Fig. 6

The tolerogenic activity of GMCSF-MOG was reflected by levels of pre-existing 2D2 Tregs. a, b 2D2-FIG mice were vaccinated with 4 nmol of GMCSF-MOG, and splenic Tregs were purified by flow sorting on day 7. Tregs were added to triplicate cultures of purified naïve splenic 2D2-FIG T responder cells that were supplemented with 10⁵ irradiated C57BL/6 splenocytes and 1 μM MOG^35–55 as designated. Cultures were pulsed with 1 μCi of [³H]thymidine during the last 24 h of a 3-day culture. Shown in a are the percent suppression of maximal MOG-stimulated proliferation and b the counts per minute (CPM) from cultures containing a 1:4 Treg/responder mixture (Treg: Tresp ratio) versus Tregs alone versus T cell responders alone. c, d C57BL/6 mice were treated with 2 nmol GMCSF-MOG (n = 8) or GMCSF-OVA (n = 7) on days − 21, − 14, and − 7. On day 0, all mice were immunized with 200 μg MOG^35–55 in CFA and 400 ng of Pertussis toxin i.p. on days 0 and 2. Shown in c are the daily mean clinical EAE scores and d the normalized mean body weight through the end of the experiment on day 40. e–h 2D2-FIG or 2D2-FIG-Rag1^−/− mice were treated with 4 nmol GMCSF-MOG, GMCSF-NFM, “GMCSF-MOG + GMCSF-NFM” (SC in saline), or with saline on days 0 and day 20 (arrow). Shown in e are the daily mean clinical EAE scores. Note that all open symbols (4 groups) and closed-square symbols (1 group) are included in the graph and are overlapping, reflecting no disease from days 0–41. Also shown in f, g, and h are the mean normalized body weight through the end of the experiment on day 41. a, b Statistical significance was analyzed using a two-tailed t test. Error bars represent standard deviation. c–h Differences between groups were analyzed using a two-way repeated measures ANOVA. Error bars represent SEM. c–h Significant differences between groups were c, d (a) vs (b); e (c) vs (e–i) and (d) vs (e-i); f (c) vs (f); and g (d) vs (g). c, e See Table 1 for clinical signs of EAE. These data are representative of (c–h) three or (a, b) two independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001)