Fig. 5.

RTL-specific Fab blocks RTL342M-induced downregulation of CD74 expression on monocytes and neutralizes therapeutic activity in EAE. a) Fab1B11 specific for two-domain RTL342M (DR2/mMOG-35-55) but not FabD2 specific for a different RTL (DR4/GAD555-567) blocks binding to and expression of CD74 on CD11b⁺ cells in vitro within the monocyte gate defined in Fig. 1a above in PBMC collected from naïve DR*1501-Tg mice. To neutralize RTL342M binding, 1 μg RTL was incubated at a 1:1 molar ratio (2 μg) of Fab1B11 or FabD2 for 2 h at room temperature prior to incubation with cells. Figure (left) shows mean fluorescent intensity (MFI) of CD74 after pre-incubation of RTL342M with Fab1B11 or control FabD2 or with no RTL or Fab1B11 alone. *p < 0.05; **p < 0.005; ***p < 0.0005. b) DR*1501-Tg mice with EAE were treated s.c. daily for 3 days at onset of clinical EAE with vehicle, 20 μg RTL342M alone, 20 μg RTL342M incubated for 2 h at a 1:1 (40 μg) or 1:2 molar ratio (80 μg) with Fab1B11 or 20 μg control FabD2 prior to injection or with 40 μg Fab1B11 alone. Top left: Daily mean clinical EAE disease scores. *p < 0.05, RTL342M + Fab1B11 vs. RTL342M + FabD2; #p < 0.0005, RTL342M vs. vehicle, RTL342M + Fab1B11 and Fab1B11 alone; RTL342M vs. RTL342M + FabD2 = ns. Top right: Statistical comparisons of cumulative disease indices (CDI): *p < 0.05; ***p < 0.0001. Bottom: Group EAE summary table. Daily mean scores were analyzed by Mann Whitney U and mean CDI by one way ANOVA with Tukey post-test, and nonparametric one way Kruskal–Wallis ANOVA with Dunn’s multiple comparisons post-test.