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[Abstract]

Human Dendritic Cells: Cell Culture Models for Studies of Particulate Antigen Formulations in vitro

Camilla Foged

Vaccine development is challenged by the fact that many new subunit vaccines based on peptides, proteins and DNA are poorly immunogenic and fail to fully mobilize immune responses. Adjuvants are therefore needed in the vaccine formulation to help eliciting an adequate immune response. However, few adjuvants are approved for use in humans, and there is thus an unmet medical need for the development of new adjuvants. Particulate antigen delivery systems are attractive alternatives to traditional adjuvants by being stable carriers and effective adjuvants. It is believed that passive targeting of particulate antigens to antigen presenting cells causes part of the adjuvant effect. Dendritic cells (DCs) are specialized antigen presenting cells with a well documented and superior ability to stimulate specific major histocompatibility complex (MHC)- restricted immune responses.

The overall aim of the present studies was to examine interactions between DCs and particulate antigen delivery systems in vitro through the establishment of appropriate cell culture models. The first purpose was to describe kinetic and functional aspects of general particulate physiochemical characteristics such as size and charge upon the interaction in vitro with DCs. DCs were generated from human peripheral blood mononuclear cells and characterized with respect to expression of surface markers associated with maturation and antigen presentation. Fluorescent E.coli and polystyrene microspheres were used as model particle structures, and the surface of the spheres was modified by covalent attachment of peptides and proteins. Interaction with DCs was studied by flow cytometry and confocal microscopy. DCs were able to take up particles in the tested size range of 0.04-15 µm in diameter. Microspheres of the size 1-15 µm interacted with a low percentage of the DCs while optimal size range for fast and efficient acquisition by a substantial percentage of the DCs was observed for particles below 0.5 µm. Negatively charged particles were generally taken up to a relatively small extent while positively charged particles interacted much more efficiently with DCs.

Surface characteristics were next shown to be equally important for the interaction between DCs and a potential vaccine formulation, exemplified by antigen-containing liposomes, through investigations of how liposome bilayer composition affected the interaction. Anionic liposomes with a bilayer composition of phosphatidylcholine, cholesterol and phosphatidylglycerol or phosphatidylserine interacted with a limited fraction of the total DC population in case of human monocyte-derived cells as well as murine bone marrow-derived DCs. Inclusion of mannosylated phosphatidylethanolamine for targeting to the mannose receptor increased the interaction of negatively charged liposomes with both human and murine DCs. This increase could be blocked in human DCs by addition of the polysaccharide mannan indicating that uptake might be mediated by the mannose receptor. Cationic liposomes containing trimethyl ammonium propane interacted with a very high percentage of both DC types and could be detected in substantial amounts intracellularly.

From these results it can be concluded that uptake of particles by DCs largely depends on particle size, surface charge and, in case of antigen-containing liposomes, bilayer composition. Such variables might be critical for the vaccination outcome.

Activation of T-cells is the functional read-out of particle-mediated antigen delivery to DCs and this parameter is therefore maybe the most important to assess for vaccine formulations. The final aim was to explore the potential of using human DC-T-cell co-culture systems to evaluate functional T-cell responses upon particle-mediated antigen delivery to DCs in vitro. DCs were pulsed with particle-associated tetanus toxoid (TT) or free TT. DC maturation was induced by addition of tumor necrosis factor a, and antigen-loaded DCs were subsequently co-cultured with autologous, carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled peripheral blood mononuclear cells. T-cell proliferation was measured by determination of CFSE-fluorescence intensity in CD4+ and CD8+ T-cells by flow cytometry. Pulsing of DCs with free TT induced proliferation of CD4+- and CD8+ T-cells in an experimentally reproducible manner. The extent of CD4 versus CD8 T-cell proliferation varied between individuals. Increased background proliferation was observed if fetal calf serum was supplied to the cultures, which could be reduced by supplementing autologous serum instead of fetal calf serum. The MHC-restriction of the CD4+ T-cell anti-TT response could be determined by MHC class II blocking experiments. DCs pulsed with particle-bound, as compared to free TT, induced individually variable T-cell responses, for one individual CD4+ T-cell division was enhanced by associating TT to particles. In conclusion the method allows for a determination of frequencies of responding specific CD4+ and CD8+ T-cells upon antigen loading of autologous DCs, thus provides a tool for analyses and comparisons of functional human T-cell responses to different antigen formulations.

The established methods enable the human, pre-clinical in vitro study of the induction of several crucial events during vaccination (antigen uptake, presentation and T-cell stimulation) caused by the interaction between DCs and particulate antigen delivery systems, for which the composition appears to be essential. Such methods will become useful for modulating and optimizing the interaction between DCs and particulate antigen delivery systems, among that for the evaluation of vaccination strategies that actively target antigens to DCs.

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