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Journal of Virology, January 2006, p. 383-394, Vol. 80, No. 1
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.1.383-394.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Prostate Tumor Cells Infected with a Recombinant Influenza Virus Expressing a Truncated NS1 Protein Activate Cytolytic CD8+ Cells To Recognize Noninfected Tumor Cells{dagger}

Clay L. Efferson,1,{ddagger} Naotake Tsuda,1,{ddagger} Kouichiro Kawano,1 Estanislao Nistal-Villán,2,3 Shankhar Sellappan,4 Dihua Yu,4 James L. Murray,5 Adolfo García-Sastre,2* and Constantin G. Ioannides1

Department of Gynecologic Oncology, Surgical Oncology, Breast Medical Oncology and Immunology, M. D. Anderson Cancer Center, Houston, Texas 77030,1 Department of Microbiology,2 Microbiology Graduate School Training Program, Mount Sinai School of Medicine, New York, New York 100293

Received 14 April 2005/ Accepted 28 September 2005


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ABSTRACT
 
Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-{alpha}/ß) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-{alpha}. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-{alpha}/ß response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-{gamma} in PBMC. The levels of IFN-{gamma} were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.


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INTRODUCTION
 
The delivery of a virus into malignant cells affords the opportunity of partial or complete tumor eradication (for excellent reviews, see references 21, 40, and 42). One of the principles of cancer virotherapy is based on the findings that certain viruses preferentially replicate in tumor cells that lack either antiviral mechanisms or cellular inhibitors of virus replication (1, 5, 16, 45). The antiviral mechanisms include the induction of alpha/beta interferon (IFN-{alpha}/ß) production by the infected cell, followed by the transcriptional activation of the IFN-responsive antiviral genes. One of the key elements of the IFN-mediated antiviral response is the IFN-inducible, double-stranded RNA-activated protein kinase R (PKR). Tumor cells containing the activated Ras oncogene or Ras oncogene-activated mitogen-activated protein kinase (MAPK) pathway produce inhibitors of PKR (1, 5, 30). Lack of active PKR in tumor cells results in increased sensitivity to viral replication and virus-induced lysis. Furthermore, activation of the immune system by infection of tumor cells with oncolytic viruses reportedly eliminated tumor cells (6, 17). An important issue when considering virotherapy of cancer remains its applicability to tumor cells that lack constitutively activated Ras oncogenes or constitutively activated MAPK. Prostate tumors in American men infrequently express mutated or activated Ras (25). In addition, the consequences of tumor apoptosis for activation of host defense by immune cells against human prostate tumors are unclear. The significance of the antitumor immune responses induced by virus-infected tumor cells has not been investigated in humans.

Influenza A viruses (IAV) are prototypes of the family Orthomyxoviridae and possess a genome of eight single-stranded RNA segments of negative polarity. It is known that IAV infection results in the induction of apoptosis of the host cells, both in cell culture and in vivo (23, 48). The fact that wild-type IAV can infect and replicate in both tumor and untransformed cells makes its use as oncolytic virus unlikely. IAV encodes a nonstructural protein (NS1) involved in virulence and inhibition of the IFN-{alpha}/ß system during virus infection (20). Interestingly, mutant IAV lacking the NS1 gene ({Delta}NS1) or expressing a truncated NS1 protein (TR-NS1) replicate poorly in normal cells but replicate significantly better in tumor cells containing activated Ras or dysregulated IFN responses (8, 32). Growth factors, such as epidermal growth factor (EGF), mediate Ras activation by signaling through the EGF receptor 1 (EGFR1)/EGFR2->Raf->Ras pathway. This pathway further activates MAPK kinases, extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal protein kinase. It is unknown whether the presence of a Raf-Ras-activated system by growth factors allows infection and mediation of antitumor effects by influenza viruses with disabled or attenuated NS1.

To identify whether a recombinant influenza A/PR8/34 virus expressing a truncated NS1 protein of 126 amino acids (TR-NS1 virus) mediates inhibition of proliferation of human tumor cells lacking constitutively activated ERK, we investigated the outcome of infection of the human prostate cancer cell line LNCap by this virus. LNCap cells are androgen-dependent cells that lack constitutively activated ERK-1 in our experiments but proliferate in response to EGF and dihydroxytestosterone (7, 22) and activate ERK in response to EGF (7, 49). For studies of the immune responses induced by virus infection, these cells possess the advantages that they express the oncogenic Her-2/neu proto-oncogene (HER-2) protein and the histocompatibility antigen HLA-A2. This not only allows characterization of the responses of cytotoxic T lymphocytes (CTL) to defined epitopes on a known tumor antigen (Ag) but also makes the findings relevant for the large number of patients who express HLA-A2. Inhibition of proliferation of LNCap cells following infection with TR-NS1 virus was dose dependent but was only transient. LNCap cells surviving the antiproliferative effects of TR-NS1 virus expanded in culture. However, the TR-NS1 virus-resistant tumor cells were completely eliminated after coculture with peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12). This effect was at least partly mediated by lytic effectors, including tumor-specific CD8+ cytolytic T cells, induced by TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) that recognized Ag on noninfected tumor cells. Our results indicate that TR-NS1 virus inhibited proliferation of a subpopulation of tumor cells lacking phospho-ERK (P-ERK), but in synergy with IL-12, activated cellular immunity to the tumor, which resulted in elimination of tumor cells.


