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320 Characterization of Live Cell Antigen Acquisition by Dendritic Cells: Implications for Expanding and Regulating Immune Responses
L. A. Harshyne*, S. C. Watkins, A. Gambotto, and S. M. Barratt-Boyes
Graduate Sch. of Publ. Hlth., Univ. of Pittsburgh, PA, USA
Background: Dendritic cells (DC) are sentinels of the immune system that survey peripheral tissues for antigen. To facilitate this function DC acquire antigen from a variety of sources; soluble protein, particulate antigen, or antigen released from cells undergoing apoptosis or necrosis. We have recently identified a novel process by which DC acquire antigen from other live cells for cross-presentation to T cells and sought to further characterize this form of antigen uptake.
Methods: Fluorescent dyes were used to label membrane and cytoplasmic proteins, which were then tracked within recipient DC using various methods of fluorescent imaging including; live cell microscopy, confocal microscopy, and flow cytometry. Inhibitors of endocytosis, monoclonal antibodies, and fluorescent conjugates were used to delineate various mechanisms of antigen uptake and subcellular localization.
Results: Real-time video microscopy showed that live-cell antigen acquisition required intimate cell contact and occurred within 4 hours. We determined that live-cell antigen acquisition by DC requires actin modulation, Na+/H+ antiporters, and is Ca+ dependent. In contrast, live-cell antigen uptake does not require microtubule restructuring, is alpha-integrin independent, and is not inhibited by mannan. Furthermore, removal of membrane proteins abrogates live-cell antigen acquisition by DC. This profile implicates a receptor-mediated form of phagocytosis. Finally, we show that acquired antigen is localized both at the plasma membrane, and within the endosomal pathway.
Conclusions: We show that DC acquire antigen from healthy cells utilizing a form of phagocytosis distinct from macropinocytosis/apoptotic uptake and route it into endocytic vesicles. We suggest that by defining molecules and mechanism(s) involved in live-cell antigen acquisition and cross-presentation to T cells, we can expand our basic knowledge of DC immunobiology, thereby enabling us to develop better vaccines to infectious agents, including HIV.
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