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

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

Liposome-coupled peptides induce long-lived memory CD8 T cells without CD4 T cells.

CD8(+) T cells provide broad immunity to viruses, because they are able to recognize all types of viral proteins. Therefore, the development of vaccines capable of inducing long-lived memory CD8(+) T cells is desired to prevent diseases, especially those for which no vaccines currently exist. However, in designing CD8(+) T cell vaccines, the role of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells remains uncertain. In the present study, the necessity or not of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells was investigated in mice immunized with liposome-coupled CTL epitope peptides. When OVA-derived CTL epitope peptides were chemically coupled to the surfaces of liposomes and inoculated into mice, both primary and secondary CTL responses were successfully induced. The results were further confirmed in CD4(+) T cell-eliminated mice, suggesting that CD4(+) T cells were not required for the generation of memory CD8(+) T cells in the case of immunization with liposome-coupled peptides. Thus, surface-linked liposomal antigens, capable of inducing long-lived memory CD8(+) T cells without the contribution of CD4(+) T cells, might be applicable for the development of vaccines to prevent viral infection, especially for those viruses that evade humoral immunity by varying their surface proteins, such as influenza viruses, HIV, HCV, SARS coronaviruses, and Ebola viruses

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

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

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

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

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

Cytokine production by spleen cells from mice with ovalbumin-specific, IgE-selective unresponsiveness induced by ovalbumin-liposome conjugate

Ovalbumin coupled with liposomes (OVA–liposome) induced selective unresponsiveness of anti-OVA IgE antibody production in BALB/c mice, whereas OVA adsorbed with aluminum hydroxide (OVA–alum) induced a substantial amount of anti-OVA IgE antibody production. Ovalbumin–liposome and OVA–alum predominantly induced IgG2a and IgG1 anti-OVA production, respectively. These results suggest that OVA–liposome and OVA–alum induce type 1 and type 2 T helper (Th) immune responses, respectively. To further investigate this issue, we examined the cytokine production induced by these two distinct adjuvants. Spleen cells taken from mice immunized with either OVA–liposome or OVA–alum were cultured in vitro with OVA and the cytokine production from each culture was analyzed. It was demonstrated that spleen cells from mice immunized with OVA–liposome produced more interferon (IFN)-γ than those immunized with OVA–alum and, furthermore, interleukin (IL)-4 was produced only by spleen cells from mice immunized with OVA–alum. These results favor the notion that OVA–liposome and OVA–alum induce Th1 and Th2 cytokines, respectively. Interestingly, the production of IL-2, a Th1 cytokine, was higher in the OVA–alum-immunized group and the production of IL-10, a Th2 cytokine, remained at low levels in both groups after primary immunization; levels of IL-10 increased in the OVA–liposome-immunized group after secondary immunization. These results do not agree with the above notion and, thus, suggest that it may be important to consider the balance between IFN-γ-producing cells and IL-4-producing cells rather than that between Th1 and Th2 cells for the regulation of IgE antibody production

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

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