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MATERIALS AND METHODS
 
Chemicals, cytokines, and antibodies. Inhibitors of kinase pathways, U0126, PD098059 (ERK inhibitor), SB2035830 (p38 MAPK inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), emodine (tyrosine kinase inhibitor), farnesyl transferase (FT1) inhibitor (Ras inhibitor), and bis-indolyl-maleimide (BIM) (PKC inhibitor), were obtained from Calbiochem, Inc., and used at the indicated concentrations (1, 16). EGF and Neu differentiation factor (NDF) were obtained from R&D Systems (Minneapolis, MN). IFN-{alpha}, IFN-{gamma}, IL-12, and IL-2 were obtained from Biosource (Camarillo, CA). IFN-{alpha}, IL-12, IL-2, IFN-{gamma}, and IL-12 (p70) detection kits were obtained from BD Pharmingen (San Diego, CA). Synthetic peptides E75 (HER-2 369-377), G60 (HES-1/, amino enhancer of split AES-1 125-135) (4, 18), and M58 (influenza virus matrix protein M1 58-66), were prepared by the M. D. Anderson Peptide Synthesis Facility. Antibodies to ERK (p44/p42), p38, and AKT and corresponding antibodies to phospho-p44/p42, phospho-p38, and phospho-Akt were obtained from Cell Signaling Technology, Inc. (39). Antibodies against {alpha}-tubulin and against ß-actin were obtained from Sigma Chemical Co. (St. Louis, Mo). The caspase inhibitors Z-IETD-fluoromethyl ketone (fmk) (specific for caspase-8), Z-LEHD-fmk (specific for caspase-9), and EDVE-fmk (specific for caspase-3) were purchased from R&D Systems. Empty recombinant soluble dimeric HLA-A2-immunoglobulin G1 (IgG1) molecules, designated as "dimers," monoclonal antibodies (MAb) used for detecting surface CD8, isotype MAb controls, fluorescein isothiocyanate (FITC)- and phycoerythrin-conjugated MAb, and all specific Ig controls were obtained from BD Pharmingen. Ab against IAV hemagglutinin (HA) was purchased from the NIH repository.

Cell culture. The prostate cancer cell lines LNCap and PC-3 (7, 22) were obtained from ATCC. Ovarian lines SKOV3, 2774, and the breast cancer line SKBR3 have been described in our previous papers (4, 18). Cells were grown in the recommended culture medium with 10% fetal calf serum (FCS). PBMC were isolated from healthy donors and separated into the plastic-adherent and non-plastic-adherent fractions. The non-plastic-adherent fraction contained 95% CD3-positive cells. The plastic-adherent PBMC were cultured in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days. Culture in GM-CSF and IL-4 resulted in immature monocyte-derived dendritic cells (MDC) as we described previously (14, 31). Unfractionated and non-plastic-adherent PBMC were stored frozen in liquid nitrogen until use.

Viruses. The generation, growth, and titration of TR-NS1 and {Delta}NS1 viruses were performed according to the methods previously described (14, 46). TR-NS1 and {Delta}NS1 viruses and their parental wild-type influenza A/PR8/34 virus (PR8) were grown in the allantoic cavities of 7-day-old embryonated chicken eggs.

Characterization of the virus effects on proliferation of tumor cell lines. Tumor cells were plated at various numbers in 24- or 48-well plates. They were infected either with parental PR8 or with TR-NS1 viruses at various multiplicities of infection (MOIs) in serum-free medium. After 2 h of infection, the medium was removed and cells were covered with fresh medium containing 10% FCS. To identify live cells at indicated time points, cells were washed, detached with trypsin, and counted in the presence of trypan blue. To determine the sensitivity of LNCap cells to cytokines, they were incubated either in 24- or 96-well plates with various concentrations of IFN-{alpha} (4,000 and 1,000 U/ml), IL-12 (200 IU/ml), and IFN-{gamma} (400 and 2,000 pg/ml).

Inhibition of tumor cell proliferation was determined with the classical 3-(4,5-dimethylthriazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) assay as previously described (19) using an MTT cell proliferation assay kit from Molecular Probes (Eugene, OR). Live cells reduce MTT to a strongly pigmented formazan product, whereas dead cells do not. Inhibition of proliferation of tumor cells by TR-NS1 virus was determined from the equation percent inhibition of optical density at 570 nm (OD570) = (A – B)/(A x 100), where A is the optical density of wells containing tumor cells that were not infected with virus and B is the optical density of wells treated with virus.

