Alexander Y. Rudensky
ProfessorDepartment of Immunology
Memorial Sloan-Kettering Cancer Center
1275 York Ave.
New York, NY 10065
1993 Searle Scholar
Intracellular assembly and transport of endogenous peptide:MHC class II complexes.MHC class II molecules present peptide fragments, resulting from proteolytic degradation of protein antigens in the endocytic compartment of antigen-presenting cells, to CD4 T cells. Class II a and b chains form heterodimers in the absence of peptide and associate with a chaperone molecule invariant chain (Ii) in the endoplasmic reticulum (ER). Ii has an endosomal targeting signal and prevents the binding of peptides to MHC class II molecules until it is cleaved by endosomal proteases. A number of intracellular organelles, including early and late endosomes, lysosomes and in some experimental models even ER, were indirectly implicated in intracellular assembly of peptide/class II complexes.
We have studied intracellular sites and kinetics of assembly and trafficking of endogenous peptide/class II complexes in mouse and human B cells using biochemical and cell biological approaches. We found that the 80-104 region of Ii chain, termed CLIP serves as an MHC-binding site and binds within the peptide binding groove of class II molecules. CLIP remains associated with class II molecules after the rest of Ii is cleaved off. A monoclonal antibody recognizing CLIP bound to the class II molecule I-Ab was recently developed in our laboratory. Previously, we characterized another monoclonal antibody specific for an abundant endogenous complex of I-Ab bound to a peptide derived from the a chain of a second class II molecule I-E or HLA-DR (DRa62-68). These two unique reagents were used for detection of the corresponding complexes.
Subcellular fractionation, in combination with biosynthetic pulse chase labeling, showed that newly synthesized class II molecules are sorted to lysosome-like endocytic MIIC vesicles relatively late after synthesis. We showed that the CLIP/class II complex is formed in the MIIC. A MHC class II-like molecule, HLA-DM, localized to the MIIC, is required for rapid conversion of CLIP/class II complexes into class II complexes bound to antigenic peptides such as DRa62-68 peptide. We co-localized the latter complex and DM molecules to MIIC vesicles by immunoelectron microscopy. All three types of MIIC, namely, multilaminar, multivesicular and intermediate MIIC, contain this complex. Kinetic analyses suggested that DRa62-68/I-Ab complexes are probably formed in denser MIIC (multilaminar) and are exported to the cell surface via lighter MIIC (multivesicular) structures.
A potential role of the multivesicular MIIC in transport of peptide/class II complexes was further supported by our analyses of intracellular trafficking of ER-formed peptide/class II complexes. This was achieved by expression of a construct encoding the I-Ab molecule, covalently attached to the DRa52-68 peptide, in human B cells. Unlike wild type class II, these complexes do not associate with Ii chain. They exhibit greatly accelerated egress from the ER and rapid arrival at the plasma membrane after transient accumulation in endosomes. Importantly, these complexes were localized exclusively to multivesicular but not to multilaminar or mixed type MIICs. These results suggest a unique role of multivesicular and multilaminar MIICs in intracellular assembly and sorting of peptide/MHC class II complexes.
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