Copolymer 1 acts against the immunodominant epitope 82-100 of myelin basic protein by T cell receptor antagonism in addition to major histocompatibility complex blocking

Abstract

The synthetic random amino acid copolymer Copolymer 1 (Cop 1, Copaxone, glatiramer acetate) suppresses experimental autoimmune encephalomyelitis, slows the progression of disability, and reduces relapse rate in multiple sclerosis (MS). Cop 1 binds to various class II major histocompatibility complex (MHC) molecules and inhibits the T cell responses to several myelin antigens. In this study we attempted to find out whether, in addition to MHC blocking, Cop 1, which is immunologically cross-reactive with myelin basic protein (MBP), inhibits the response to this autoantigen by T cell receptor (TCR) antagonism. Two experimental systems, "prepulse assay" and "split APC assay," were used to discriminate between competition for MHC molecules and TCR antagonism. The results in both systems using T cell lines/clones from mouse and human origin indicated that Cop 1 is a TCR antagonist of the 82-100 epitope of MBP. In contrast to the broad specificity of the MHC blocking induced by Cop 1, its TCR antagonistic activity was restricted to the 82-100 determinant of MBP and could not be demonstrated for proteolipid protein peptide or even for other MBP epitopes. Yet, it was shown for all the MBP 82-100-specific T cell lines/clones tested that were derived from mice as well as from an MS patient. The ability of Cop 1 to act as altered peptide and induce TCR antagonistic effect on the MBP p82-100 immunodominant determinant response elucidates further the mechanism of Cop 1 therapeutic activity in experimental autoimmune encephalomyelitis and MS.

Figure 1

Antagonistic effect of Cop 1 in a prepulse assay on murine T cell lines and clones. (A) MHC blocking of SJL/J MBP p82–100 clone 3. (B) TCR antagonism of SJL/J MBP p82–100 clone 3. (C) MHC blocking of SJL/J PLP p139–151 clone III. (D) TCR antagonism of SJL/J PLP p139–151 clone III. (E) MHC blocking of B10.PL MBP p1–11 clone 2. (F) TCR antagonism of B10.PL MBP p1–11 clone 2. Irradiated (3,000 rad) spleen cells (3 × 10⁶/ml) were incubated with a suboptimal dose of the antigen (2–10 μg/ml) at 37°C. After 3 h, cells were washed and recultured in individual wells of 96-well microculture plates (5 × 10⁵/well) with T cells (1.5 × 10⁴/well). Cop 1 or control peptide inhibitor were either preincubated in the first stage with the antigen to inhibit MHC binding or added in the second stage with the T cells to inhibit binding to the TCR. Results are expressed as mean cpm thymidine incorporation for triplicate cultures ×10⁻³ and represent one of five independent experiments.

Figure 2

Antagonistic effect of Cop 1 in a prepulse assay on human T cell clones. (A) MHC blocking of SS-GP-25 clone specific to p82–100 of MBP. (B) TCR antagonism of SS-GP-25 clone specific to p82–100 of MBP. (C) MHC blocking of SS-GP-42 clone unresponsive to p82–100 of MBP. (D) TCR antagonism of SS-GP-42 clone unresponsive to p82–100 of MBP. Irradiated (10,000 rad), EBV-transformed cells (0.3 × 10⁶/ml) were incubated with: peptide 82–100 of MBP [3 μM (□)] and [6 μM (○)] or MBP [1.5 μM (•)]. After 3 h, cells were washed extensively and recultured in individual wells of 96-well microculture plates (5 × 10⁴/well) with T cells (1.5 × 10⁴/well). Cop 1 was either preincubated in the first stage with the antigen to inhibit MHC binding or added in the second stage with the T cells to inhibit binding to the TCR. Results are expressed as mean cpm thymidine incorporation for triplicate cultures ×10⁻³ and represent one of three independent experiments.

Figure 3

Antagonistic effect of Cop 1 in a split APC assay on SS-GP-25 human T cell clone specific to MBP p82–100. (A) MHC blocking. (B) TCR antagonism. Peptide 82–100 of MBP [3 μM (□) and 6 μM (○)] and Cop 1 (3–12 μM) was either coincubated on the same APC (EBV-transformed cells, 0.3 × 10⁶/ml), competing for MHC binding, or presented on different APC, which were mixed only when added to the T cells, competing on binding to the TCR. After 3 h of incubation, the EBV cells were washed and added to the T cells (1.5 × 10⁴/well). Cultures were pulsed with 1 μCi [³H]thymidine at the end of 48 h of incubation. Results are expressed as mean cpm thymidine incorporation for triplicate cultures ×10⁻³ obtained in one of two independent experiments.