Soluble MOG35-55/I-Ab Dimers Ameliorate The MOG35-55Induced Experimental Autoimmune Encephalomyelitis In C57BL/6Mice

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS mediated by autoreactive T cells directed against myelin antigens, which serves as an animal model of human multiple sclerosis (MS). In this work, a soluble divalent MOG35-55/I-Ab fusion protein (MOG35-55/I-Ab dimer) which targets specific CD4+T cells was constructed and prepared. Their inhibition effects on MOG35-55specific T cells were studied in the MOG35-55induced-EAE model in which the T cell epitope was clear. In vitro and in vivo researches demonstrated that the soluble MOG35-55/I-Ab dimer was able to inhibit the MOG35-55-specific T cells by reducing proliferation of CD4+T cells and decreasing the percentage of Thl and Th17cells, and therefore was very effective in the suppression of EAE in C57BL/6mice induced by MOG35-55. The mechanisms involved in this antigen-specific dimer-mediated suppression were found to be downregulated TCR-CD3expression as well as upregulated expression of membrane-bound TGF-β3(mTGF-β1) and IL-10suppressive 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 disease.1,Constructio,expression and identification of soluble I-Ab/FcOn the basis of our previous work, the I-Ab/Fc recombinant gene without MOG35-55peptide covalently attaching to the C terminal of the I-Abβ chain was constructed through overlapping PCR, and a kozak sequence was added at the upstream of the initiation codon. The fusion protein I-Ab/Fc (I-Ab dimer) was expressed by the Bac-to-Bac (Bacteria-to-Baculovirus) expression system, and purified through the straphylococcal protein A (SPA) affinity chromatography. The results of sandwich ELISA and Western blot showed that the I-Ab dimer has the intact conformation and.the expected molecule weight. The expression level was higher than our previous MOG35-55-I-Ab/Fc molecule with MOG35-55covalently attached.2,Soluble MOG35-55/I-Ab dimer can inhibit MOG35-55specific T cells in vitroIn this work, splenocytes from the MOG35-55-immunized wild type C57BL/6mice (WT EAE) and2D2transgenic mice were used as the MOG35-55-specific autoreactive T cells to evaluate the peptide-specific inhibition effect of MOG35-55-loaded I-Ab dimer (MOG35-55/I-Ab dimer) in vitro. The resluts showed that the dimer was able to suppress over90%proliferation of the MOG35-55-specific T cells and inhibit the peptide-induced expansion of Thl and Th17cells at the concentration of1.2nM. Further study using2D2T cells suggested that the mechanisms involved in the suppression would be downregulated TCR-CD3expression and upregulated membrane TGF-β (mTGF-β) and IL-10.3,The theraputic effect of soluble MOG35-55/I-Ab dimer on the MOG35-55induced EAEIn EAE model, the administration of MOG35-55/I-Ab dimer resulted in a delayed onset and a significant amelioration of the disease severity at clinical levels and showed protection of the CNS from inflammatory damage at histological levels, whereas the empty I-Ab without peptide replacing did not show the theraputic effect, suggesting the soluble I-Ab dimer acted in an antigen-specific manner. In addition, not only the pathogenic Thl and Th17cells but also IFN-γ-and IL-17-secreting CD8+T cells in peripheral lymphoid organs were observed significantly reduced by MOG35-55/I-Ab dimer treatment, suggesting that the MOG35-55/I-Ab dimer targets CD4+T cell also influence the function of CD8+T cells, probably through regulating the CD4+Th cell network.