To eliminate the effects of tumor cell stimulation by growth factors present in the FCS, in some experiments, cells were treated by first decreasing serum concentration to 5%, followed by culture in low FCS concentration (0.3 to 0.5%) for 16 to 20 h before the addition of virus, growth factors EGF and NDF, or tyrosine kinase inhibitors as described previously (10). For analysis of formation of tumor cell-leukocyte clusters, 5 x 104 infected or mock-infected tumor cells were incubated with 3 x 106 PBMC. Cultures were examined with an inverted microscope (Nikon TMS-F) and recorded using a NIKON Cool Pix 995 digital camera. Tumor cells were considered to induce clusters only when they were completely surrounded by leukocytes.

In vitro induction of CTL by TR-NS1 virus-infected tumor cells. LNCap, SKOV3, and SKBR3 cells were plated at a density of 5 x 104 cells/well in 24-well plates, allowed to attach for 2 to 3 h, and then infected with TR-NS1 virus. Two hours later, the infection medium was removed, the cells were rinsed with phosphate-buffered saline two times, and RPMI 1640 medium containing 10% FCS (complete RPMI 1640 medium) was added to the wells. A total of 3 x 106 freshly isolated PBMC were added 2 h later, followed by IL-12 at 100 pg/ml and/or EGF plus NDF at 60 ng/ml as indicated (10). IL-2 at 150 IU/ml was added 24 h later. When MDC were used as antigen-presenting cells (APC), they were added to the TR-NS1 virus-infected tumor cells before addition of T cells. In some experiments, tumor necrosis factor alpha was added to stimulate MDC maturation (2, 10). Cells were maintained in culture for 6 to 8 additional days. The presence of T cells expressing T cell receptors (TCR) specific for the CTL epitopes from HER-2 (E75), AES-1/2 (G60), and influenza virus matrix (M58) was determined using HLA-A2-peptide (dE75, dG60, and dM58) dimers on day 6 as described previously (26). Negative-control dimers consisted of HLA-A2-IgG1 dimeric molecules that were not pulsed with peptide before they were used for staining. Negative-control dimers were designated dNP.

CTL assays were performed on day 8 after priming as previously described using 51Cr-labeled tumor cells as the targets (2). Ag specificity of CTL was determined using inhibitory targets in the same experiment. Inhibitory targets were T2 cells pulsed with peptide E75 or not pulsed with peptide and were designated T2-E75 and T2-NP, respectively.

Immunoblot analysis. TR-NS1 virus was added at a final MOI of 3 PFU/cell on monolayers of LNCap cells and incubated with tumor cells for 60 min. Control cultures were mock infected and treated under the same conditions as the cultures of infected cells. Cells were then washed in cold phosphate-buffered saline and lysed in lysis buffer. Cell lysates were adjusted to the same protein concentration and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nylon membranes, and blotted with corresponding antibodies as previously described (26, 39). Scanning densitometry was performed as previously described using a Hewlett-Packard scanner. The results were analyzed using Scion Image software for Windows (Scion Corporation, Frederick, MD).

Cell cycle and apoptosis analysis. Uninfected LNCap cells or cells infected with TR-NS1 or PR8 virus were stained with annexin and propidium iodide using the TACS-annexin V-FITC apoptosis detection kit and were analyzed by flow cytometry using a Becton Dickinson FACSCalibur with Cell Quest software (Becton Dickinson) (26, 39).


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RESULTS
 
TR-NS1 virus inhibited proliferation of LNCap cells. To address whether TR-NS1 virus inhibited the proliferation of prostate cancer line LNCap, LNCap cells were infected with TR-NS1 virus over a range of viral doses. Inhibition of proliferation of LNCap cells was viral dose dependent and was as high as 85% (Fig. 1A). Addition of EGF after TR-NS1 virus infection slightly decreased the virus-induced inhibition of tumor cell proliferation. LNCap cells resistant to TR-NS1 virus infection at 48 h expanded; therefore, the apparent inhibitory effects of TR-NS1 virus were weaker at 120 h than at 48 h (compare Fig. 1A and B). To distinguish whether the effects of TR-NS1 virus infection differed in P-ERK+ PC-3 cells, the experiments were repeated with PC-3 cells under the same conditions. PC-3 cells are androgen independent but express a constitutively activated MEK->ERK pathway. TR-NS1 virus inhibited PC-3 cell proliferation by more than 90% in the presence of EGF (data not shown). The results of these experiments together show that TR-NS1 virus infection eliminated the majority of prostate tumor cells. Although only a small part (5 to 15%) of LNCap cells survived TR-NS1 virus infection, they expanded in culture. Exogenous EGF and NDF did not render the LNCap cells more susceptible to virus-mediated death. To the contrary, they induced a slight increase in resistance of LNCap cells to TR-NS1 virus-mediated death. This observation raised the question of how prostate tumor cells were eliminated.



