Biochemical evidence for the rapid assembly and disassembly of processed antigen-major histocompatibility complex class II complexes in acidic vesicles of B cells

Helper T cell recognition of antigen requires that it be processed within antigen-presenting cells (APC) to peptide fragments that subsequently bind to major histocompatibility complex (MHC) class II molecules and are displayed on the APC surface. Heretofore, processed antigen-MHC class II complexes have been detected by functional assays, measuring the activation of specific T cells. We now report direct, biochemical evidence for the assembly of processed antigen-MHC class II complexes within splenic B cells as APC. The I-Ek MHC class II molecules were immunoprecipitated from B cells that had processed the model protein antigen cytochrome c radiolabeled across its entire length by reductive methylation of lysine residues and covalently coupled to Ig-specific antibodies, allowing internalization after binding to surface Ig. Our previous studies showed that I-Ek immunoaffinity purified from B cells that had processed cytochrome c contains functional processed antigen--MHC class II complexes and that approximately 0.2% of the I-Ek molecules are specifically associated with one of two predominant processed antigenic fragments. Here we show that these complexes are rapidly assembled, within 30-60 min after antigen binding to surface Ig on splenic B cells. Maximal numbers of complexes are assembled by 2 h in a process that is sensitive to acidic vesicle inhibitors but not to inhibitors of protein synthesis. The processed antigen-I-Ek complexes have a relatively short half-life of 2- 4 h and are disassembled or degraded within 8 h after antigen is first internalized. The disassembly or degradation of the processed antigen-I- Ek complexes requires acidic vesicle function, and in the presence of an acidic vesicle inhibitor the complexes are long lived. Thus, using a biochemical assay to monitor processed antigen-I-Ek complexes, we find that, in B cells, processed antigen is relatively rapidly associated in acidic vesicles with preexisting MHC class II molecules, and the complexes are disassembled 4-6 h later in processes that also require acid vesicle function.