Mannan-MOG35-55 Reverses Experimental Autoimmune Encephalomyelitis, Inducing a Peripheral Type 2 Myeloid Response, Reducing CNS Inflammation, and Preserving Axons in Spinal Cord Lesions.

CNS autoantigens conjugated to oxidized mannan (OM) induce antigen-specific T cell tolerance and protect mice against autoimmune encephalomyelitis (EAE). To investigate whether OM-peptides treat EAE initiated by human MHC class II molecules, we administered OM-conjugated murine myelin oligodendrocyte glycoprotein peptide 35-55 (OM-MOG) to humanized HLA-DR2b transgenic mice (DR2b.Ab°), which are susceptible to MOG-EAE. OM-MOG protected DR2b.Ab° mice against MOG-EAE by both prophylactic and therapeutic applications. OM-MOG reversed clinical symptoms, reduced spinal cord inflammation, demyelination, and neuronal damage in DR2b.Ab° mice, while preserving axons within lesions and inducing the expression of genes associated with myelin (Mbp) and neuron (Snap25) recovery in B6 mice. OM-MOG-induced tolerance was peptide-specific, not affecting PLP178-191-induced EAE or polyclonal T cell proliferation responses. OM-MOG-induced immune tolerance involved rapid induction of PD-L1- and IL-10-producing myeloid cells, increased expression of Chi3l3 (Ym1) in secondary lymphoid organs and characteristics of anergy in MOG-specific CD4+ T cells. The results show that OM-MOG treats MOG-EAE in a peptide-specific manner, across mouse/human MHC class II barriers, through induction of a peripheral type 2 myeloid cell response and T cell anergy, and suggest that OM-peptides might be useful for suppressing antigen-specific CD4+ T cell responses in the context of human autoimmune CNS demyelination.

Mannan-conjugated myelin peptides prime non-pathogenic Th1 and Th17 cells and ameliorate experimental autoimmune encephalomyelitis.

Antigen presenting cells (APC) are critical for regulating immune responses. We tested mannan-peptide conjugates for targeting myelin peptides to APC to induce T cell tolerance and resistance to experimental autoimmune encephalomyelitis (EAE). Myelin peptides conjugated to mannan in oxidized (OM) or reduced (RM) forms protected mice against EAE in prophylactic and therapeutic protocols, with OM-conjugated peptides giving best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation, but not alterations in Th1, Th17 and Treg cell differentiation or T cell apoptosis compared to EAE controls. Bone marrow-derived dendritic cells (DC) loaded with OM-MOG showed up-regulated expression of co-stimulatory molecules, reduced PD-L1 expression and enhanced CD40-inducible IL-12 and IL-23 production compared to MOG DC, features consistent with immunogenic DC. OM-MOG induced active T cell tolerance because i.d. administration or passive transfer of OM-MOG DC suppressed ongoing EAE, while OM-MOG-vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. As in vivo, MOG-specific T cells cultured with OM-MOG DC showed reduced proliferation and equal Th1 and Th17 cell differentiation compared to those with MOG DC, but surprisingly cytokine production was unresponsive to CD40 engagement. Impaired effector T cell function was further evidenced in spinal cord sections from OM-MOG-vaccinated EAE mice, where markedly reduced numbers of CD3(+) T cells were present, restricted to leptomeninges and exceptional parenchymal lesions. Our results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and APC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells.

Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

A novel pathogenic peptide of thyroglobulin (2208-2227) induces autoreactive T-cell and B-cell responses in both high and low responder mouse strains.

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208-2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (A(b)), or susceptibility (A(s), E(k)) to EAT. We therefore immunized LR BALB/c (H-2(d)) and C57BL/6 (H-2(b)) strains, as well as HR CBA/J (H-2(k)) and SJL/J (H-2(s)) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1-3), as well as in SJL/J mice (two of six mice, infiltration index 1-2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.