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  		FIG. 1. (A and B) Dose-dependent inhibition of LNCap cell proliferation by TR-NS1 virus. EGF ({blacksquare}), at a final concentration of 60 ng/ml, was added 1 h after virus infection to some cells ({square}, no EGF added). The percent decrease in OD570 indicates inhibition of proliferation by virus infection relative to uninfected tumor cells by the MTT assay. Tumor proliferation was determined 48 h (A) or 120 h (B) after infection. (C and D) TR-NS1 virus mediates oncolysis of LNCap cells. LNCap cells were infected with the indicated amounts of TR-NS1 and PR8 viruses. The number of live tumor cells (Live Cell No) was determined before infection (Pre) and 48 h and 72 h after infection with PR8 and TR-NS1 virus or mock infection (none). Note the rapid decrease in LNCap cell numbers at 48 h after infection with TR-NS1 virus. (E to G) Both TR-NS1 and parental PR8 viruses infected the majority (>90%) of LNCap cells. Infected LNCap cells were stained with a specific antibody against HA, followed by FITC-conjugated rabbit anti-goat antibody, and analyzed by flow cytometry. The numbers of live gated cells out of total cells were as follows: 3,229/3,555 for no virus, 1,192/2,430 for PR8, and 862/2,205 for TR-NS1. PR8 virus replicated better than TR-NS1 virus in surviving LNCap cells, according to the geometric mean MFI, indicative of the higher density of HA in PR8 virus-infected cells than in TR-NS1 virus-infected cells.

In order to compare wild-type and TR-NS1 viruses with respect to their ability to inhibit tumor cell proliferation, the experiments were repeated with TR-NS1 and parental PR8 viruses in parallel. Figure 1C shows that TR-NS1 virus had stronger oncolytic effects than parental PR8 virus did. Figure 1D shows that at 50 PFU per cell and EGF, the increase in the number of TR-NS1 virus-infected cells was smaller (1.3-fold) than those of PR8 virus-infected cells and noninfected LNCap cells. To address whether these differences were due to differences in infectivity, LNCap cells were infected with PR8 and TR-NS1 viruses (50 PFU/cell) and expression of viral HA was determined 18 h later. Figure 1E, F, and G show high and similar numbers of LNCap cells infected with PR8 and TR-NS1 viruses. Thus, despite high levels of virus infection, a percentage of tumor cells survived and proliferated. Of note, PR8 virus-infected cells have higher levels of HA than TR-NS1 virus-infected cells (see the mean fluorescence intensity [MFI] values for HA staining), indicating that the wild-type virus has higher levels of replication in LNCap cells. These results are consistent with the known role of NS1 in enhancing viral protein expression (13, 15, 38).

To address whether IAV infection modified the cell cycle of the LNCap cells, we performed cell cycle analysis of uninfected, PR8 virus-infected, and TR-NS1 virus-infected LNCap cells. The results in Fig. 2A, B, C, and D show that infection of LNCap cells with 3 and 10 PFU/cell of TR-NS1 virus increased the number of apoptotic cells (pre-G1) by 2.25-fold and 3.28-fold, respectively, compared with uninfected cells. The effect of PR8 virus at 10 PFU/cell was similar to that of TR-NS1 virus at 3 PFU/cell. The apoptotic cells in TR-NS1 virus-infected LNCap cells represented 18.6% of the tumor population compared with 5.6% of uninfected tumor cells. At the same time, TR-NS1 virus inhibited the progression of the cell cycle from G1 to S phase. Similar results were also obtained with SKBR3 cells, which express constitutively activated HER-2 and ERK (data not shown).



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  		FIG. 2. (A to D) Cell cycle analysis of TR-NS1 virus-infected cells shows increases in apoptotic cells and lower numbers of dividing cells. LNCap cells were mock infected or infected with the indicated viruses. Twenty hours postinfection, the number of cells in the G1, S, and G2/M phases and the number of apoptotic cells (pre-G1) were determined by propidium iodide staining and fluorescence-activated cell sorting analysis. (E to G) Infection with TR-NS1 virus results in higher numbers of annexin-positive cells than infection with wild-type PR8 virus. LNCap cells were mock infected or infected with 100 PFU per cell of the indicated viruses. Flow cytometry analysis of cells stained 24 h after infection with annexin V-FITC and propidium iodide is shown. (H) Treatment of LNCap cells with PKC and MEK->ERK inhibitors reduced inhibition of proliferation of LNCap cells by TR-NS1 infection. LNCap cells were treated with 10 µM caspase-3 inhibitor EDVE-fmk (Cas-3), 10 µM caspase-9 inhibitor Z-LEHD-fmk (Cas-9), 40 µM PKC inhibitor BIM, or 60 µM MEK/ERK inhibitor U0126, and infected with 16 PFU/cell of TR-NS1 virus. Cell viability was determined 48 h postinfection. The percent decrease in OD570 indicates inhibition of proliferation by virus infection relative to uninfected tumor cells in the MTT assay. (I) TR-NS1 virus activated ERK-1 in LNCap cells. LNCap cells were mock infected or infected with 3 PFU/cell of TR-NS1 virus. One hour postinfection, cell lysates were made and analyzed by Western blotting using antibodies against the indicated proteins. The numbers to the right side of the bands indicate (top to bottom) the following ratios: P-ERK-1/ERK-1, P-ERK-2/ERK-2, ERK-1/actin, and ERK-2/actin.

