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Regulation of Immune Responses by Genetically-Engineered Dendritic Cells and Exosomes

Ruffner, Melanie Anne (2010) Regulation of Immune Responses by Genetically-Engineered Dendritic Cells and Exosomes. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Approximately 5-8% of Americans are affected by some type of autoimmune disease. The current standard of care for these patients involves pharmacologic therapy to induce systemic immunosuppression, which carries significant side effects. There is a need for therapies that effectively restore immune-mediated tolerance towards the body, but do not cause inappropriate immunosupression.The following dissertation details the synthesis, characterization, and performance of tolerogenic dendritic cells (DC), as well as the exosomes that they secrete. Exosomes are small (40-100 nm) membrane-bound microvesicles produced by reverse budding of the membrane of the multivesicular endosome in DC, as well as many other cell types. DC were transduced with adenoviral vectors expressing target genes in order to manipulate their function. After transduction, DC and their exosomes were examined for the ability to modulate disease-induced inflammation in the NOD mouse model of type 1 diabetes and the delayed-type hypersensitivity (DTH) model.DC transduced with an IL-4 expressing vector are capable or preventing the onset of hyperglycemia when administered to 12-weeks-old and 12-16 week-old prediabetic mice. Treated mice demonstrate modulation of T-cell mediated β-cell autoimmunity, including reduced insulitis, increased FoxP3 expression in the islet-draining lymph nodes, and Th2 skewing of islet-antigen induced cytokine secretion profile. We further demonstrate that transduction of DC with adenoviral vectors expressing indoleamine 2,3-diooxygenase (IDO) and CTLA-4-Ig are efficient means to generate IDO+ DC in vitro, and that these DC effectively reduce paw swelling in the DTH model of antigen-specific inflammation. The exosomes secreted by these IDO+ DC contain IDO protein, and suppress DTH responses at a level comparable to their parental DC in a manner dependent on B7-1 and B7-2. To confirm the role of B7 costimulatory molecules on tolerogenic DC and exosomes, we further demonstrate that B7-1 and B7-2, but not PD-L1 or PD-L2, are required for in vivo suppressive activity of rIL-10 treated DC and their secreted exosomes.This endeavor has increased the knowledge regarding therapeutic DC and exosomes. We have demonstrated strategies to generate tolerogenic DC and exosomes, as well as factors that are required for their activity in vivo.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Ruffner, Melanie Annemer26@pitt.eduMER26
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRobbins, Paul Dprobb@pitt.eduPROBB
Committee MemberMorelli, Adrian Emorelli@pitt.eduMORELLI
Committee MemberHuard, Johnnyjhuard@pitt.eduJHUARD
Committee MemberLotze, Michael Tlotzemt@upmc.eduMTL5
Committee MemberAtaai, Mohammad Mataai@engr.pitt.eduATAAI
Date: 26 January 2010
Date Type: Completion
Defense Date: 5 August 2009
Approval Date: 26 January 2010
Submission Date: 26 August 2009
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Dendritic Cells; Exosomes; Hypersensitivity; Interleukin-4; Type 1 Diabetes; Adenoviral Gene Transfer; Immunoregulation
Other ID:, etd-08262009-165551
Date Deposited: 10 Nov 2011 20:01
Last Modified: 19 Dec 2016 14:37


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