Fig. 4.

Tolerogenic Liposomes Prevent EAE Disease Progression via Antigen Specific Tolerance. (A) C57BL/6 mice (14–15 mice per group) were injected on consecutive days to induce EAE following procedure in methods section. Following final injection, the disease was allowed to progress for 5 days, at which point mice were injected with treatment candidates on day 4 and 5, allowed to rest for 48 h then treated again on day 7 and 8. Treatment groups were: PBS (on both days), Free I, Lipo^I, or Lipo^{TLR + I}. For all TLR containing formulations, the FLA formulation was given first (e.g day 4), then 24 h later the CpG formulation was administered (e.g. day 5). All formulations (except PBS) contained 10 μg of MOG peptide in each 100 μL injection. and MOG_35–55 peptide was fully encapsulated in liposomal formulations. After final treatment, mice were monitored for 37 days and disease progression tracked. Error bars represent ±SEM of disease score. (B) Groups of 5 mice were treated similarly to part A, but sacrificed on day 14 following EAE induction. Serum was taken and analyzed for and MOG_35–55 peptide specific IgG levels via ELISA. UT denotes an “untreated” mouse, a naïve C57BL/6 mouse without and MOG_35–55 exposure (C) Lymph nodes from mice in part B were dissociated and lymph cells were stained for analysis of T-cells. The number of EAE peptide tetramer positive CD4⁺ activated T cells is shown. A representative flow plots of CD45⁺, CD3⁺, CD4⁺ cells show a distribution of CD4 (y axis) and MOG_35–55 peptide tetramer (x axis). (D) and MOG_35–55 peptide tetramer positive T reg cells from lymph nodes in part C. Below is representative flow plots of CD45⁺, CD3⁺, CD4⁺ cells showing distribution of MHCII-MOG peptide tetramer (y axis) and FoxP3 (x axis). (E–G) C57Bl/6 mice (N = 10) were similarly treated as in part A with Lipo^{TLR + I}, but with treatment starting on day 8 after EAE induction. Mice were then injected i.p with PBS or antibodies against mouse (E) PD-L1, (F)PD-1 or (G) CTLA-4. Mice were injected with 500 μg antibody on day 7 and day 10 and 250 μg antibody on day 14 and day 17. Statistical significance of AUC differences of EAE disease development curves was assessed by using one-way ANOVA and Tukey’s multiple comparison test in (A) and (E–G). Significance for B-D was determined by a two-way ANOVA with Tukey post hoc test for multiple comparisons. *p < 0.5, **p < 0.01, ***p < 1 × 10⁻⁴, ****p < 1 × 10⁻⁵..