Frequency of CD4+ T-cells is significantly reduced in the blood of mice treated with RTL551.

<p>Mice were euthanized at the indicated time points after onset and isolated blood cells were lysed, stained with fluorescent antibodies and analyzed by flow cytometry. Representative dot plot for CD4 staining in blood on D1 is shown in panel A. Sequential changes in CD4+ T-cell frequencies in the blood of mice with EAE are shown in panel B. Data in panel B are mean±SD values from 8 mice per group except for naïve which had 5 mice. Significant differences between the groups (p≤0.05) were determined using Student's <i>t</i> test and are indicated by an asterisk.</p

Treatment with RTL551 significantly reduces the percentages of CD226 and T-bet expressing CD4+ T-cells compared to treatment with Vehicle or RTL550 in the blood of mice with EAE early after treatment initiation.

<p>Representative dot plots for CD226 and T-bet staining, respectively, on CD4 T-cells in blood are shown in panels A & B. Sequential changes in the expression of CD226 and T-bet on CD4 T-cells in the blood of mice from the three groups during EAE are shown in panels C & D. Data in panels C & D are mean±SD values from 8 mice per group except for naïve which had 5 mice. Significant differences between the groups (p≤0.05) were determined using Student's <i>t</i> test and are indicated by an asterisk.</p

RTL551 treated mice have less infiltration of immune cells in the CNS.

<p>Panel A represents H&E staining on paraffin embedded spinal cord sections from Vehicle, RTL550 or RTL551 treated C57BL/6 mice at indicated time points after EAE onset. Spinal cords from control groups of mice (Panel A) showed dense mononuclear infiltration (arrows) and sections from RTL551 treated mice showed reduction in inflammatory cells. Magnification: A, 5X; insets, 20X. Panel B is the mean±SD values of cells isolated from the CNS of mice from the three groups (8 mice per group) at indicated time points. Significant differences between the groups (p≤0.05) were determined using Student's <i>t</i> test and are indicated by an asterisk.</p

RTL551 treated mice had reduced frequencies of IL-17 and IFNγ secreting cells in the spleen D3 post onset.

<p>Splenocytes were stimulated with leukocyte activation cocktail and were stained with respective antibodies and subjected to flow cytometry. Representative dot plots for IL-17 and IFN-γ staining (respectively) on CD4+ T-cells are shown in panels A & B respectively. Cells were gated on CD4+ cells for this analysis. IL-17 staining on gated CD8+ T-cells is shown in panel C. Data are representative of 3 mice per group.</p

Treatment of EAE mice with RTL551 inhibits expansion of CD44 expressing CD4+ T-cells in the periphery compared to treatment with Vehicle or RTL550.

<p>Splenocytes and lysed blood cells were stained with respective antibodies and subjected to flow cytometry. Dot plot for CD44 staining on blood CD4+ T-cells on day 3 post onset is shown in panel A. Cells were gated on live CD4+ cells for this analysis. Values in panels B & C are representative of 8 mice per group on D3 after onset of clinical signs of EAE except for naïve which had 5 mice. Significant differences between the groups (p≤0.05) were determined using Student's <i>t</i> test and are indicated by brackets.</p

EAE Mice treated with RTL551 at onset have significantly reduced clinical scores as compared to vehicle or RTL550 treated mice.

<p>Male C57BL/6 mice were immunized with MOG-35-55/CFA/Ptx. Mice were scored for clinical signs of EAE as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021868#s2" target="_blank"><i>Materials and Methods</i></a>. Data presented are the mean±SD disease scores of 8–10 mice per group. Significant differences between the groups were determined using Mann Whitney <i>U</i> test.</p

Selective reduction of T-bet expressing CD4 T-cells from the CNS of EAE mice treated with RTL551.

<p>T-bet expressing CD4+ T-cells were increased in the CNS of control groups of mice with EAE when compared to onset. One injection of RTL551 induced significant reduction and recruitment of CD4+ T-cells into the CNS. Representative dot plots for CD4+ and T-bet staining on the cells isolated from CNS of mice are shown in panels A & D. Sequential changes in mean±SD absolute numbers of CD4+ and CD8+ cells in the CNS of control and RTL551 treated mice are shown in panels B & C. Percentages are demonstrated in panel E and panel F shows absolute numbers of T-bet expressing CD4+ T-cells in the CNS of mice D1 post onset. There were 8 mice per group and significant differences between the groups (p≤0.05) were determined using Student's <i>t</i> test and are indicated by an asterisk (panels C and D) or by brackets (panels E and F).</p

Additional file 1: Figure S1. of Sex-dependent treatment of chronic EAE with partial MHC class II constructs

EAE disease course in female and male (A) DR*1501-Tg and (B) C57BL/6 mice, immunized with mMOG-35-55 peptide. Figure S2. Days 20 and 63 p.i. spinal cord lumbar sections from EAE female DR*1501-Tg mice were stained with luxol fast blue (LFB) and analyzed for demyelination, toluidine blue for spinal cord damage, and CD4+ cell frequency in the spinal cords of RTL342M- vs. vehicle-treated mice. (PDF 231 kb