(HT) † These authors contributed equally to this work. Running title: LBP mediates LPS-induced TLR4 internalization and signalingKeywords: CD14, cell surface receptor, endotoxin, innate immunity, LPS, LPS-binding protein (LBP), pathogen-associated molecular pattern (PAMP), pattern recognition receptor (PRR), TIR-domaincontaining adapter-inducing interferon-β (TRIF), Toll-like receptor 4 (TLR4), AbstractToll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKε, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NFκB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces LBP mediates LPS-induced TLR4 internalization and signaling 1 http://www.jbc.org/cgi

Summary Immune‐checkpoint blockade antibodies have been approved for the treatment of cancer. However, poorly immunogenic tumours are less responsive to such therapies. Agonistic anti‐Toll‐like receptor 4 (TLR4) monoclonal antibodies (mAbs) activate only cell‐surface TLR4; in contrast, lipopolysaccharide (LPS) activates both TLR4 and intracellular inflammatory caspases. In this study, we investigated the adjuvant activity of an anti‐TLR4 mAb in T‐cell‐mediated antitumour immunity. The anti‐TLR4 mAb induced the activation of antigen‐specific T‐cells in adoptive transfer studies. The growth of ovalbumin (OVA)‐expressing tumours was significantly suppressed by administration of OVA and the anti‐TLR4 mAb in combination, but not individually. The antitumour effect of anti‐PD‐1 mAb was enhanced in mice administered with OVA plus the anti‐TLR4 mAb. The OVA‐specific IFN‐γ‐producing CD8 T‐cells were induced by administration of OVA and the anti‐TLR4 mAb. The suppression of tumour growth was diminished by depletion of CD8, but not CD4, T‐cells. The inflammatory response to the anti‐TLR4 mAb was of significantly lesser magnitude than that to LPS, as assessed by NF‐κB activation and production of TNF‐α, IL‐6 and IL‐1β. Administration of LPS (at a dose that elicited levels of proinflammatory cytokines comparable to those by the anti‐TLR4 mAb) plus OVA induced no or less‐marked activation of OVA‐specific T‐cells and failed to suppress tumour growth in mice. In conclusion, the agonistic anti‐TLR4 mAb induces potent CD8 T‐cell‐dependent antitumour immunity and an inflammatory response of lesser magnitude than does LPS. The agonistic anti‐TLR4 mAb has potential as an adjuvant for use in vaccines against cancer.