Kinetics and Intracellular Pathways Required for MHC Ii-Peptide Loading and Surface Expression of a Hapten-Protein Conjugate in Murine Macrophage
type: CreativeWork
description: Unlike B lymphocytes which recognize antigenic proteins in native form, CD 4+ T lymphocytes recognize fragments of foreign proteins in the context of the major histocompatibility complex class II molecule (MHC II). The sequence of events that lead to the formation of these MHC II-peptide complexes has been termed antigen processing, and several specific questions related to this pathway remain unanswered. Therefore, a novel fluorescent hapten-protein antigen consisting of the small hapten, fluorescein, conjugated to the protein, bovine serum albumin, was constructed. In the globular form, the antigenic probe is relatively non-fluorescent due to the short Stoke's shift of fluorescein and the close spatial proximity of the fluorescein molecules. However, upon unfolding or proteolytic degradation of the carrier, the spatial constraint is alleviated and a significant increase in fluorescence intensity results. For this reason, the probe is sensitive to several intracellular events involved in antigen processing. By combining this novel antigenic protein with several techniques including flow cytometry and subcellular fractionation, new insights into the cell biology of antigen processing in murine macrophage were revealed. Specifically, the proteolytic enzymes involved in the cleavage of the antigen act at different points within the endocytic system providing a vast array of peptides for MHC II binding. Subcellular fractionation experiments demonstrated that MHC II-peptide complexes form in multiple subcellular organelles analogous to late endosomes and lysosomes and that MHC II-peptide complexes reach the surface of the cell via the existing endocytic machinery in transferrin receptor positive compartments. These studies also suggested that a novel macrophage surface receptor specific for polyaromatic compounds affected the processing of the probe by increasing the concentration of the probe 100-fold relative to the extracellular media and by influencing the subcellul…
abstract: Unlike B lymphocytes which recognize antigenic proteins in native form, CD 4+ T lymphocytes recognize fragments of foreign proteins in the context of the major histocompatibility complex class II molecule (MHC II). The sequence of events that lead to the formation of these MHC II-peptide complexes has been termed antigen processing, and several specific questions related to this pathway remain unanswered. Therefore, a novel fluorescent hapten-protein antigen consisting of the small hapten, fluorescein, conjugated to the protein, bovine serum albumin, was constructed. In the globular form, the antigenic probe is relatively non-fluorescent due to the short Stoke's shift of fluorescein and the close spatial proximity of the fluorescein molecules. However, upon unfolding or proteolytic degradation of the carrier, the spatial constraint is alleviated and a significant increase in fluorescence intensity results. For this reason, the probe is sensitive to several intracellular events involved in antigen processing. By combining this novel antigenic protein with several techniques including flow cytometry and subcellular fractionation, new insights into the cell biology of antigen processing in murine macrophage were revealed. Specifically, the proteolytic enzymes involved in the cleavage of the antigen act at different points within the endocytic system providing a vast array of peptides for MHC II binding. Subcellular fractionation experiments demonstrated that MHC II-peptide complexes form in multiple subcellular organelles analogous to late endosomes and lysosomes and that MHC II-peptide complexes reach the surface of the cell via the existing endocytic machinery in transferrin receptor positive compartments. These studies also suggested that a novel macrophage surface receptor specific for polyaromatic compounds affected the processing of the probe by increasing the concentration of the probe 100-fold relative to the extracellular media and by influencing the subcellul…
author: Weaver, Donald John, Jr
language: eng
keywords: Biology, Microbiology
date published: 2015-09-28T15:17:46Z
date modified: 2026-07-22T22:26:27Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/86742