PEPTIDE BINDING TO SURFACE CLASS-II MOLECULES IS THE MAJOR PATHWAY OFFORMATION OF IMMUNOGENIC CLASS-II-PEPTIDE COMPLEXES FOR VIABLE ANTIGEN-PRESENTING CELLS

Although studies on fixed APCs have demonstrated that peptide can bind to cell surface class II molecules, the mechanisms by which peptide-c lass II complexes are formed in viable cells is largely unexplored. To explore the possibility that peptide loading of class II molecules wa s occurring after endocytosis of peptides as well as by surface bindin g, we utilized an immunogenic hemagglutinin peptide (HAP 128-145) from the influenza strain A/Japan/57, and studied the appearance of surfac e complexes of HAP 128-145 bound to HLA-DRw11 molecules on human B-lym phoblastoid cells (BLCLs). Detection of the bound peptide was made pos sible by a rabbit antiserum (alphaHAP) raised against HAP 128-145, whi ch recognizes both the free peptide as well as peptide bound to DRw11 on living APCs. Pretreatment of the BLCLs with a variety of inhibitors of protein synthesis and intracellular trafficking failed to decrease the levels of HAP 128-145/DRw11 surface complexes. However, significa nt inhibition in the appearance of these complexes was caused by a dec rease in the temperature at which the cells were incubated with peptid e. Temperature-specific inhibition was also observed for fixed DRw11-p ositive APCs and purified DRw11 molecules indicating that the effect o f temperature was directly on the class II molecules. We conclude that surface binding of peptide by class II molecules on human B cells is a major pathway of formation of immunogenic class II-peptide complexes for at least some soluble antigenic peptides, and that endocytosis of soluble peptides with subsequent binding of peptide by intracellular class II molecules plays little if any role in the formation of such c omplexes. Moreover, class II molecules have evolved to stably bind pep tide optimally at physiologic temperatures, independent of cell metabo lism.