The molecular events that regulate MHC restricted antigen presentation are poorly understood at this time. This proposal is based on the hypotheses that MHC Class II-peptide interactions are initiated in an intracellular compartment and that the intracellular trafficking patterns that lead to expression of an MHC-antigen complex at the cell surface are regulated events. We will use molecular and biochemical approaches to define the intracellular sorting events that lead to Class II localization in specific intracellular compartments in the endocytic pathway. Specifically, we will test our hypothesis that sequences within the Class II molecule (beta 80-84) control localization within late endosomal or lysosomal compartments by transferring the Class II sequence to a Class I molecule and determining if this altered Class I sequence is localized in the endocytic pathway. In addition, we will use a mutant class II molecule that lacks endosomal localization in the absence of Ii, but is dramatically localized in these compartments in the presence of Ii, to dissect the mechanism by which invariant chain enhances the expression of Class II molecules in the endocytic compartments of APC. In addition to these genetic techniques, two different methods (vectorial tagging of endosomal proteins and endosome vesicle purification using colloidal iron and electromagnetic isolation) will be established to selectively isolate proteins expressed in the endocytic pathway. These techniques, coupled with biochemical analyses of MHC and associated molecules will be used to define the sequence of events that leads to selective trafficking of MHC Class II and invariant chain molecules into the endocytic compartments, localization in specific subcompartments in this pathway and egress from these compartments for expression at the cell surface. Finally, because the goal of this work is to identify the importance of intracellular activities of Class II with antigen presentation, we will perform functional analyses on APC that have been constructed to vary in the sites of Class II localization. These APC will be used in antigen presentation assays designed to both concentrate and localize antigens in subsites within the endocytic pathway. The results of our analyses will allow us to understand the molecular events that regulate formation of peptide:Class II complexes at the cell surface for recognition by CD4 T cells leading to protective immune responses or to autoimmune phenomena.
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