Fig. 1: HBeAg induces liver sinusoidal endothelial cell activation to promote intrahepatic CD8 T cell immunity and HBV clearance.

A LSECs from mice that were hydrodynamically injected with pSM2 plasmid (HBV) or PBS (HDI Ctrl) were isolated 14 days postinjection (dpi) and cocultured with polyclonally stimulated splenocytes at a ratio of 1:2 (LSECs: splenocytes). Anti-CD3/anti-CD28–stimulated splenocytes were used as a responder control (RC). Unstimulated splenocytes were used as a negative control (NC). IFNγ production was measured after 48 h. CFSE-labeled polyclonally stimulated splenocytes were cocultured with LSECs, and after 72 h, CD8 T cell proliferation was analyzed by flow cytometry. B Naïve mouse or primary human LSECs were treated with recombinant HBeAg (rHBeAg) or left untreated (Ctrl) for 24 h, washed and cocultured with polyclonally stimulated splenocytes or PBMCs at a ratio of 1:2. LSECs from mice that were hydrodynamically injected with pcDNA3.1/HBeAg or the pcDNA3.1/null plasmid at 2 dpi were isolated and cocultured with polyclonally stimulated splenocytes at a ratio of 1:2. IFNγ production was measured after 48 h. Anti-CD3/anti-CD28–stimulated splenocytes were used as a responder control (RC). Unstimulated splenocytes were used as a negative control (NC). C LSECs from naïve mice were mock-treated or treated with rHBeAg, and total RNA was extracted from the cells 6 h later for RNA-seq analysis. Log2-fold changes in the top 20 differentially expressed genes (DEGs), KEGG pathway enrichment analysis and gene set enrichment analysis (GSEA), as determined by RNA-seq analysis. D Supernatants of rHBeAg-LSECs or HBV-LSECs were analyzed for TNF α and IL-27. E Polyclonally stimulated splenocytes were cocultured with rHBeAg-LSECs or HBV-LSECs, and then 10 μg/ml anti–IL-27 Abs (aIL-27) or anti–TNFα Abs (aTNFα) were added to the cocultures, with untreated cocultures as the control (Ctrl). IFNγ production was measured by ELISA after 48 h. F Polyclonally stimulated splenocytes were cocultured with LSECs from naïve mice, and 50 ng/ml rIL-27 or 100 ng/ml rTNFα was added to the cocultures. IFNγ production was measured by ELISA after 48 h. Anti-CD3/anti-CD28-stimulated splenocytes only were used as a responder controls (RC). Unstimulated splenocytes were used as a negative control (NC). Unpaired t-test or one-way ANOVA is used. Error bars, mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001.