Early endosomes are required for major histocompatiblity complex class II transport to peptide-loading compartments

Antigen presentation to CD4(+) T lymphocytes requires transport of newly sy nthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii c hain, which directs the MHC class II-Ii complexes from the trans-Golgi netw ork (TGN) to endosomes. The subcellular machinery responsible for the speci fic targeting of MHC class II molecules to the endocytic pathway, as well a s the first compartments these molecules enter after exit from the TGN, rem ain unclear. We have designed an original experimental approach to selectiv ely analyze this step of MHC class II transport. Newly synthesized MHC clas s II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19 degrees C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor-containi ng endosomes using Tf-HRP complexes and the HRP-insoluble substrate diamino benzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the c ell surface. Thus, early endosomes appear to be required for delivery of MH C class II molecules to the endocytic pathway. Under cross-linking conditio ns, most alpha beta Ii complexes accumulated in tubules and vesicles devoid of gamma-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-in dependent pathway for the delivery of newly synthesized MHC class II molecu les from the TGN to endosomes.