Fig 1: Antigen-specific tolerance induction and protection from CD4 T cell-mediated autoimmune disease using LSEC-targeting nanoparticles. (A) Experimental setup testing protection from MOG-induced experimental autoimmune encephalomyelitis (EAE) in fibrotic Mdr2-knockout or non-fibrotic C57BL/6 control mice. LSEC-targeting nanoparticles conjugated with MOG peptides (MOG-NP) or unconjugated control nanoparticles (NP) were administered intravenously, one day later followed by EAE induction through immunization to MOG; EAE symptoms were then scored for 30 days. (B) Disease course was plotted by a clinical EAE score in Mdr2-knockout or control mice, comparing MOG-NP and NP (left). Cumulative disease score for each animal over the 30-day observation period is shown on the right. (C) Experimental setup testing protection from MOG-induced EAE in fibrotic or non-fibrotic C57BL/6 mice; fibrosis was induced by administration of carbon tetrachloride over four weeks. LSEC-targeting nanoparticles conjugated with MOG peptides (MOG-NP) or unconjugated control nanoparticles (NP) were then administered intravenously, and one day later, EAE was induced by immunization to MOG, followed by three additional carbon tetrachloride administrations in the first one and a half weeks after MOG immunization; EAE symptoms were scored for 30 days following MOG immunization. (D) Disease course was plotted by a clinical EAE score in fibrotic vs. non-fibrotic mice, comparing MOG-NP and free NP treatment (left). Cumulative disease score for each animal over the 30-day observation period is shown on the right. E) MOG-specific 2D2 T cells (5x106 per mouse) were transferred 1 day after EAE induction, retrieved at day 7 and analyzed by flow cytometry. By staining for TCR Va3.2 and TCR Vb11 chains (right), transferred 2D2 cells could be identified against the background without T cell transfer (left). (F) Increase of MOG-specific Tr1-like cells following MOG-NP treatment in livers of Mdr2KO mice (left) or CCl4-treated mice (right). Tr1-like cells were defined as CD49b+ LAG3+ IL10+ TCRVa3.2+ TCRVb11+ CD4+ T cells. (G) MOG-NP treatment induced an increase in PD-1 expression on MOG-specific CD4 T cells, both in livers of Mdr2KO mice (left) or CCl4-treated mice (right). (H) MOG-NP treatment induced an increase in TIM3 expression on MOG-specific CD4 T cells, both in livers of Mdr2KO mice (left) or CCl4-treated mice (right). ns = non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001.