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Published on: October 30, 2015
Kinetics and intracellular pathways required for major histocompatibility complex II-peptide loading and surface
1Department of Microbiology, University of Illinois at Urbana-Champaign, IL, USA.
Insights
Macrophages utilize multiple vesicles for major histocompatibility complex (MHC) class II peptide loading and transport, not just specific organelles. This antigen processing occurs in both lysosomes and transferrin receptor-positive endosomes.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Antigen processing is crucial for adaptive immunity.
- Understanding the intracellular pathways of major histocompatibility complex (MHC) class II loading is essential.
- Murine macrophages play a key role in antigen presentation.
Purpose of the Study:
- To investigate the kinetics and intracellular pathways of antigen processing in macrophages.
- To elucidate the role of different vesicular compartments in MHC class II-peptide loading and transport.
- To utilize a novel fluorescent antigen probe for detailed mechanistic studies.
Main Methods:
- Construction of a fluorescent antigen probe (FITC10BSA).
- Application of flow cytometry and subcellular fractionation techniques.
- Analysis of antigen processing within murine macrophage populations.
Main Results:
- Macrophage antigen processing involves multiple vesicles, not limited to specific organelles.
- MHC class II-peptide loading occurs in both lysosomes and transferrin receptor-positive endosomes.
- Fluorescently labeled MHC II-peptide complexes were observed in transferrin receptor-positive organelles, indicating a retrograde pathway.
Conclusions:
- Macrophages employ a complex vesicular network for MHC class II-peptide loading and transport.
- Antigen processing and loading are not restricted to canonical lysosomal pathways.
- MHC II-peptide complexes may utilize retrograde transport pathways for cell surface presentation.
Abstract:
A fluorescent antigen, FITC10BSA, that is sensitive to several of the biochemical processes involved in antigen processing was constructed. In combination with both flow cytometry and subcellular fractionation, the unique probe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggested that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II-peptide loading and transport. Although newly formed MHC II-peptide complexes were detected in cathepsin D-positive, lysosomal associated membrane glycoprotein (LAMP-1)-positive lysosomes, MHC II-peptide loading also occurred in transferrin receptor-positive endosomes. Interestingly, MHC II-fluoresceinated complexes were only observed in transferrin receptor-positive organelles as opposed to MHC II-unlabelled peptide complexes which were detected in traditional early lysosomal compartments. More importantly, MHC II-peptide complexes were monitored in light transferrin receptor-positive fractions following their initial appearance in dense endosomal/lysosomal fractions. Control experiments suggested that these complexes represented intermediates in the process of migrating to the cell surface through a retrograde pathway within the macrophage.