We have also determined the percentage of apoptotic LNCap cells after infection with wild-type PR8 and TR-NS1 viruses by annexin V staining (Fig. 2E, F, and G). The number of annexin-positive cells in early apoptosis increased by 97% in cultures infected with TR-NS1 virus but only by 50% in cultures infected with PR8 virus compared with noninfected cells. Our results suggest that the TR-NS1 virus induced higher apoptosis than the wild-type PR8 virus, consistent with the proposed attenuation of apoptosis by NS1 during virus infection (50). In summary, TR-NS1 virus infected the majority of tumor cells, induced apoptosis in a subpopulation of these cells, and inhibited tumor cell proliferation. However, these effects were transient and surviving cells expanded.

TR-NS1 virus activated ERK-1 in LNCap cells. To identify signaling pathways mediating inhibition of proliferation, LNCap cells were pretreated with the PKC inhibitor BIM, the MEK-ERK inhibitor U0126, and caspase inhibitors at the time of infection with 16 PFU of TR-NS1 virus per cell (9). Pretreatment with BIM resulted in less inhibition of proliferation by TR-NS1 virus, as detected by the MTT assay at 48 h postinfection, indicating a role for PKC activation in LNCap infection. The MEK->ERK inhibitor U0126 had similar (although weaker) blocking of inhibitory effects than the PKC inhibitor did. The caspase-3 and caspase-9 inhibitors were also able to reduce the antiproliferative effects of TR-NS1 virus infection, but they were significantly less efficient than U0126 (Fig. 2H). Thus, maximum antiproliferative effects mediated by TR-NS1 virus require PKC and MEK-ERK activities, as well as the induction of caspase pathways.

It has previously been reported that IAV infection induces ERK activation (35). The results in Fig. 2I show that interaction of TR-NS1 virus with LNCap cells for 1 h activated ERK in these cells. The levels of phosphorylation of ERK-1 were significantly higher than the levels of phosphorylation of ERK-2, suggesting that virus infection preferentially activated ERK-1. This is evidenced by the phospho-ERK (P-ERK)/ERK ratios. The P-ERK-1/ERK-1 ratios in uninfected and infected cells were 0.026 and 0.746, respectively, an increase of more than 20-fold. The P-ERK-2/ERK-2 ratios in uninfected and infected cells were 0.235 and 0.815, respectively, which represented an increase of less than fourfold. The total amounts of ERK-1 and ERK-2 were similar in uninfected and TR-NS1 virus-infected cells compared with actin.

Human PBMC and IL-12 induced complete elimination of TR-NS1 virus-infected LNCap cells. Since the inhibition of proliferation of LNCap cells by TR-NS1 virus was transient, the elimination of tumor cells by direct viral infection alone was not effective. In addition to directly lysing tumor cells, oncolytic viruses are known to activate specific immune responses with antibody responses to the virus being dominant. Induction of cellular responses to tumor-specific antigens would require the presence of proinflammatory cytokines, such as IL-12, which is essential for CD8+ cell differentiation and natural killer (NK) cell activation. To address whether TR-NS1-LNCap cells stimulated release of endogenous IL-12 in PBMC, LNCap cells were infected with increasing doses of TR-NS1 virus and incubated with either freshly isolated PBMC (containing M{phi} and DC) or nonadherent PBMC (lacking M{phi} and DC) at two stimulator/responder ratios. M{phi} and DC are the main producers of IL-12. The results in Fig. 3A and B show that TR-NS1-LNCap cells did not induce an increase in the IL-12 level above the background level at 24 or 48 h, indicating that these infected cells were weak inducers of IL-12. Similar IL-12 levels were observed for TR-NS1-infected PBMC (Fig. 3C). The same PBMC responded with high levels of IL-12 to positive-control inducers, such as muramyl dipeptide and staphylococcal peptidoglycan (Fig. 3D).



