Anti-OVA CTL response in mice injected with BV-OVA is long-lasting and not affected by repeated immunization with the same vector.

<p>(A, B) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. One hundred and ten days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes. Twenty hours later, spleen cells were recovered to determine (A) the percentage of specific <i>in vivo</i> killing by flow cytometry and (B) IFN-γ content by ELISA in supernatants of spleen cells from immunized mice, cultured for 48 hours in the presence of OVA protein. (C) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT or PBS. Seven and fourteen days later, spleen cells were recovered and cultured with BV-WT (MOI 5) for 48 hours. The IFN-γ content in supernatants was determined by ELISA (D) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. Fifteen days later, mice received a second i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS, combined as shown in the figure. Seven days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes. Twenty hours later, spleen cells were recovered and the percentage of specific <i>in vivo</i> killing was determined by flow cytometry. *, p<0.05; ***, p<0.001. Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 5).</p

Anti-OVA CTL response in mice injected with BV-OVA.

<p>C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA, BV-OVAsur, BV-WT + OVA (30 ng or 1 mg), OVA alone (1 mg) or PBS. Seven days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes as target cells. (A) A representative histogram of remaining CFSE^high and CFSE^low cells in control and BV-OVA immunized mice 20 hours after injection of target cells is shown. (B) Percentage of specific <i>in vivo</i> killing of one representative experiment. (C) IFN-γ content in supernatants of spleen cells from immunized mice determined by ELISA. Spleen cells were recovered and cultured for 48 hours in the presence of OVA protein or BV-WT. As control, spleen cells without stimulus were also cultured. *, p<0.05; **, p<0.01. (D, E) IFN-γ intracellular staining on splenocytes from immunized mice with 30 ng OVA+BV-WT, BV-WT, BV-OVA, OVA alone or PBS, as indicated above. Spleen cells were recovered and cultured for 12 hours in the presence of OVA protein or OVA_256-264, and incubated in the presence of brefeldin A for 6 additional hours. Then, cells were labeled for CD4 or CD8 markers and intracellular IFN-γ. (D) A representative dot plot is shown. (E) Percentage of IFN-γ+ CD4 or CD8 T cells. **, p<0.01; ***, p<0.001. (F) Comparison of specific <i>in vivo</i> killing between mice immunized with BV-OVA vs BV-OVAsur. ***, p<0.001. (G) Comparison of the frequency of IFN-γ-producing CD8 T cells in splenocytes from BV-OVA vs BV-OVAsur immunized mice. **, p<0.01. Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 4).</p

BV induce BMDCs maturation.

<p>(A) Maturation of BMDCs by BV. BMDCs were incubated for 18 hours with BV-WT or mock. Then, cells were stained with anti-CD11c and one of the following antibodies: anti-CD40, CD86 or I-A^b, and analyzed on a FACSCanto II. In all cases, a minimum of 2×10⁵ events was acquired. Results are representative of two independent experiments and are expressed as the geometric mean of the fluorescence intensity (MFI) for each indicated molecule in total DCs. (B) Production of inflammatory cytokines. BMDCs from C57BL/10 ScCr (TLR4 deficients) and C57BL/6 (TLR4 competents) were incubated for 18 hours with BV-WT, or a supernatant from mock infected Sf9 cells (SN), and then levels of IL-6 and IL-12 p40 were determined in supernatants of BMDCs by ELISA. Results are representative of two independent experiments.</p

BV-OVA induces CD8 T cell activation.

<p>Splenic CD11c+ cells (2×10⁵) purified from C57BL/6 mice were incubated with BV-WT, BV-OVA or BV-OVAsur at the indicated multiplicity of infection (MOI) for 120 minutes and washed twice. Then, DCs were cultured for 3 days with CFSE-labeled CD8 T cells from OT-I mice (having a transgenic T cell receptor which recognizes OVA_257-264 in the context of H-2K^b). T cell proliferation and CD25 expression were analyzed by flow cytometry, after labeling cells with an anti-CD3 antibody and exclusion of dead cells with 7AAD. (A) A representative dot plot overlay of cell proliferation vs CD25 expression of CD8 T cells cultured with DCs, which were preincubated with BV-OVA (black dots) or BV-WT (gray dots) at a MOI of 30. (B) Percentage of CD8 T cells undergoing one or more rounds of proliferation and (C) IFN-γ content, assessed by ELISA, in supernatants of culture of CD8 T cells with DCs preincubated with BV-OVA or BV-WT. (D) T cell proliferation and (E) CD25 expression of CD8 T cells co-cultured with DCs preincubated with BV-OVA (▪), BV-OVAsur (▴) or BV-WT (○). Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 4).</p

Estimation of the number of CpG motifs in AcNMPV genome.

<p>The frequency at which each CpG hexamer appeared in the BV genome was determined by using the GenBank accession number for the complete genome of AcNPV NC 001623.</p

Prophylactic and therapeutic vaccinations with BV-OVA.

<p>(A, B) Prophylactic vaccination. Groups of eight C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. Seven days later, immunized mice were challenged with a s.c. injection of 1×10⁵ syngenic OVA-expressing melanoma cells (MO5 cells). (A) Progression of tumor volumes and (B) survival are shown. (C, D) Therapeutic vaccination. Groups of eight C57BL/6 mice were challenged with an s.c. injection of 1×10⁵ MO5 cells. Seven days later, mice showing palpable tumors were treated with 5×10⁷ PFU BV-WT, BV-OVA or PBS by i.v. injection. At days 11 and 17 post-tumor cell inoculation, mice were s.c. injected near to the tumors with 1×10⁷ PFU BV-WT, BV-OVA or PBS respectively. At day 21 post-tumor cell inoculation, mice were intratumorally injected with 1×10⁷ PFU BV-WT, BV-OVA or PBS respectively. (C) Progression of tumor volumes and (D) survival are shown. Results are representative of three independent experiments and are expressed as mean+/− SEM (n = 8). *, p<0.05; **, p<0.01.</p

Innate immune response in mice injected with BV.

