Levine, Timothy Paul; (1993) Aspects of the cell biology of antigen processing for MHC II. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Antigen processing for presentation by antigen presenting cells (APCs) expressing class II major histocompatibility complex molecules (MHC II) involves partial digestion of antigen, binding of antigen-derived peptides to MHC II, and intracellular trafficking of both antigen and MHC II. In this thesis, two major aspects of these intracellular events have been studied. A flow cytometric assay was developed to measure fluid-phase endocytosis by dendritic cells, potent APCs which have been shown to have poor lysosomal function, and which have been suggested to have a low level of endocytic activity. The assay used a two compartment model to measure the separate activities of early endosomes and late endosomes. Comparison of dendritic cells with different B lymphocyte APCs showed that endocytic traffic through late endosomes, some of which are thought to be related to the site of peptide loading, was similar in dendritic cells and B cells. Thus, the low endocytic activity reported elsewhere was not detected in this project. Organelles containing MHC II were studied in a cell-free system using membranes from disrupted APCs. These organelles were studied by density gradient centrifugation and separated from total membrane by immuno-isolation on magnetic beads. Density gradient centrifugation demonstrated that organelles containing newly synthesized MHC II had a marginally higher density than early endosomes. Immuno-isolated MHC II was found to co-isolate with endocytic markers, in particular markers of early endosomes. This indicates that MHC II is widely distributed throughout the endocytic pathway, including early endosomes, even though it is not widely thought that antigen binds MHC II in early endosomes. In addition, immuno-isolated MHC II was enriched for newly synthesized molecules, which indicates that the organelles where peptide loading is thought to occur were immuno-isolated. In initial experiments, immuno-isolated newly synthesised MHC II was used to perform some of the biochemical events of antigen processing.

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