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  		FIG. 3. (A to D) TR-NS1-LNCap cells and TR-NS1 virus are weak inducers of IL-12 in PBMC. LNCap cells were infected with TR-NS1 virus at the indicated MOI for 4 h. Freshly isolated PBMC (A) or nonadherent PBMC (B) were added at a stimulator/responder ratio of 1:16 (circles) or 1:32 (squares). IL-12 production was determined 24 h (open symbols) and 48 h after stimulation (closed symbols). The number of responders in each well was kept the same at 500,000 PBMC. (C) PBMC were incubated directly with TR-NS1 virus using the same conditions as in panel A, but in the absence of LNCap cells. (D) For a positive control, the PBMC were induced by muramyl dipeptide (MDP) and staphylococcal peptidoglycan (PGN). (E and F) TR-NS1-LNCap cells attract leukocytes from PBMC. Uninfected LNCap cells (F) and TR-NS1-LNCap cells (E) are shown. (G) Addition of IL-12 and PBMC completely eliminated TR-NS1-LNCap cells. LNCap cells were infected with 3 PFU per cell of TR-NS1 virus where indicated, washed, and distributed in equal numbers (50 x 104) per well in a 24-well plate in complete RPMI 1640 medium. A total of 3 x 106 of PBMC from the same donor were added in each well where indicated. IL-12 was added at 200 pg/ml where indicated. The percent live cells was determined by dividing the number of live tumor cells from each culture 48 h after infection by the number of originally plated tumor cells.

Despite weak or no induction of IL-12, TR-NS1-LNCap cells attracted leukocytes, which formed large clusters with tumor cells within 24 h (Fig. 3E). Cluster formation was not observed with uninfected tumor cells (Fig. 3F). Therefore, chemoattraction and cell aggregate formation were due exclusively to virus infection. In separate experiments, we also observed the formation of clusters between MDC infected with PR8 virus or other influenza virus variants and leukocytes (data not shown). To address whether this effect was amplified by IL-12, IL-12 was added to all cultures (uninfected and infected). IL-12 did not induce cluster formation in cultures of uninfected LNCap cells and PBMC (data not shown). These results were confirmed with three additional healthy donors and with ovarian (SKOV3) and breast (SKBR3) cells (data not shown). It should be noted that LNCap cells are HLA-A2+ (12), but not all donors were HLA-A2+.

To determine whether the formation of clusters was correlated with a reduction in the number of tumor cells, we counted the live surviving tumor cells in each culture. The results are shown in Fig. 3G. The number of TR-NS1-LNCap cells incubated with PBMC decreased to 20% (48 h) of the uninfected cells. The number of uninfected LNCap cells incubated with IL-12 and PBMC also decreased compared to control LNCap cultures, but 35% of tumor cells were alive 48 h later. In contrast, TR-NS1-LNCap cells incubated with PBMC and IL-12 were completely eliminated 48 h later. Therefore, leukocytes together with IL-12 completely eliminated tumor cells infected with TR-NS1 virus.

TR-NS1-LNCap cells induced IFN-{gamma} in human PBMC. IFN-{gamma} is the prototype Th1 cytokine whose synthesis is activated by Ag plus IL-12 in T cells and by IL-12 alone in NK cells (47). To address whether TR-NS1-LNCap cells activated induction of IFN-{gamma} in PBMC, we analyzed early IFN-{gamma} induction in response to stimulation by uninfected LNCap and TR-NS1-LNCap cells. Because EGF plus NDF induced resistance of tumor cells to death by infection with TR-NS1 virus, they were included in the experiment. Uninfected LNCap cells induced low and insignificant levels of IFN-{gamma} in PBMC (Table 1). The level of IFN-{gamma} increased by more than 3-fold when TR-NS1-LNCap cells were used as stimulators and by more than 12-fold when IL-12 was added to the cultures. Therefore, infection of LNCap cells with TR-NS1 virus activated IFN-{gamma} production in PBMC even in the presence of EGF plus NDF. Exogenous IL-12 acted as a cofactor to enhance IFN-{gamma} production. The results were confirmed with PBMC from a second donor. Figure 3A shows that the amount of IL-12 produced by PBMC in response to high numbers of TR-NS1-LNCap cells was borderline (10 pg/ml). Thus, it is likely that exogenous IL-12 at 200 pg/ml compensated for the low endogenous production of IL-12 in response to virus. To address whether IFN-{gamma} and IL-12 could eliminate tumor cells, IFN-{gamma} at the concentration detected to be produced by the same number of PBMC (400 pg/ml) and IL-12 at the concentration used as a cofactor for induction of IFN-{gamma} (200 pg/ml) were added to uninfected LNCap cells. The effects of cytokines on the proliferation of tumor cells were determined 3 days later. Figure 4A shows that these cytokines together eliminated less than 50% of the tumor cells. It should be mentioned that the IFN-{alpha}-mediated decrease in the number of LNCap cells (10 to 20%) was smaller than the decrease mediated by IFN-{gamma}. These results indicated that IL-12 and IFN-{gamma} mediated only partial tumor elimination.


