ANALYSIS OF LYMPHOCYTIC CHORIOMENINGITIS VIRUS ANTIGENS PRESENTATION BY MACROPHAGES

Abstract

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 iii responses via cross-presentation, and characterized how different epitopes from LCMV are cross presented in vitro and in vivo.