Antigen processing and intracellular Ia. Possible roles of endocytosis and protein synthesis in Ia function.
Anti-I-A mAb and monovalent Fab fragments were used to explore the cellular distribution and endocytosis of I-A in peritoneal exudate cells (PEC) and TA3 B lymphoma-hybridoma cells. TA3 cells contained 1.6 x 10(5) I-A sites/cell, 22 to 35% of which were intracellular. This intracellular pool was cycloheximide resistant. PEC contained 1.8 x 10(5) I-A sites/cell, 25 to 40% of which were intracellular. Upon adherence, however, the intracellular pool of I-A in PEC dropped to 2 to 11% of the total cellular I-A. Ag processing by TA3 cells was unaffected 3 h after abrogation of protein synthesis with cycloheximide, suggesting that newly synthesized I-A is not necessary for Ag processing in TA3 cells (post-synthetic processing and transport of I-A to the plasma membrane were complete by 2 h in TA3 cells with or without cycloheximide, as assessed by sequential immunoprecipitation of surface and intracellular I-A). In adherent PEC, however, cycloheximide markedly inhibited Ag processing, suggesting depletion of factors necessary for Ag processing. Ag processing may involve binding of processed Ag peptides to intracellular Ia derived to varying degrees from both endocytosis and new biosynthesis. To explore the possibility of I-A recycling, I-A endocytosis was demonstrated using mAb and monovalent Fab probes; internalization occurred within 5 min and peaked by 10 to 15 min with 15 to 35% of bound antibody in an intracellular compartment, resistant to an acid wash. Subcellular density gradient fractionation demonstrated that I-A and transferrin were processed exclusively in an endosomal fraction of relatively light density, whereas ligands of the mannose receptor were processed in light endosomes and in a distinct, denser population of endosomes, and accumulated in lysosomes. Thus, I-A appears to be internalized into a specific population of endosomes that may play a central role in Ag processing.
Harding
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Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Unanue
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001202:Ascitic Fluid / Q000378:metabolism; D001692:Biological Transport; D001826:Body Fluids / Q000378:metabolism*; D002451:Cell Compartmentation; D003513:Cycloheximide / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D000949:Histocompatibility Antigens Class II / Q000276:immunology / Q000378:metabolism*; D006825:Hybridomas / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008808:Mice, Inbred CBA; D009113:Muramidase / Q000378:metabolism; D014176:Protein Biosynthesis; D013347:Subcellular Fractions / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D007140:Immunoglobulin Fab Fragments; D003513:Cycloheximide; D009113:Muramidase