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  		TABLE 1. Induction of IFN-{gamma} in PBMC by TR-NS1-LNCap cellsa



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  		FIG. 4. (A) IFN-{alpha}, IFN-{gamma}, and IL-12 mediate incomplete elimination of uninfected LNCap cells. LNCap cells were incubated with IL-12, IFN-{gamma}, or IL-12 plus IFN-{gamma} and as a negative control with IFN-{alpha} at 1,000 U/ml (bar 1) or 4,000 U/ml (bar 2). Cytokines were not added to the medium of some cells (none). The number of live cells was determined 48 h later. (B and C) CD8-mediated lysis of tumor cells by PBMC stimulated with TR-NS1-LNCap cells. Lysis of uninfected and TR-NS1 virus-infected LNCap cells (32 PFU/cell) by PBMC stimulated with TR-NS1-LNCap is partially inhibited by CD8-specific antibody but not by isotype control antibody, indicating that one population of effectors was CD8 dependent. Effectors were generated by stimulation with uninfected LNCap cells (LNCap-ST) or by stimulation with TR-NS1-LNCap cells (TR-NS1-ST). The effector-to-target cell (E:T) ratio is shown. The percent specific lysis in the presence of isotype control Ab (black columns), anti-CD8 Ab (gray columns), and anti-CD3 Ab (white columns) is indicated. The percentage of death due to CD8 T cells is indicated over the columns in panel B. In panel B, significant differences in the percentages of specific lysis are indicated by an asterisk (P < 0.05). NS, not significantly different.

CD8+ cells activated by TR-NS1-LNCap cells lysed uninfected LNCap cells. IAV is known to activate natural killer cells, which mediate lysis of virus-infected cells. This process involves recognition of HA expressed on targets by NK cells (29). In addition, we wanted to know whether TR-NS1-LNCap cells activated CD8+ cells that recognized antigens presented by the tumor cells. For this purpose, we performed cytolytic assays using noninfected LNCap cells as the targets. To account for lytic effects mediated by CD8+ cells, inhibition of tumor lysis by CD8-specific Ab was determined in parallel. Effectors were HLA-A2+ PBMC stimulated with uninfected LNCap (designated LNCap-ST) and with TR-NS1-LNCap (designated LNCap-TR-NS1-ST). Lysis of LNCap cells by LNCap-ST effectors was not inhibited by CD8-specific MAb. Lysis of LNCap cells by TR-NS1-LNCap-ST effectors was partially inhibited by anti-CD8 Ab (Fig. 4B).

To verify that LNCap-TR-NS1-ST effectors recognized Ag generated by the endogenous pathway, we used TR-NS1-LNCap cells as the targets. LNCap-TR-NS1-ST effectors mediated higher levels of lysis of TR-NS1-LNCap cells than LNCap-ST effectors at the same effector-to-target cell ratio (Fig. 4C). Compared with LNCap-ST effectors, the higher levels of lysis of TR-NS1-LNCap cells by LNCap-TR-NS1-ST effectors were all due to the CD8+ effectors. In summary, the results are consistent with both uninfected and TR-NS1 virus-infected LNCap-mediated activation of a significant number of tumor-lytic NK and NK-T cells; however, only LNCap-TR-NS1-ST cells activated additional cytolytic CD8+ cells, which lysed more tumor cells. The results also indicate that cytolysis was not due only to alloreactive effectors activated by partially HLA-matched tumor cells, since LNCap cells (uninfected and infected) were used for activation of effectors from the same donor.

TR-NS1-LNCap cells expanded Ag-specific CD8+ cells. To determine whether Ag-specific CD8+ T cells expanded upon stimulation with TR-NS1-LNCap cells, we determined the presence of E75-TCR+ cells and M58-TCR+ cells in PBMC cultures stimulated by TR-NS1-LNCap cells. E75-TCR+ cells were expected to expand if peptides from HER-2 protein in LNCap cells were presented to CD8+ cells. Presentation can occur either by the tumor cells through the endogenous pathway or by the APC through the exogenous pathway/cross-priming. Similarly, M58-TCR+ cells were expected to expand if the immunodominant CTL epitope of IAV, represented by the influenza virus matrix M58-66 peptide was presented to CD8+ cells by either pathway. Figure 5A shows that the number of E75-TCR+ cells increased by more than 3.0-fold in PBMC stimulated by TR-NS1-LNCap cells compared with cultures stimulated with uninfected LNCap cells. Thus, TR-NS1-LNCap cells expanded HER-2/neu-specific CD8+ cells. In contrast, the expansion of M58-TCR+ cells was smaller (only twofold), and M58-TCR+ cells represented 1.25% of CD8+ cells (Fig. 5B).



