Amino acid copolymers that alleviate experimental autoimmune encephalomyelitis in vivo interact with heparan sulfates and glycoprotein 96 in APCs.

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS. One approved treatment for relapsing forms of MS is YEAK, a random copolymer of the amino acids tyrosine, glutamic acid, alanine, and lysine. YFAK, a second-generation copolymer composed of tyrosine, phenylalanine, alanine, and lysine, is more successful in treating experimental autoimmune encephalomyelitis, a mouse model of MS. Although originally designed and optimized based on the autoantigen myelin basic protein (MBP) and the MBP-derived peptide MBP85-99 presented to the MS-associated class II MHC molecule HLA-DR2, YEAK and YFAK also stimulate cytokine and chemokine production in APCs that lack class II MHC products. How YEAK and YFAK copolymers interact with APCs remains enigmatic. We used biotinylated YFAK to affinity-purify YFAK-interacting proteins from RAW264.7 cells and tested APCs from mice deficient in several of the newly identified interactors for their capacity to secrete CCL22 in response to YEAK and YFAK. We propose that initial contact of YFAK with cells is mediated mainly by electrostatic interactions, and find that interaction of YFAK with host proteins is strongly dependent on ionic strength. Cells deficient in enzymes involved in sulfation of proteins and proteoglycans showed strongly reduced binding of biotinylated YFAK. Lastly, cells stimulated with YFAK in the presence of heparin, structurally similar to heparan sulfates, failed to produce CCL22. We conclude that charge-dependent interactions of copolymers that alleviate MS/experimental autoimmune encephalomyelitis are critical for their effects exerted on APCs and may well be the main initial mediators of these therapeutically active copolymers.

Novel synthetic amino acid copolymers that inhibit autoantigen-specific T cell responses and suppress experimental autoimmune encephalomyelitis.

Copolymer 1 (Cop 1, Copaxone [Teva Marion Partners, Kansas City, Missouri, USA]), a random amino acid copolymer of tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K), reduces the frequency of relapses by 30% in relapsing-remitting multiple sclerosis (MS) patients. In the present study, novel random four-amino acid copolymers, whose design was based on the nature of the anchor residues of the immunodominant epitope of myelin basic protein (MBP) 85-99 and of the binding pockets of MS-associated HLA-DR2 (DRB1*1501), have been synthesized by solid-phase chemistry. Poly (Y, F, A, K) (YFAK) inhibited binding of the biotinylated MBP 86-100 epitope to HLA-DR2 molecules more efficiently than did either unlabeled MBP 85-99 or any other copolymer including Cop 1. Moreover, YFAK and poly (F, A, K) (FAK) were much more effective than Cop 1 in inhibition of MBP 85-99-specific HLA-DR2-restricted T cell clones. Most importantly, these novel copolymers suppressed experimental autoimmune encephalomyelitis, induced in the susceptible SJL/J (H-2(s)) strain of mice with the encephalitogenic epitope PLP 139-151, more efficiently than did Cop 1. Thus, random synthetic copolymers designed according to the binding motif of the human immunodominant epitope MBP 85-99 and the binding pockets of HLA-DR2 might be more beneficial than Cop 1 in treatment of MS.

Inhibition of experimental autoimmune uveitis by amino acid copolymers.

Glatiramer acetate (GA), a synthetic random amino acid copolymer, poly(Y, E, A, K)n, is widely used for treatment of multiple sclerosis. It inhibits experimental autoimmune encephalomyelitis (EAE) in mice by competition with the antigen and by induction of regulatory T cells. A novel copolymer, poly (F, Y, A, K)n , designated FYAK, was more effective than GA in its immunomodulatory activity in EAE. Here, FYAK and GA were compared in the amelioration of another disease model in mice, experimental autoimmune uveoretinitis (EAU). When tested by co-immunization with an uveitogenic antigen, FYAK was superior to GA in its capacity to inhibit EAU induction, as well as immune processes related to this condition. Further, regulatory T-cell lines specific to FYAK were more immunosuppressive than GA-specific lines in the EAU model. The superiority of FYAK-specific lines was accompanied by higher production of Th2 cytokines. These data thus demonstrate that FYAK, a novel copolymer, is superior to GA in its capacity to inhibit immunopathogenic processes in a non-central nervous system tissue.

CD1d and CD1d-restricted iNKT-cells play a pivotal role in contact hypersensitivity.

CD1d-restricted T-cells are activated by glycolipids presented by the major histocompatibility complex class-Ib molecule CD1d, found on the surface of antigen-presenting cells (APC). This interaction between APC, most notably dendritic cells (DC), and CD1d-restricted T-cells is an important regulatory step in the initiation of adaptive immune responses. It is well known that DC play a crucial role in the induction of contact hypersensitivity (CHS), a frequently studied form of in vivo T-cell-mediated immunity. In this study, we show that CD1d-restricted T-cells are also necessary for CHS, because both wild-type mice treated systemically or topically with CD1d glycolipid antagonists and CD1d-restricted T-cell-null mice have markedly diminished CHS responses. Thus, pharmacologic antagonists of CD1d can be used as effective inhibitors of CHS, a prototype for a variety of delayed-type tissue hypersensitivity responses.