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Characterization of dendritic cell handling of cell-associated membrane and cytoplasmic proteins from live and apoptotic cells

Gleason, Sherrianne M. (2008) Characterization of dendritic cell handling of cell-associated membrane and cytoplasmic proteins from live and apoptotic cells. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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 cell's viability and that the DCs ability to capture cell-associated protein would vary based on the protein's 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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Gleason, Sherrianne
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBarratt-Boyes, Simon Msmbb@pitt.eduSMBB
Committee MemberRinaldo, Charles Rrinaldo@pitt.eduRINALDO
Committee MemberHendricks, Robert Lrlh13@pitt.eduRLH13
Committee MemberSalter, Russell Drds@pitt.eduRDS
Committee MemberWatkins, Simon Cswatkins@pitt.eduSWATKINS
Date: 29 September 2008
Date Type: Completion
Defense Date: 31 July 2008
Approval Date: 29 September 2008
Submission Date: 30 July 2008
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Infectious Diseases and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: protein uptake; antigen uptake; endocytosis
Other ID:, etd-07302008-103641
Date Deposited: 10 Nov 2011 19:55
Last Modified: 15 Nov 2016 13:47


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