Soluble MOG35-55/I-A^b Dimers Ameliorate Experimental Autoimmune Encephalomyelitis by Reducing Encephalitogenic T Cells

<div><p>The MOG35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice is a useful animal model to explore therapeutic approaches to T cell-mediated autoimmune diseases because the dominant T-cell epitope(s) have been defined. It is rational that antigen-specific immunosuppression can be induced by using MHC-peptide complexes as specific TCR ligand(s) that interact with autoreactive T cells in the absence of co-stimulation. In this study, a soluble divalent MOG35-55/I-A^b fusion protein (MOG35-55/I-A^b dimer) was constructed to specifically target the autoreactive CD4⁺ T cells in the EAE mouse. Intraperitoneal administration of the MOG35-55/I-A^b dimer significantly delayed and ameliorated EAE symptoms by reducing EAE-related inflammation in the mouse CNS and reducing encephalitogenic Th1 and Th17 cells in the peripheral lymphoid organs. We observed that dimer intervention at a concentration of 1.2 nM suppressed MOG35-55 peptide-specific 2D2 transgenic T cells (2D2 T cells) proliferation by over 90% after <em>in vitro</em> activation with MOG35-55 peptide. The mechanisms involved in this antigen-specific dimer-mediated suppression were found to be downregulated TCR-CD3 expression as well as upregulated expression of membrane-bound TGF-β (mTGF-β) and IL-10 suppressive cytokines by the autoreactive CD4⁺ T cells. Collectively, our data demonstrates that soluble divalent MHC class II molecules can abrogate pathogenic T cells in EAE. Furthermore, our data suggests that this strategy may provide an efficient and clinically useful option to treat autoimmune diseases.</p> </div

MOG35-55/I-A^b dimer delays onset and reduces severity of EAE <i>in vivo</i>.

<p>Female C57BL/6 wild-type mice were immunized with MOG35-55 to induce paralyzed EAE. PBS, excess MOG35-55 peptide, Mulv env145-158/I-A^b dimer (1 µg per mouse) or MOG35-55/I-A^b dimer (1 µg per mouse) was administered i.p. starting at day 9 post-immunization. Treatments lasted 4 days. (A) Data are expressed as mean clinical scores ± SD (n = 10 for PBS and MOG35-55/I-A^b dimer-treated groups, n = 8 for excess MOG35-55 and Mulv env145-158/I-A^b dimer-treated groups). Black arrows indicate i.p. injection. The mean clinical score in MOG35-55/I-A^b dimer-treated group is lowest (<i>P<</i>0.05), whereas there is no difference among PBS, excess MOG35-55 and Mulv env145-158/I-A^b dimer-treated groups. (B–C) Spinal cord sections obtained from the above four groups at day 25 post-immunization were analyzed for degree of inflammation by H&E and for demyelination by LFB (original magnification 200×). (B) The MOG35-55/I-A^b dimer-treated group showed minimal inflammatory cell infiltration and demyelination. One representative sample from each group is depicted. (C) Semi-quantitative analyses of inflammation and demyelination in spinal cords from four groups were conducted. Histopathological scores were determined as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047435#s4" target="_blank">Materials and Methods</a></i> and are presented as mean ± SD from 4 mice. Data presented are representative of two independent experiments. Differences between groups were assessed by Mann-Whitney U test. *, <i>P<</i>0.05; **, <i>P<</i>0.01 and n.s., nonsignificant.</p

MOG35-55/I-A^b dimer modulates the expression of CD3 and TCR as well as the mTGF-β1 and IL-10 suppressive cytokines in MOG35-55-specific T cells.

<p>(A–D) The 2D2 splenocytes were stimulated with MOG35-55 (25 µg/mL) <i>in vitro</i> in the presence of MOG35-55/I-A^b dimer (1.2 nM) or PBS. After 5 days culture, cells were harvest and stained with anti-CD4-FITC mAb, anti-CD3-PE-Cy7 mAb, anti-TCR-Vα3.2-APC mAb, anti-mTGF-β1-PE and/or anti-IL-10-PE mAb. (A–B) The MFIs of (A) PE-cy7-CD3 and (B) APC-TCR Vα3.2 on CD4⁺ cells were analyzed by flow cytometry and expressed as the mean ± SD from 6 mice in each group. (C-D) MOG35-55/I-A^b dimer intervention induced a population of CD3^lowTCR^lowCD4⁺ cells with high level of (C) mTGF-β1 and (D) IL-10 expression. The right panel represents the mean ± SD of MFI of PE-mTGF-β1 and PE-IL-10 on CD3^highTCR^highCD4⁺/CD3^lowTCR^lowCD4⁺ cells in dimer intervention group (n = 3). (E) Splenocytes were isolated from mice treated with PBS or MOG35-55/I-A^b dimer on day 18 post-immunization and stained with anti-CD3-APC, anti-CD4-FITC, anti-TGF-β1-PE, and/or anti-IL-10-PE Abs. CD4⁺ cells were gated for analysis by flow cytometry. (a–b) The mean percentage of CD3^lowCD4⁺ cells and the MFI of APC-CD3 on CD4⁺ cells from 6 mice in each group are represented as the mean ± SD. (c) The MFIs of PE-mTGF-β1 and PE-IL-10 on CD3^lowCD4⁺/CD3^highCD4⁺ cells in dimer treated-group are expressed as the mean ± SD from 6 mice respectively. *, <i>P<</i>0.05; **, <i>P<</i>0.01; ***, <i>P<</i>0.001 and n.s., nonsignificant.</p

MOG35-55/I-A^b dimer treatment abrogates autoreactive Th1 and Th17 expansion <i>in vivo</i>.

<p>MOG35-55 immunized mice were treated with PBS, excess MOG35-55 peptide, Mulv env145-158/I-A^b dimer or MOG35-55/I-A^b dimer. On day 18 post-immunization, cells were isolated from spleen (SPN) and lymph nodes (LN) for IFN-γ⁺ Th1 and IL-17⁺ Th17 cells detection by intracellular staining. (A) Gated for CD3⁺CD4⁺ (B) Gated for CD3⁺CD8⁺. The left panel shows one representative sample from each group. The right panel represents the mean ± SD of percentage of Th1 or Th17 cells with differences (n≥4). *, <i>P<</i>0.05; **, <i>P<</i>0.01; ***, <i>P<</i>0.001 and n.s., nonsignificant.</p