Figure 1.
DEX particles are of spherical appearance and uniform in size.
(A) Shape and size distribution of DEX(−) dispersed in PBS was studied by electron microscopy. (B) Hydrodynamic radii of Dextran T500 and derived DEX particle formulations as function of q2 in DPBS buffer (0.33 mg/ml) were determined by DLS (see Methods). Graphs denote the angular dependency of the apperent diffusion coefficient of the different dextran solutions in buffer solution. (C) To assess cytotoxic effects of DEX particles on BM-DC viability, cells (2.5×105) were seeded into wells of 96 well cell culture plates in a volume of 50 µl in triplicates, and DEX(−) particles (20 mg/ml) were added at different amounts as indicated. One day later, viability of BM-DCs was assessed as described (see Methods). The viability of untreated BM-DCs was set to 100% (dashed line).
Figure 2.
BM-DCs engulf DEX particle formulations in an OVA-dependent manner, mainly via the MR.
Aliquots of unstimulated day 6 BM-DCs (5×105 cells; C57BL/6) were left untreated (−) or were coincubated with FITC-labeled DEX particles in duplicates as indicated (each 50 µl). (A) Aliquots were removed after the indicated period of time, and the frequencies of FITC+CD11c+ BM-DCs were assessed by flow cytometry. Data represent mean±SEM of duplicates and are representative of three independent experiments. Statistical significant differences between OVA-containing DEX particles (DEX[OVA], DEX[OVA+LPS]) and the corresponding control group (DEX[−], DEX[LPS]) are indicated for each time point (*p<0.05, **p<0.01). (B) Cellular uptake of FITC-labeled DEX(OVA) by BM-DCs, stained with anti-CD11c antibody (red) and DAPI (blue), was assessed by confocal laser scanning microscopy 4 h (left panel) and 24 h (right panel) after the onset of coincubation. (C) In parallel cultures, aliquots of BM-DCs were left untreated or were incubated with mannan at high dose (200 µg/ml) for 30 min. Afterwards, DEX(OVA + LPS) was added to either group. Aliqouts of BM-DCs were harvested at the indicated time points, stained for CD11c, and analyzed by flow cytometry. Data represent mean±SEM of duplicates and are representative of three independent experiments. (D) Spleen cells derived from C57BL/6 mice (2×106 cells/200 µl) were left untreated (data not shown) or were coincubated with FITC-labeled DEX particles as indicated (each 30 µl) for 24 h. Afterwards, the cells were stained with either of the indicated cell lineage markers (DCs: CD11c-PE-Cy7, Macrophages: F4/80-eFlour405, B cells: CD19-APC, T cells: CD3-PE), and were analyzed by flow cytometry for the frequency of FITC+ cells within either lineage. Numbers in brackets indicate the overall frequency of either cell lineage within the spleen cell suspension. Data represent mean±SEM of three independent experiments. Statistical significant differences between groups are indicated (***p<0.001).
Figure 3.
BM-DCs are strongly activated by LPS-containing DEX particle formulations, and codelivery of OVA results in robust antigen-specific CD4+ T cell activation.
(A) Aliquots of unstimulated day 6 BM-DCs (106 cells; C57BL/6) were left untreated (−), stimulated with LPS (100 ng/ml), or were coincubated with DEX particles (100 µl) as indicated for 24 h. Afterwards, expression of CD40, CD80, and CD86 was assessed by flow cytometry. Graphs denote mean fluorescence intensities (MFI) ± SEM of three experiments. Statistical significant differences between groups are indicated (**p<0.01, ***p<0.001). (B) Unstimulated day 6 BM-DCs (106 cells; C57BL/6) were treated with soluble OVA (2 µg) or as described in A for 24 h. Titrated numbers of BM-DCs were cocultured with sorted CD4+ OT-II T cells in triplicates for 3 days at the ratios indicated. T cell proliferation was assessed as incorporation of 3H-thymidine added for the last 16–18 h. Data represent mean±SEM of triplicates and are representative of three independent experiments. Statistical significant differences between DEX(OVA-LPS) versus OVA plus LPS and DEX(OVA) versus OVA for each T cell/BM-DC ratio are indicated (*p<0.05, **p<0.01).
Figure 4.
DEX particles containing OVA and LPS induce strong activation of CD4+ antigen-specific T cells when applied in vivo.
(A) Mice (C57BL/6) received CFSE-labeled OVA-specific OT-II T cells (107/mouse) i.v. Two days later, mice (three animals per group) were immunized i.v. with OVA (4 µg per mouse), LPS (0.4 µg), and DEX particles (each 200 µl) as indicated. After another three days, frequencies of CD4+CD45.1+Vα2+ OT-II T cells in spleen cell suspensions were analyzed by flow cytometry. Data represent mean±SEM of two independent experiments. The frequencies of proliferating CD4+ OT-II T cells in either treated group were signifiantly higher than in the non-immunized control group. (B) CD4+ DO11.10 T cells (5×106) were transferred to BALB/c mice (3 animals per group). One day later, OVA323–339 peptide (40 µg), LPS (100 ng), or different DEX particle formulations (each 40 µg) were injected as indicated. For challenge, LPS-stimulated BM-DCs were pulsed with OVA323–339 peptide (0.1 µg/ml), and were injected into either hind foot pad of pretreated BALB/c mice. Food pad thickness was recorded daily. Data represent mean±SEM of six recordings per group. Statistical significant differences between any group versus the control group (DEX[−]) are indicated (*p<0.05, **p<0.01, ***p<0.001).
Figure 5.
The nanovaccine DEX(OVA+LPS) induces profound activation of antigen-specific CD8+ T cells in vivo.
C57BL/6 mice received CFSE-labeled, OVA-specific OT-I T cells (107) i.v. Two days later, groups of mice (each five animals) were either left untreated or were immunized with OVA (4 µg per mouse), LPS (4 µg), and DEX particle formulations (each 200 µl) as indicated. (A) On day 5, the frequencies of proliferating CD8+CD45.1+Vα2+ OT-I cells were determined in spleen cell suspensions by flow cytometry. (B) In the same experiments, the frequencies of IFNγ+ OT-I T cells were assessed by flow cytometry. (A,B) Data represent mean±SEM of two independent experiments each. The frequencies of proliferating and IFN-γ producing CD8+ OT-I T cells in either group were signifiantly higher than in the non-immunized control group. Other statistical significant differences between groups are indicated (*p<0.05, **p<0.01, ***p<0.001). (C) On day 4 after immunization, mice were injected with CFSElow target cells (loaded with OVA257–264) and CFSEhigh control cells (each 107 cells) derived from syngeneic Ly-5.1+ mice. 4 h later, splenocytes were isolated and frequencies of CFSE-labeled cell populations were assessed by flow cytometry. Upper panel: Data represent mean±SEM of two independent experiments. Statistical significant differences between groups are indicated (*p<0.05, **p<0.01). Lower panel: Frequencies of Ly-5.1+ target cells (CFSElow) and control cells (CFSEhigh) in spleen cell suspensions derived from one mouse of either group are shown as histograms. Graphs are representative of two independent experiments.
Figure 6.
DEX particles containing OVA elicit a specific Th2-biased humoral immune response, augmented upon codelivery of LPS.
Naive C57BL/6 mice (two mice per group) were immunized with DEX particle formulations as indicated. On days 7 (upper panel) and 14 (lower panel), mice were bled and derived sera were used for detection of OVA-specific IgG1 and IgG2a antibody titers. Data represent mean±SEM of two sera per group.