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 i n suppression of experimental allergic encephalomyelitis (EAE) and mye lin basic protein (MBP), was shown to bind extensively and promiscuous ly to the class II MHC molecules on antigen-presenting cells (APC) wit hout prior processing. In the case of human APC, binding has earlier b een 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 o n 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 C op 1 or MBP were incubated with the APC, intensity of cell staining wi th anti-DR, but not with anti-DQ or anti-class I antibodies, was signi ficantly increased, compared to the staining of control APC not reacte d 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 inhib itor, had no effect on the enhanced staining intensity with anti-DR an tibody of cells treated with Cop 1 or MBP, whereas it inhibited the en hanced staining of both DR and DQ molecules caused by the respective a ntibodies, in the absence of these antigens. Brefeldin A, a protein tr ansport inhibitor, lowered the levels of staining intensity with anti- DR and anti-DC antibodies in both cases, with and without antigen adde d to the APC. Fluorescence microscopic analysis revealed that cells in cubated with Cop 1 or MBP, but not with p84-102 or OVA, exhibit both b right staining of the cell membrane and clusters produced by the aggre gation of DR molecules with these antigens. Taken together, these obse rvations indicate that Cop 1 and MBP, due to their polyvarent characte r, lead to increased fluorescence intensity of their complexes with HL A-DR, possibly due to recruitment and clustering of previously synthes ized DR molecules. explain the efficient binding of these antigens to the MHC class II molecules.