Abstract 529: Characterization of a humanized TLR8 mouse model and its application in immuno-oncology drug development

Abstract

Abstract Background: TLR8 belongs to the Toll-like receptor family, serving as endosomal sensors of uridine rich single strand viral or endogenous RNA and several types of small molecule agonist. In humans, TLR8 is primarily expressed on monocytes, macrophages and myeloid dendritic cells (DCs), while in murine it was originally reported that TLR8 has no function due to the lack of five amino acids that recognize the endogenous RNA ligand. Increasing evidence suggests that mouse TLR8 may function differently from its human counterpart, including ligand binding and signaling through pathways other than NK-κB. Therefore, a humanized TLR8 mouse model is pivotal for mechanistically understanding TLR8 innate immune regulation and pharmacologically targeting TLR8 in various malignancies and inflammatory diseases. Methods: Exon 3 of mouse Tlr8 was replaced with the corresponding human region to generate humanized TLR8 knock-in mice. hTLR8+/- mice (n=5) and age matched naïve C57BL/6 mice were maintained until 27 weeks of age. Liver, spleen, kidney and pancreas were collected for pathological characterization. Serum cytokines levels were analyzed by MSD, and hTLR8 expression pattern and immune cells were analyzed in the spleen by flow cytometry. Comparisons of anti-PD-1 response in TLR8 knock-in mice and naïve C57BL/6 mice bearing various syngeneic tumors were also conducted. Results: We systemically characterized the phenotypic changes between age-matched 27-week-old C57BL/6 and hTLR8 mice. A high percentage of hTLR8 expression was detected on CD11b+ F4/80+ macrophages, CD11b+Gr-1+ myeloid cells and CD11c+ DCs. Hepatomegaly, splenomegaly, and nephromegaly were observed in aged hTLR8 mice. Histology analysis revealed a prominent portal inflammation with fibrosis and bile duct hyperplasia in liver; a decreased cellularity in periarteriolar lymphoid sheath and increase of histiocytes in the red pulp of spleen; and inflammatory immune cell infiltration in the kidneys. Analysis of serum cytokines showed a significant increase of KC/GRO (CXLCL1), TNF-α and IL-10 in the peripheral blood of the aged hTLR8 mice. PBMC from hTLR8 mice responded to a specific RNA-ligand based human TLR8 agonist (ORN-8L) in vitro. We are now profiling different syngeneic and genetically engineered murine tumor models to fully characterize the potential adjuvant-like function of hTLR8 in different tumor models. Conclusion: Expression of humanized TLR8 leads to auto-activation of TLR8 signaling in mice. This pivotal model system enables us to study the function of human TLR8 and pharmacology of its agonists and antagonists in vivo. These knock-in mice may also serve as an interesting host strain for syngeneic tumor studies, potentiating immune responses to therapies. Citation Format: Hongjuan Zhang, Ruilin Sun, Annie X. An, Henry Q.x Li, Davy Xuesong Ouyang. Characterization of a humanized TLR8 mouse model and its application in immuno-oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 529.