Binding of copolymer 1 and myelin basic protein leads to clustering of class II MHC molecules on antigen-presenting cells

Copolymer 1 (Cop 1), a synthetic copolymer of amino acids, effective in suppression of experimental allergic encephalomyelitis (EAE) and myelin basic protein (MBP), was shown to bind extensively and promiscuously to the class II MHC molecules on antigen-presenting cells (APC) without prior processing. In the case of human APC, binding has earlier been demonstrated to DR but not DQ or class I molecules. In the present study, we examined whether binding of Cop 1 and MBP affects MHC class II expression on the cell membrane. Biotinylated derivatives of these antigens were used to monitor their direct binding to MHC molecules on living APC by flow cytometry using phycoerythpin-streptavidin, while the levers of MHC surface expression were monitored by staining with FITC-conjugated anti-class I- and class II-specific antibodies. When Cop 1 or MBP were incubated with the APC, intensity of cell staining with anti-DR, but not with anti-DQ or anti-class I antibodies, was significantly increased, compared to the staining of control APC not reacted with these antigens. In contrast, staining intensity was unaffected when p84-102, a human immunodominant epitope of MBP, or ovalbumin (OVA), a protein which undergoes proteolytic degradation prior to binding, were incubated with the APC. Cycloheximide, a protein synthesis inhibitor, had no effect on the enhanced staining intensity with anti-DR antibody of cells treated with Cop 1 or MBP, whereas it inhibited the enhanced staining of both DR and DQ molecules caused by the respective antibodies, in the absence of these antigens. Brefeldin A, a protein transport inhibitor, lowered the levels of staining intensity with anti-DR and anti-DC antibodies in both cases, with and without antigen added to the APC. Fluorescence microscopic analysis revealed that cells incubated with Cop 1 or MBP, but not with p84-102 or OVA, exhibit both bright staining of the cell membrane and clusters produced by the aggregation of DR molecules with these antigens. Taken together, these observations indicate that Cop 1 and MBP, due to their polyvarent character, lead to increased fluorescence intensity of their complexes with HLA-DR, possibly due to recruitment and clustering of previously synthesized DR molecules. explain the efficient binding of these antigens to the MHC class II molecules.