title: Tapasin-/- and TAP1-/- macrophages are deficient in vacuolar alternate class I MHC (MHC-I) processing due to decreased MHC-I stability at phagolysosomal pH.
abstract: Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin(-/-) cells and experiments with tapasin(-/-) and TAP1(-/-) macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin(-/-) cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin(-/-) cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin(-/-) and TAP1(-/-) macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin(-/-) and TAP1(-/-) macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin(-/-) or TAP1(-/-) cells at 26 degrees C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.
authors.affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Chefalo
authors.forename: Andres G
authors.initials: AG
authors.lastname: Grandea
authors.forename: Luc
authors.initials: L
authors.lastname: Van Kaer
authors.forename: Clifford V
authors.initials: CV
authors.lastname: Harding
mesh_terms: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000143:Acids; D000818:Animals; D017951:Antigen Presentation / Q000235:genetics*; D000942:Antigens, Bacterial / Q000276:immunology / Q000378:metabolism; D017920:Antiporters / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D004527:Egg Proteins / Q000276:immunology / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006183:H-2 Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007136:Immunoglobulins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010047:Ovalbumin / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments; D010588:Phagosomes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D013696:Temperature; D014617:Vacuoles / Q000276:immunology / Q000378:metabolism
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D000143:Acids; D000942:Antigens, Bacterial; D017920:Antiporters; D004527:Egg Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; C110366:OVA-8; D010446:Peptide Fragments; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; C107762:tapasin; D010047:Ovalbumin