Figure 6
Coimmunization prevents autoimmune encephalomyelitis induced by amyloid-β42 protein. (A) Schematic representation of amyloid-β42 autoimmune encephalomyelitis (Aβ42-EAE) induction and vaccination. EAE was induced by immunization with Aβ42 protein + complete Freund’s adjuvant (CFA) + pertussis toxin (PT) (Aβ42-EAE model) on day 0, and then, on day 1, PT was injected a second time. The EAE-induced mice were further coimmunized or protein-immunized on days 2 and 14. T cells infiltrating the brain were assessed by isolating T cells from the brain on day 21. (B) Single-cell suspensions of brain cells taken from EAE-induced animals were isolated with or without vaccination or from naïve C57BL/6 mice and then stained on day 21. FSC, Forward scatter; SSC, Side scatter. (C) The cells were immunostained for CD3 and CD4 and analyzed by fluorescence-activated cell sorting (FACS). The upper left panel shows cells from naïve mice with no antibody staining, followed by antibody-stained cells from the naïve mice, from the myelin oligodendrocyte glycoprotein (MOG)–induced EAE (MOG-EAE) model, from the Aβ42-induced EAE model, from the Aβ42-induced EAE mice vaccinated with Aβ42 protein vaccine or from the coimmunized mice. (D) Graph showing the number of CD4+ T cells in brain. The number of double-positive CD3+ and CD4+ T cells per 106 brain cells from the groups of six animals represented in (C) were statistically analyzed and plotted. (E) Histopothological examination of brain cortex on the sixth day after EAE model induction, stained with hematoxylin and eosin. The naïve tissue section was taken from naïve mice without any treatment or induction. The results shown are representative of three independent experiments. Statistical analysis was performed using parametric one-way analysis of variance, and t-tests were used to compare two groups. The arrows point to the lymphocytes areas. The bars represent 50 micrometer in length. co, Coimmunization; pro, Protein immunization. All data are presented as mean ± SD. ****P < 0.0001 compared with protein-vaccinated Aβ42-EAE mice.