Dendritic cells (DCs) are a heterogeneous population of immune cells that influence a wide variety of immune responses, including immunity to infectious diseases and malignant tumors, and in the generation of tolerance. In their immature state, DCs are highly specialized at capturing and internalizing exogenous antigens. Cell-associated antigens are of special interest because they play a role in both the induction of immunity and tolerance. This study aimed to add to the field of DC biology by further describing how DCs handle cell-associated proteins from both live and apoptotic cells. We hypothesized that the DCs ability to capture, internalize, and process integral membrane proteins would vary based on the target cells viability and that the DCs ability to capture cell-associated protein would vary based on the proteins intracellular localization. To quantitatively and qualitatively characterize uptake, we created a biologically relevant system using the Epstein Barr virus latent membrane protein 2 and the melanoma protein gp100, each fused to the enhanced green fluorescent protein (EGFP) and expressed at the outer plasma membrane of a tumor cell line, along with a cell line expressing EGFP in the cytoplasm. We found (1) DCs captured integral membrane proteins but not cytoplasmic protein from live cells; (2) DCs captured membrane and cytoplasmic proteins from apoptotic cells more efficiently and at a faster rate than from live cells; (3) during direct physical interactions DCs transiently surveyed live cells capturing small quantities of membrane, but stayed in prolonged contact with apoptotic cells while continuously internalizing membrane fragments; (4) DC internalization of membrane protein from live cells was clathrin-dependent while uptake from apoptotic cells was clathrin- and caveolae-dependent; and (5) internalized membrane protein from both live and apoptotic cells was found in early endosomes, late endosomes, and lysosomes. This work has potential broad public health implications as it is important to understand all aspects of DC biology when developing vaccines for both chronic and acute diseases. We hope that by uncovering the intricacies of DC handling of cell-associated proteins we will gain a better understanding of how to possibly manipulate DCs in order to influence the immune response.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07302008-103641 |
| Date | 29 September 2008 |
| Creators | Gleason, Sherrianne M. |
| Contributors | Charles R. Rinaldo, Robert L. Hendricks, Simon M. Barratt-Boyes, Simon C. Watkins, Russell D. Salter |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf, video/x-msvideo |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07302008-103641/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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