefore, an optimized oxoadenine TLR7/8 agonist that demonstrated maximal activity in the in vitro assays was further assessed in a vaccine study with the CRM197 antigen in a porcine model. Antigen-specific antibody production was greatly enhanced in a dose dependent manner, with antibody titers increased 800-fold compared to titers from pigs vaccinated with the non-adjuvanted vaccine. Moreover, pigs vaccinated with antigen containing the highest dose of adjuvant promoted a 13-fold increase in the percentage of antigen-specific CD3(+)/CD8(+) T cells over pigs vaccinated with antigen alone. Together this work demonstrates the promise of these novel TLR7/8 agonists as effective human vaccine adjuvants.