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, 5 (12), e15225

Liposome-coupled Antigens Are Internalized by Antigen-Presenting Cells via Pinocytosis and Cross-Presented to CD8 T Cells

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Liposome-coupled Antigens Are Internalized by Antigen-Presenting Cells via Pinocytosis and Cross-Presented to CD8 T Cells

Yuriko Tanaka et al. PLoS One.

Abstract

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8+ T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8+ T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8+ T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8+ T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates.
A, DQ-OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages expressing DM-DsRed (class II) or labeled with red fluorescein (class I), as described in Materials and Methods. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage DM or class I); cell only, macrophages without co-culture with DQ-OVA-coupled liposomes. B, the green- and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in Materials and Methods. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig. 1A. Asterisk, significant (p<0.01) difference of samples.
Figure 2
Figure 2. Uptake of liposome-coupled OVA by macrophages.
Alexa-labeled OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages as described in Materials and Methods. Thirty minutes after the onset of the culture, macrophages were recovered and analyzed using flow cytometry.
Figure 3
Figure 3. Influence of inhibitors for uptake of OVA coupled to oleoyl liposomes by macrophages.
Alexa- or DQ-labeled OVA was coupled to oleoyl liposomes and added to the culture of macrophages as described in Materials and Methods. Treatment of macrophages with cytochalasin B or DMA was done 60 minutes prior to the addition of OVA-liposome conjugates.
Figure 4
Figure 4. Intracellular localization of liposomal antigens taken up by macrophages.
A, DQ-OVA was coupled to oleoyl liposomes and added to the culture of cloned macrophages of which endosomal marker EEA1-positive compartments, or lysosomal marker LAMP-1-positive compartments were stained as described in Materials and Methods. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage EEA1 or LAMP-1); cell only, macrophages without co-culture with DQ-OVA liposomes. B, the green- and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in Materials and Methods. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig, 4A. Asterisk, significant (p<0.01) difference of samples.
Figure 5
Figure 5. IFN-γ production by splenic CD4/CD8+ T cells of mice immunized with OVA after co-culture with CD11c+ cells pulsed with OVA coupled to oleoyl liposomes.
Splenic CD4/CD8+ T cells were taken from mice immunized with OVA and were cultured with CD11c+ cells pulsed with OVA coupled to oleoyl liposomes with or without inhibitors as described in Materials and Methods. IFN-γ production of T cells in the supernatants in the absence of inhibitors was normalized to 100%. Data represent the mean values ± SD of triplicate culture. Asterisk, significant (p<0.01) difference as compared with the ‘no inhibitor’ group.

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