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.
Highlights
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TapasinϪ/Ϫ and TAP1Ϫ/Ϫ macrophages are deficient in alternate MHC-I Ag processing, but still present exogenous peptide, suggesting a deficit in MHC-I stability and function specific to the vacuolar processing environment
Macrophages were incubated with bacterial Ag (E. coli HB101.Crl-OVA) to assess vacuolar alternate MHC-I Ag processing or with exogenous SIINFEKL peptide (OVA257–264) to assess peptide-receptive MHC-I at the cell surface
Summary
MHC-I, class I MHC; BFA, brefeldin A; BMM, bone marrow-derived macrophages; CBS, citrate-buffered saline; ER, endoplasmic reticulum; PEM, peritoneal exudate macrophages; PeM, peritoneal macrophages. The present studies provide the first examination of alternate MHC-I Ag processing in tapasinϪ/Ϫ cells Macrophages from both TAP1Ϫ/Ϫ and tapasinϪ/Ϫ mice have MHC-I molecules that are poorly loaded, i.e., contain peptides that are not bound with high affinity. In addition to defects in conventional MHC-I Ag processing, TAP-deficient cells may have decreased levels of postGolgi peptide-receptive MHC-I molecules that are needed for vacuolar alternate MHC-I Ag processing [8, 18, 38] These deficits are reversed by enhancing the expression of post-Golgi peptide-receptive MHC-I molecules by incubation at 26°C, addition of 2-microglobulin or addition of exogenous stabilizing peptide [8, 9, 18, 38]. Our observations suggest that MHC-I presentation of peptides derived from exogenous Ag requires stable precursor peptide:MHC-I complexes that enter the phagosome, where acidic pH facilitates peptide exchange to form complexes of MHC-I with antigenic peptides from exogenous Ag
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