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. 2011 Feb 21:10:20.
doi: 10.1186/1476-4598-10-20.

Pro-inflammatory cytokine/chemokine production by reovirus treated melanoma cells is PKR/NF-κB mediated and supports innate and adaptive anti-tumour immune priming

Lynette Steele  1 , Fiona Errington, Robin Prestwich, Elizabeth Ilett, Kevin Harrington, Hardev Pandha, Matt Coffey, Peter Selby, Richard Vile, Alan Melcher
Affiliations

Pro-inflammatory cytokine/chemokine production by reovirus treated melanoma cells is PKR/NF-κB mediated and supports innate and adaptive anti-tumour immune priming

Lynette Steele et al. Mol Cancer. .

Abstract

Background: As well as inducing direct oncolysis, reovirus treatment of melanoma is associated with activation of innate and adaptive anti-tumour immune responses.

Results: Here we characterise the effects of conditioned media from reovirus-infected, dying human melanoma cells (reoTCM), in the absence of live virus, to address the immune bystander potential of reovirus therapy. In addition to RANTES, IL-8, MIP-1α and MIP-1β, reovirus-infected melanoma cells secreted eotaxin, IP-10 and the type 1 interferon IFN-β. To address the mechanisms responsible for the inflammatory composition of reoTCM, we show that IL-8 and IFN-β secretion by reovirus-infected melanoma cells was associated with activation of NF-κB and decreased by pre-treatment with small molecule inhibitors of NF-κB and PKR; specific siRNA-mediated knockdown further confirmed a role for PKR. This pro-inflammatory milieu induced a chemotactic response in isolated natural killer (NK) cells, dendritic cells (DC) and anti-melanoma cytotoxic T cells (CTL). Following culture in reoTCM, NK cells upregulated CD69 expression and acquired greater lytic potential against tumour targets. Furthermore, melanoma cell-loaded DC cultured in reoTCM were more effective at priming adaptive anti-tumour immunity.

Conclusions: These data demonstrate that the PKR- and NF-κB-dependent induction of pro-inflammatory molecules that accompanies reovirus-mediated killing can recruit and activate innate and adaptive effector cells, thus potentially altering the tumour microenvironment to support bystander immune-mediated therapy as well as direct viral oncolysis.

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Figures

Figure 1
Figure 1
Melanoma cell lines produce eotaxin, IP-10 and IFN-β in response to reovirus infection. Melanoma cell lines were treated with 0 (open bars), 1 (pale grey bars) or 5 (dark bars) pfu/cell reovirus and supernatants were collected after 48 hours and assayed for eotaxin (A), IP-10 (B) and IFN-β (C). Results shown are representative of 3 independent experiments.
Figure 2
Figure 2
Reovirus infection activates NF-κB in melanoma cells to induce cytokine secretion. (A) Melanoma cell lines were seeded in 100 mm dishes and treated with 10pfu/cell reovirus. At 4, 8, 12, 16, 20 and 24 hours post-infection whole cell lysates were prepared and I-κB assessed by western blot. (B) Melanoma cell lines were seeded as in (A), and nuclear fractions were prepared and western blotted for NF-κB p65. Densitometry data is shown underneath each blot. (C) Melanoma lines were seeded in 24 well plates and pre-treated with 50 μM CAPE, or equivalent DMSO solvent concentrations, for 2 hours prior to addition of reovirus at the indicated doses. Supernatants were collected after 48 hours and IL-8 levels determined using ELISA. Data are representative of at least 3 independent experiments. * indicates P < 0.05, by Student's t-test.
Figure 3
Figure 3
PKR mediates cytokine production by reovirus treated melanoma cells. (A) Melanoma cell lines were seeded in 24 well plates and pre-treated with 2.5 mM 2-AP or equivalent PBS controls, for 2 hours prior to addition of reovirus at the indicated doses. Supernatants were collected after 48 hours and IL-8 levels determined. Data are representative of at least 3 independent experiments. * indicates P < 0.05, by Student's t-test. (B) Mel-624 tumour cells were seeded in 100 mm dishes and transfected with 100 nM PKRV or irrelevant control siRNA. Cell lysates were prepared and western blotted for total PKR, with β-actin used to confirm equal track loading. (C) Mel-624 cells were seeded in 24 well plates and transfected with 100 nM PKRV or irrelevant control siRNA; reovirus was then added at the indicated doses. Supernatants were collected 24 and 48 hours later and IL-8 levels determined. Data are representative of two independent experiments. * indicates P < 0.05 by Student's t-test.
Figure 4
Figure 4
Isolated NK cells, DC and CTL migrate toward virus filtered reoTCM. 5 × 105 NK cells (A), DC (B) or CTL (C) were resuspended in RPMI + 0.5% human AB serum + 1% L-Glutamine and placed in a 5 μm (NK + CTL) or 8 μm (DC) Thincerts™ to separate cells from virus filtered reoTCM/non-reoTCM. Migration was assessed after 3 hours by labelling cells with CD11c-PE (DC), CD56-PE/CD3-FITC (NK cells) or CD3-FITC/CD8-PerCP (CTL) and then using Trucount™ tubes to provide an internal counting control. A cell:bead ratio was determined for each tube and a migration index calculated by normalising this ratio to those of non-reoTCM controls. Data represents means of triplicate wells +/- SEM and is representative of 4 independent donors. * indicates P < 0.05 by Student's t-test.
Figure 5
Figure 5
NK cells cultured in reoTCM are activated against melanoma cell targets. (A) NK cells were cultured in the presence of virus-filtered reoTCM/non-reoTCM overnight and CD69 expression determined. (B) NK cells were cultured in the presence of filtered reoTCM or non-reoTCM for 48 hours and then co-cultured with Mel-888 tumour targets prior to CD107 and intracellular IFNγ assessment. Results shown are gated on CD56+ve/CD3-ve cell populations and are representative of 4 independent donors.
Figure 6
Figure 6
ReoTCM effectively supports priming of specific CTL by tumour cell-loaded DC. PBMC were incubated with autologous DC that had been cultured overnight with Mel-888 tumour cells in the presence of reoTCM/non-reoTCM. The PBMC were restimulated 7 days later and assayed at 14 days. (A) Lymphocyte proliferation was determined via trypan blue exclusion (2 representative donors are shown). (B) IFN-γ levels in CTL supernatants were determined by ELISA (2 representative donors are shown). (C) Cytotoxicity of lymphocytes primed in the presence of reoTCM versus non-reoTCM was determined by 51Cr release assay using Mel-888 tumour cells as specific targets and SKOV-3 as irrelevant controls. One donor is shown as representative of 2 independent experiments. * indicates P < 0.05 by Student's t-test. (D) CTL as in (C) were further assayed for CD107 degranulation and intracellular IFN-γ on co-culture with Mel-888 targets. The results of one donor are shown, representative of at least four independent experiments.
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