Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-alpha and IFN-gamma. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials

Cytokine release from dog PBDC cultures.

<p>Peripheral blood cultures were collected after 7 days in culture with recombinant canine IL-4 and GM-CSF, or Ad.hFlt3L conditioned medium and incubated for additional 24 h without added cytokines in the absence (mock) or presence of CpG₂₂₁₆ or LPS. Release of IL-6 (<b>A</b>), TNF-α (<b>B</b>) and IFN-γ (<b>C</b>) was determined by ELISA. *p<0.05 vs mock (Two-way ANOVA followed by Tukey's Multiple Comparison test). Scatter plots show values from individual dog blood cultures (n = 4–12).</p

Phenotypic characterization of dog peripheral blood DC cultures.

<p>Peripheral blood cultures were collected after 7 days of culture with recombinant canine IL-4 and GM-CSF or Ad.hFlt3L conditioned medium. Immune cell populations in the culture were identified using antibodies against CD14 (monocytes), CD11c (dendritic cells) and CD18 (macrophages). Numbers in representative dot plots indicate the percentage of positive cells. Approximately 12,000 events were collected from each sample. Dot plots are representative of 4 dogs. SSC: Side Scatter; FSC: Forward Scatter.</p

Allogeneic mixed-lymphocyte reaction (MLR).

<p>DCs (stimulators, <b>A</b>) from dog peripheral blood monocytes cultured with either Ad.hFlt3L conditioned media (purple dots) or IL-4+GM-CSF (orange dots) were incubated with allogeneic spleen T cells (responders, <b>B</b>) purified by Fluorescence Activated Cell Sorting (FACS) from the spleen of a second dog. Dot plots show the percentage of CD11c⁺-cells (<b>A</b>) and the percentage of CD8⁺ and CD4⁺ T cells collected from dog spleen by FACS (<b>B</b>). SSC: Side Scatter; FSC: Forward Scatter. <b>C</b>, T cell proliferation was determined by BrdU incorporation after 4 days in co-culture. Additional controls included DCs incubated without T cells (broken lines). *p<0.05 vs. DCs no T cells (Randomization test).</p

DC phagocytic activity.

<p>Peripheral blood cultures were collected after 7 days in culture with recombinant canine IL-4 and GM-CSF or Ad.hFlt3L conditioned medium and incubated for additional 24 h without added cytokines in the absence (mock) or presence of CpG₂₂₁₆ or LPS. Activated dog DCs were mixed with 1.0 µm yellow-green fluorescent FluoSpheres for 4 hrs at 4°C or 37°C. <b>A</b>, Confocal microphotographs show CD11c⁺ and MHCII⁺ cells (red) containing yellow-green beads in their cytoplasm. Nuclei were stained with DAPI (blue). Scale bars: 10 µm. <b>B</b>, Scatter plot shows the quantification of CD11c⁺ DCs containing yellow-green fluorescent FluoSpheres in their cytoplasm. *p<0.05 vs mock ∧ p<0.05 vs. IL-4/GM-CSF (Two-way ANOVA followed by Tukey's Multiple Comparison test).</p

Morphology of peripheral blood-derived dog dendritic cells cultured with canine IL-4+GM-CSF or Ad.hFlt3L conditioned medium.

<p>White cells from dog peripheral blood were purified using Lymphocyte Separation Media and cultured in the presence of recombinant canine IL-4 (50 ng/ml) and GM-CSF (33 ng/ml) or Ad.hsFlt3L conditioned medium (10 ng/ml). The images show the appearance of the culture after 7 days in culture. Low magnification images (20×) show typical spheres formed in culture supplemented with canine IL-4+GM-CSF (<b>A</b>) or hsFlt3L (<b>B</b>). Insets show higher magnification images of cells that exhibit typical long cytoplasmic processes characteristic of dendritic cell morphology. Scale bars: 10 µm. <b>C</b>, After 7–9 days in culture non-adherent cells were collected and counted. The graph shows the yield of cells per ml of blood obtained from 27 dogs after culture with hsFlt3L or canine-IL-4/GM-CSF.</p