Internalization of oncolytic reovirus by human dendritic cell carriers protects the virus from neutralization - PubMed (original) (raw)

. 2011 May 1;17(9):2767-76.

doi: 10.1158/1078-0432.CCR-10-3266. Epub 2011 Mar 9.

Montserrat Bárcena, Fiona Errington-Mais, Stephen Griffin, Kevin J Harrington, Hardev S Pandha, Matthew Coffey, Peter J Selby, Ronald W A L Limpens, Mieke Mommaas, Rob C Hoeben, Richard G Vile, Alan A Melcher

Affiliations

Internalization of oncolytic reovirus by human dendritic cell carriers protects the virus from neutralization

Elizabeth J Ilett et al. Clin Cancer Res. 2011.

Abstract

Purpose: Dendritic cells (DC) may be the most effective way of delivering oncolytic viruses to patients. Reovirus, a naturally occurring oncolytic virus, is currently undergoing early clinical trials; however, intravenous delivery of the virus is hampered by pre-existing antiviral immunity. Systemic delivery via cell carriage is a novel approach currently under investigation and initial studies have indicated its feasibility by using a variety of cell types and viruses. This study addressed the efficacy of human DC to transport virus in the presence of human neutralizing serum.

Experimental design: Following reovirus-loading, DC or T cells were cocultured with melanoma cells with or without neutralizing serum; the melanoma cells were then analyzed for cell death. Following reovirus loading, cells were examined by electron microscopy to identify mechanisms of delivery. The phagocytic function of reovirus-loaded DC was investigated by using labeled tumor cells and the ability of reovirus-loaded DC to prime T cells was also investigated.

Results: In the presence of human neutralizing serum DC, but not T cells, were able to deliver reovirus for melanoma cell killing in vitro. Electron microscopy suggested that DC protected the virus by internalization, whereas with T cells it remained bound to the surface and hence accessible to neutralizing antibodies. Furthermore, DC loaded with reovirus were fully functional with regard to phagocytosis and priming of specific antitumor immune responses.

Conclusions: The delivery of reovirus via DC could be a promising new approach offering the possibility of combining systemic viral therapy for metastatic disease with induction of an antitumor immune response.

©2011 AACR.

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Figures

Figure 1

Figure 1

(a) FACS analysis for reovirus retention on iDC, mDC and T cells, plots are representative of 4 independent experiments. (b) freeze/thaw preparations of reovirus loaded cells were used in a standard plaque assay and reovirus retention as a % of the loading dose was calculated. Graph shows mean ± SE of data from 6 independent experiments. (c) FACS analysis for Jam-1 expression on iDC, mDC and T cells; representative of 4 independent experiments. (d,e) FACS plots showing reovirus retention following blocking of the carrier cells with anti-Jam-1 antibody prior to reovirus loading (d), and removal of sialic acid from the carrier cells prior to loading (e); representative of 4 independent experiments.

Figure 2

Figure 2

(a) Mel-888 cells were cultured with reovirus at 10 pfu/cell ± human serum at the dilutions shown. After 24 and 48 h the cells were harvested and PI stained to determine cell death. (b) iDC, mDC and T cells were loaded with reovirus at the MOI indicated and cultured ± human blocking serum at 1:50 dilution, with target Mel-888 or MeWo cells. At 72 h the cells were harvested and stained with PI for FACS analysis. Graphs show mean ± SE of data from 3 independent experiments; formula imageformula image denotes significance p < 0.01 between iDC or mDC vs T cells or neat reovirus, all at MOI 10. (c) MeWo cells were cultured for 48 h, with reovirus-loaded iDC, mDC or T cells, or neat virus at the MOI indicated, ± blocking serum. Carrier cells were removed, fresh PBS was added to the wells and phase-contrast images were taken. Images shown are representative of 3 independent experiments. (d) Carrier cells loaded with reovirus, were cultured with Mel-888 or MeWo cells in the presence of 30% blocking serum for 72 h. Graph shows mean ± SE of data from 2 independent experiments; formula image denotes significance p < 0.05 between iDC or mDC vs T cells or neat reovirus. (e) FACS analysis showing Jam-1 expression by Mel-888 cells (solid line) and MeWo cells (broken line). (f) MeWo cells were incubated with 10 μg/ml anti-JAM-1 prior to the addition of reovirus or carrier cells loaded at the MOI shown. After 48 h culture the cells were harvested and stained with PI. Graph shows mean ± SE for 2 independent experiments; formula image denotes significance p < 0.05.

Figure 3

Figure 3

iDC (a), mDC (c,d) and T cells (b) were loaded ± reovirus. The cells were then washed, re-suspended in medium and placed at 37°C for 6 h to allow internalization of the virus to take place, after which time the cells were fixed, immunogold labelled and analyzed by Electron Microscopy. Scale bars all 500 nm. N, nucleus; M, mitochondria; G, Golgi.

Figure 4

Figure 4

(a) iDC (top row) or mDC (bottom row) were loaded ± 10 pfu/cell reovirus, and cultured with Mel-888 cells in the presence of blocking serum; after 48 h the cells were harvested, labelled with CD11c and analyzed for the maturation markers indicated. Plots shown are representative of 3 independent experiments. (b) PFA-fixed or live DC were loaded as in (a) and cultured with Cell Tracker Green-labelled Mel-888 cells, then harvested and labelled with CD11c. Double positive cells were identified by FACS. Plots shown are representative of 4 independent experiments. (c) reovirus-loaded cells (iDC(reo), mDC(reo)) or non-loaded cells (iDC, mDC), were cultured for 24 h with Mel-888 cells and used to prime isolated autologous T cells. Cells were harvested and a CD107 degranulation assay against Mel-888 or SKOV-3 (irrelevant) targets was performed. Plots shown are representative of 3 independent experiments; numbers indicate % of CD8 cells degranulating.

Figure 5

Figure 5

Reovirus at 0 or 10 pfu/cell, or DC or T cells loaded at 0 or 10 pfu/cell were cultured with Mel-888 cells for 24 h and used to prime autologous PBMC. CTL were then used in a CD107 assay (a); graph shows mean ± SE of data from 5 independent experiments. (b) autologous PBMC were primed as in (a) but with blocking human serum added to the Mel-888 cells during co-culture with reovirus or carrier cells. Graph shows mean ± SE of data from 5 independent experiments; formula image denotes significance p < 0.05 between iDC or mDC and T cells or reovirus. (c) Representative plots showing CD107 degranulation by CD8 cells from priming cultures, against Mel-888 (relevant) or SKOV (irrelevant) targets. (d) Supernatants from priming cultures were analyzed for IFN-γ by ELISA; graph shows mean ± SE of data from 5 independent experiments.

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