Modified amino acid copolymers suppress myelin basic protein 85–99-induced encephalomyelitis in humanized mice through different effects on T cells

Abstract

Human HLA-DR2, and specifically that encoded by the DRA͞DRB1*1501 alleles, is thought to be important in the presentation of the immunodominant epitope MBP 85-99 (9, 10). Although the association of the human MHC class II (MHC II) locus with MS has consistently been observed in genome-wide scans (11), an association of the HLA-DR2, DQ6 haplotype, including the DRB1*1501 allele, with susceptibility to MS has been suggested in several population-and family-based studies (12). The importance of the DRB1*1501-associated MBP 85-99 epitope in the pathogenesis of MS has been particularly proposed based on data that, in MS lesions, the microglia͞ macrophages were positive for the HLA-DR2͞MBP 85-99 complex detected by a monoclonal antibody specific for this complex (13). Moreover, mice transgenic (tg) for HLA-DR2 and the TCR from an MS patient develop EAE spontaneously in 6-12 months and the disease appearance can be accelerated by immunization with MBP 85-99 (14).Several clinical attempts have been made to interfere with the MHC II͞MBP 85-99͞TCR trimolecular complex with varying effectiveness (8,(15)(16)(17)(18)(19). Currently, Copolymer 1 (Cop1, Copaxone, glatiramer acetate) is used in MS patients as an immunomodulator (20)(21)(22). Cop1 is a random polypeptide synthesized from four amino acids, L-tyrosine, L-glutamic acid, L-alanine, and L-lysine [poly(Y,E,A,K)n], in a specific molar ratio of 1.0, 1.4, 4.2, and 3.4, respectively. Although the mechanism of action of Cop1 is not fully understood, several immunologic effects have been suggested (23,24). These include competition of Cop1 with the immunodominant MBP 85-99 peptide for binding to DRB1*1501 and competition of MHC II͞Cop1 and MHC II͞MBP complexes for binding to the TCR (23,(25)(26)(27)(28). Cop1 has also recently been shown to polarize T cells into a T helper 2 (Th2) phenotype. Splenocytes from Cop1-immunized mice transfer protection against EAE, and Cop1-specific Th2 cells capable of secreting IL-4 and IL-10 dominate in the CNS of protected mice (29). Similarly, MS patients treated with Cop1 have Cop1-specific T cells with a Th2 phenotype (30).Although widely used in the treatment of MS, Cop1 reduces the frequency of relapses by only Ϸ30% (21). We hypothesized that by improving the binding capacity of copolymers to HLA-DR2 relative to MBP 85-99, it may be possible to develop a more effective immunomodulator. Several random 4-aa copolymers were designed based on the knowledge of anchor residues used by the immunodominant MBP 85-99 epitope that bind to the DRB1*1501-encoded molecule. To determine the in vivo and in vitro effect of these copolymers, a humanized double-tg mouse

Peptide 15-mers of defined sequence that substitute for random amino acid copolymers in amelioration of experimental autoimmune encephalomyelitis

“…2A Left). Likewise, in the mice immunized with J3 or J5 alone a lower response was induced suggesting, by analogy to responses to copolymers (30), that the non-tg, endogenous T cells (that represent Ϸ70% of the T cells in the humanized mice) might have responded to these peptides presented by HLA-DR2. However, a negligible response to J2 (that bound weakly) was seen.…”

“…Proliferative responses were measured as described in refs. 17 (17,29,30), peptide synthesis (13), isolation of empty HLA-DR2 (12,13), inhibition by peptide 15-mers of MBP 85-99 binding to HLA-DR2 (DRA͞DRB1*1501) (13,17), sorting of tg and endogenous (non-tg) T cells from humanized mice by flow cytometry (30), cytokine measurement by ELISA (30), and detection of PLP 139-151-reactive CD4 ϩ T cells by using I-A s ͞PLP 139-151 tetramers (31,32). (11,17).…”

“…As a negative control, Nase 101-120 peptide, which also binds I-A s , was used. To determine the proliferative response of transgenic (tg) T cells expressing the HLA-DR2-restricted͞MBP 85-99-specific TCR (hV␤2 ϩ CD4 ϩ ) and non-tg T cells (hV␤2 Ϫ CD4 ϩ ), splenocytes were fractionated from double-tg humanized mice that express both HLA-DR2 and a MBP 85-99-specific hV␤2 T cell receptor from an MS patient (30) by flow cytometry and seeded into a 96-well U-bottom microtiter plate in complete medium at 4 ϫ 10 5 cells per well. Irradiated splenocytes of humanized mice, also expressing HLA-DR2, were used as antigen-presenting cells.…”

“…Cop1 binds to several human HLA-DR molecules, including HLA-DR2, and blocks modestly the presentation of MBP 85-99 (27,28). The effectiveness of amino acid copolymers with in vitro and in vivo assays was greatly improved by modifications that enhanced their binding to HLA-DR2 (29,30). We hypothesized that by similarly improving the binding of synthetic peptide mimetics (13,29,30), their effectiveness might also be greatly enhanced.…”

“…The effectiveness of amino acid copolymers with in vitro and in vivo assays was greatly improved by modifications that enhanced their binding to HLA-DR2 (29,30). We hypothesized that by similarly improving the binding of synthetic peptide mimetics (13,29,30), their effectiveness might also be greatly enhanced. Thus, T cell recognition of MBP 85-99 might be more effectively inhibited and͞or additional immunosuppressive functions of the copolymers might be enhanced by the peptide mimetics.…”