The activation of cytotoxic T-cell (CTL) responses requires antigen presentation by professional antigen presenting cells. Macrophages (MØ) can regulate CTL responses but little is known about the role played by splenic macrophages (Sp-MØ) in antigen cross-presentation.
Here, we established, and characterized, an efficient culture method for generating Sp-MØ. By monitoring MØ markers, we found that 7-days Sp-MØ resembles the red pulp macrophages (RPMØ) phenotypic characteristics. The phagocytic capacity of Sp-MØ was increased as the cells become more differentiated. Thus, increased differentiation of Sp-MØ in vitro can be macrophage-colony stimulating factor (M-CSF) driven, which allows for an optimal condition to increase the yield of the spleen-derived MØ.
As a result of examining the antigen presentation of Sp-MØ during differentiation, we reported that Sp-MØ down-regulated their ability to cross-present the cell-associated lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) but not the soluble OVA proteins without altering their capacity to directly present LCMV antigens after infection. Mechanistically, we defined the cytosolic pathway as the dominant cross-presentation pathway used by Sp-MØ. Further analysis revealed a direct relationship between Sp-MØ differentiation, phagosomal acidification, and antigen cross-presentation. As Sp-MØ become more mature, their vesicular phagosomal system acquired high acidic characteristics, which adversely affected antigen cross-presentation due to potent and enhanced antigen degradation.
We also addressed the capacity of diverse LCMV antigens, generated during virus infection, to induce LCMV-specific CTL responses via cross-presentation by employing antigen donor cells (ADCs) that provide sufficient LCMV antigens after virus inactivation with no possible direct antigen presentation. Our results demonstrated that the ADCs induced LCMV-specific CTL responses in vitro and in vivo. Out of the four CTL epitopes tested (NP396, NP205, GP33, and GP276), in vitro cross-presentation were dominated by LCMV-NP396 epitope; while the in vivo cross-priming has shifted towards LCMV-GP33 and NP396 epitopes.
Collectively, the data presented in this thesis have defined for the first time important factors that influence Sp-MØ culturing in vitro and highlighted a potential role for the Sp-MØ in regulating CTL
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responses via cross-presentation, and characterized how different epitopes from LCMV are cross presented in vitro and in vivo. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-12 15:07:38.492
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/5529 |
| Date | 13 April 2010 |
| Creators | Alatery, Attiya |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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