When the cationic lipid DOTAP (1,2-dioleyltrimethylammoniumpropane) is used to encapsulate an antigenic peptide from the human papillomavirus E7 oncogene (E7), the resultant DOTAP/E7 particles act as a therapeutic vaccine to cause tumor regression through an antigen-specific immune response when the vaccine is injected into mice bearing E7-positive TC-1 tumors. Of critical importance, the DOTAP works as both a delivery vehicle and an adjuvant without induction of a pro-inflammatory cytokine response in vivo. It is hypothesized the antigen specific immune response is mediated by dendritic cells in vivo. To that end, the interaction of murine bone marrow-derived dendritic cells (BMDC) with the vaccine in vitro was investigated. When BMDC were incubated in the presence of DOTAP or the DOTAP/E7 vaccine, there was a dose and time-dependent upregulation of co-stimulatory molecule expression, indicating that BMDC were activated by the cationic lipid DOTAP. Further experiments indicated that BMDC were capable of internalizing DOTAP liposomes through many endocytic routes and the vaccine trafficked through the vacuolar pathway. An indirect method was used validate antigen presentation by BMDC, wherein the generation of antigen specific effector cells after incubation of CD8+ T lymphocytes, purified from the spleens of naïve mice, with fixed dendritic cells that had been activated by the vaccine, was examined. Not surprisingly, the DOTAP/E7 therapeutic vaccine was capable of initiating the generation of effector CD8+ T lymphocytes in vitro through a conventional mechanism, which requires dendritic cell activation and presentation of the peptide antigen. Interestingly, it was found the same simple vaccine was capable of generating antigen specific effectors in vitro in the absence of antigen presenting cells in a novel pathway of effector generation, based on the expression of CD8 on the cell surface, T cell receptors that recognize a specific peptide antigen/MHC complex as well as an antigen-specific increase in IFN-γ production. Further studies revealed a possible mechanism for this novel pathway. Taken as a whole, these observations may lead to additional applications of DOTAP both in vitro and in vivo to modulate the immune response toward the correction of a variety of diseases.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08062009-100439 |
| Date | 07 August 2009 |
| Creators | McEwen, Lisa Marie |
| Contributors | Stefan Duensing, Leaf Huang, Paul Robbins, Louis Falo, Saleem Khan |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08062009-100439/ |
| Rights | restricted, 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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