<p>(A) Induction of inflammatory cytokines by BV. C57BL/6 mice were i.v. injected with 5×10⁷ PFU BV (•) or with supernatants of uninfected insect cells (SN, □), and at the times shown sera were collected and assayed for IL-6, IFN-γ and IL12p40 by ELISA. Sera collected before the injection were employed as control. Results are representative of two independent experiments. The line depicted in each group corresponds to the mean of each group of dots. (B, C) Activation of NK, NKT and T cells by BV. C57BL/6 mice were i.v. injected with 5×10⁷ PFU BV, and 3 hours post injection splenic populations were incubated in the presence of BFA for an additional 6 hours. Then, cells were labeled for CD49b and CD3 markers and intracellular IFN-γ. (B) A representative dot plot of the proportion of NK (CD49b+ CD3-), NKT (CD49b+ CD3+) and T (CD49b- CD3+) cells in the spleen of a control mouse and (C) the percentage of NK, NKT and T cells (left) and proportion of IFN-γ+ NK, NKT and T cells after BV injection (right) are shown. Results are representative of two independent experiments and are expressed as mean +/− SEM (n = 4). (D) <i>In vivo</i> maturation of DCs by BV. C57BL/6 mice were injected i.v. with saline, 5×10⁷ PFU BV-WT and 30 ng or 30 µg CpG-containing oligodeoxinucleotide (CpG-ODN) 1826. Eighteen hours later, their CD11c+ spleen cells were labeled with anti-CD86 and analyzed on a FACSCanto II flow cytometer. In all cases, a minimum of 2×10⁵ events was acquired. Results are representative of two independent experiments and are expressed as the geometric mean of the fluorescence intensity (MFI) and as the frequency of CD86+ cells in total DCs. Results are representative of at least two independent experiments.</p

Characterization of BV-OVA.

<p>(A) Scheme of Baculovirus (BV) vectors. The Ovalbumin (OVA) (653–1222) and enhanced Green Fluorescent Protein (GFP) (1-717) sequences were cloned 5′ of the VP39 sequence. A linker sequence (GGGGS) was added in the N-terminus of VP39 to provide distance and flexibility for the N-terminal fusion proteins to fold correctly. Production of the fusion protein is driven by the strong polyhedrin promoter. (B) Western blot analysis of purified virions. The virions were purified by ultracentrifugation for 30 min at 131,000× g onto a 25% sucrose cushion. Fusion proteins OVAVP39 (MW 64 KDa), GFPVP39 (MW 67 KDa) and OVAGP64 (MW 89 KDa) were detected with anti-OVA or anti-GFP specific polyclonal antibodies. (C) Immunoelectron microscopy of BV-OVA. Recombinant BV-OVA purified by ultracentrifugation through a 25% sucrose cushion were treated with 1% of Triton 100. Virions capsids were adsorbed to Formvar-coated grids, and the presence and localization of OVAVP39 fusion protein on the capside of the virion was detected with an anti-OVA polyclonal antibody and an anti rabbit IgG-gold conjugate. The figure is representative of all fields examined. Bar = 100 nm. (D) Uptake of BV by Bone Marrow-derived Dendritic Cells (BMDCs). The BMDCs were incubated with BV-GFP for 2 hours at 37°C or 4°C. Then, cells were washed, stained with anti-CD11c and analyzed on a FACSCanto II. In all cases, a minimum of 2×10⁵ events was acquired. Results are representative of two independent experiments and are expressed as the geometric mean of the fluorescence intensity (MFI) of the FL1 channel in total DCs or the percentage of DCs which are positive for FL1 channel. (E) <i>In vitro</i> OVA presentation by DCs infected with BV-OVA. Spleen CD11c+ cells (2×10⁵) were incubated <i>in vitro</i> with BV-OVA (•) or BV-GFP (○), and cultured overnight with 10⁵ B3Z cells/well. Then, cells were washed and the presentation of the OVA_257-264 epitope to B3Z cells was monitored by the activity of β–galactosidase with a colorimetric assay. Results are representative of two independent experiments and are expressed as mean +/− SEM of the optical density at 595 nm (OD₅₉₅, n = 3).</p

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8⁺ T Cells upon Stimulation with a TLR7 Ligand

<div><p>The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8⁺ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8⁺ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α⁺ cDCs from old mice have an impaired ability to activate naïve CD8⁺ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8⁺ T cells and the generation of effector cytotoxic T cells.</p></div

Ag degradation in cDCs is affected by aging.

<p>Persistence of OVA protein in cell lysates of total (A) or CD8α⁺ (B) cDCs purified from young and old mice was determined by Western blot after 1 hour pulse loading with 0.625 mg/mL OVA plus 20 μg/mL polyU/DO (time 0, 0h) and 4h chase. Actin was used for loading control. The control line contains total cell lysates of untouched splenic DCs. Densitometric analysis of Western blots (right) is expressed as the ratio of integrated optical density (IOD) at time 0 relative to IOD at chase time. (C) Percentages of total live and dead cDCs after 1 hour pulse loading and 4h chase. Data represent the mean ± SEM of duplicate cultures and are representative of 2 independent experiments. ***p < 0.001.</p