Secondary CTL response in mice immunized with OVA_257-264-liposome.

<p>Mice were immunized with 50 µl of OVA_257-264-liposome in the presence of 5 µg CpG, and 2, 4, 8, 16, and 20 weeks later, they received a booster ip injection with 200 µl of 1 mg/ml OVA in PBS (closed box) or no booster injection (open box). Three days after the booster injection, <i>in vivo</i> CTL assay was performed. Data represent mean percentages of cells killed and SEs of three mice per group. ND, not detected. *, significant difference (p>0.01).</p

Antigen-specific CD8⁺ T-cell proliferation assay.

<p>Mice were immunized with OVA_257-264-liposome and 1 week or 20 weeks later, CD8⁺ T cells of the immunized mice were cultured in the presence (closed box) or absence (open box) of OVA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015091#s4" target="_blank">Materials and Methods</a>. Data represents mean ³H- thymidine incorporation and SE of triplicate cultures. *, significant difference (p>0.01).</p

Kinetics of primary CTL response induced by OVA_257-264-liposome conjugates.

<p>Mice were immunized with 50 µl of OVA_257-264-liposome in the presence of 5 µg CpG; one to 5 days later, an <i>in vivo</i> CTL assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015091#s4" target="_blank">Materials and Methods</a>. The numbers for each time period indicate percentages of target cells killed. Data are representative of three individual mice in each group for which similar results were obtained.</p

Effect of <i>in vivo</i> elimination of CD4⁺ T cells on the induction of primary and secondary CTL responses by OVA_257-264-liposomes.

<p>Mice with (closed box) or without (open box) CD4⁺ T-cell elimination were immunized with 50 µl of OVA_257-264-liposome solution in the presence of 5 µg CpG, and CTL induction was monitored. <b>A</b>, CTL response 1 week after immunization. <b>B</b>, CTL response 20 weeks after immunization with or without booster injection. <i>In vivo</i> CTL assay was performed 3 days after the booster injection. Data represent mean percent killing and SE of three mice per group. ND, not detected. *, significant difference (p>0.01).</p

Dose-response of cytokine production by CD8⁺ T cell and CTL induction in mice immunized with OVA_257-264-liposome or with OVA_257-264 solution.

<p>A serial two-fold dilution of OVA_257-264-liposome (open box) and OVA_257-264 solution (closed box) were made in PBS, and mice were immunized with the diluents in the presence of 5 µg CpG. OVA_257-264 solution containing equal amounts of peptides as those in OVA_257-264-liposome. One week after the immunization, IFN-γ production by CD8⁺ T cells (<b>A</b>) and the CTL response (<b>B</b>) were monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015091#s4" target="_blank">Materials and Methods</a>. Data represent means and SE of three mice per group. *, significant difference (p>0.01).</p

Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig. 1A. Asterisk, significant (<i>p</i><0.01) difference of samples.</p

Intracellular localization of liposomal antigens taken up by macrophages.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig, 4A. Asterisk, significant (<i>p</i><0.01) difference of samples.</p

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. 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 (<i>p</i><0.01) difference as compared with the ‘no inhibitor’ group.</p

Influence of inhibitors for uptake of OVA coupled to oleoyl liposomes by macrophages.

<p>Alexa- or DQ-labeled OVA was coupled to oleoyl liposomes and added to the culture of macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015225#s4" target="_blank">Materials and Methods</a>. Treatment of macrophages with cytochalasin B or DMA was done 60 minutes prior to the addition of OVA-liposome conjugates.</p

Establishment of GM-IMs from murine BM.

<div><p>(A) BM cells were taken from two-month old C57BL/6 or EGFP mice. Suspensions of BM cells (3x10⁶ cells) were plated in ten cm diameter non-coated plastic culture dishes. They were cultured in RPMI-1640 supplemented with 10% GM-CSF-CM and 10% FBS. Lower panel shows phase contrast photograph of BM cultured with 10% GM-CSF-CM for six days or six months. Scale bars: 50 µm.</p> <p>(B) Growth curve of GM-IMs. BM cells cultured with 10% GM-CSF-CM for the indicated times, during which cells became GM-IMs. Data shown are the mean ratios ± SE of four independent experiments.</p> <p>(C) SA-β-gal staining of BM cells cultured with 10% GM-CSF-CM for the indicated times, during which cells became GM-IMs. Scale bars: 100 µm.</p> <p>(D) Expression of cellular senescent marker proteins. Cell lysates were Western blotted for p16. Β-actin was used as a control.</p> <p>(E) May-Grunwald-Giemsa staining of GM-IMs. Five months cultured GM-IMs was compared with days zero and six of BM culture in 10% GM-CSF-CM, BMMφ and peritoneal macrophages (PMφ). Scale bars: 50 µm.</p> <p>Data are representative of three independent experiments with similar results (A, C-E).</p></div