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  		FIG. 5. (A and B) Increase in E75-TCR+ cells (A) and M58-TCR+ cells (B) in PBMC stimulated with TR-NS1-LNCap compared with PBMC stimulated with uninfected LNCap. The total number of Ag-specific cells was obtained by counting the numbers of E75-TCR+ and M58-TCR+ cells in the sample used for analysis in flow cytometry and reporting the numbers of cells compared to the original number of PBMC. Cultures received IL-12 as described in Table 1, footnote a. (C) LNCap cells infected with TR-NS1, {Delta}NS1, and KIF-NS viruses are better activators of expansion of E75-TCR+ cells than LNCap cells infected with parental PR8 virus. In all cases, LNCap cells were infected with 32 PFU/cell of the indicated viruses. KIF-NS virus expresses E75 in the neuraminidase of the TR-NS1 virus (14).

To address whether poor expansion of M58-TCR+ cells was due to anergy or an absence of M58-TCR+ cell precursors, DC from the same donor were used as APC to stimulate autologous T cells. M58-TCR+ cells were present in the PBMC of this donor with a high frequency (0.70%). M58-TCR+ cells increased by 3.74-fold when stimulated by TR-NS1-DC (see Fig. S1 in the supplemental material). Therefore, the TR-NS1-LNCap cells as the APC system were less stimulatory than the TR-NS1-DC cells were.

In order to determine whether M58 was dominant compared with E75, DC from the same donor were infected with KIF-NS virus (KIF-NS-DC). KIF-NS is identical to the TR-NS1 virus, except that it expresses the E75 epitope as part of the viral neuraminidase protein (14). KIF-NS-DC were used to stimulate autologous isolated T cells. The results show that the activating ability of M58 and E75 was similar when presented by the endogenous pathway by the same virus in the same professional APC (see Table S1 in the supplemental material). We also investigated whether free peptides E75 and M58 presented by DC mediated similar effects in activation of memory cells. We stimulated in parallel freshly isolated PBMC from three additional HLA-A2+ healthy donors with these peptides in the absence of IL-2 and IL-12 to avoid activation of naïve cells. All four donors responded to M58 by producing IFN-{gamma} in the range 24 to 150 pg/ml, while responded to E75 by producing IFN-{gamma} in the range 2.5 to 7.9 pg/ml. This indicated that there is a higher frequency of IFN-{gamma}-producing memory effectors to M58 but a lower frequency of IFN-{gamma}-producing memory effectors to E75 (see Table S2 in the supplemental material). The reasons for E75 dominance in LNCap-TR-NS1 cross-priming are unknown. LNCap cells overexpress HER-2 protein (not shown). It is possible that HER-2 protein already being present in sufficiently high levels provided significantly higher levels of precursors of E75 for CTL priming than IAV matrix protein. It is also possible that a sufficient amount of IAV matrix has not been synthesized in our experimental system.

In an additional experiment, using PBMC from a second HLA-A2+ donor as responders, we compared the immunogenicity of TR-NS1 virus with {Delta}NS1 (completely lacking the NS1 gene), KIF-NS (TR-NS1 virus expressing the E75 epitope), and PR8 (wild-type) influenza viruses (Fig. 5C). Parental PR8 virus was less effective than the NS1 mutant viruses in activation of E75-TCR+cells, while KIF-NS virus was the most effective in expanding E75-specific cells. Thus, the tumor cells lysed by TR-NS1 virus in the first 20 h after infection were most likely the source of E75 peptides.

TR-NS1 virus induced death of breast and ovarian tumors expressing activated HER-2 and activated CD8+ cells recognizing CTL epitopes from the HER-2 protein. To address whether TR-NS1 virus was oncolytic for tumor cells that express activated ERK and activated HER-2, we determined the ability of this virus to eliminate tumor cells of the cell lines SKBR3 (a breast cancer line) and SKOV3 (an ovarian cancer line). Both lines overexpress HER-2 protein at high levels. SKBR3 and SKOV3 cells were more sensitive to oncolysis than LNCap cells, and they were completely eliminated 72 h after infection with TR-NS1 virus (data not shown).

The results in Fig. 4 and 5 raised the question whether CTL could have been activated by cross-priming. Since both SKBR3 and SKOV3 cells are HLA-A2–, we addressed this question by determining whether TR-NS1 virus-infected SKBR cells (TR-NS1-SKBR3) and TR-NS1 virus-infected SKOV3 cells (TR-NS1-SKOV3) (32 PFU/cell) expanded and activated indicator HLA-A2+ E75-TCR+ cells in the presence of HLA-A2+ DC. Purified HLA-A2+ DC were added to HLA-A2– TR-NS1-SKBR3 and HLA-A2– TR-NS1-SKOV3 cells. Four hours later, purified autologous HLA-A2+ T cells were added to the cultures, followed by IL-12. To determine the presence of Ag-specific cells in these cultures, for indicators, we used T cells expressing TCR specific for the HER-2 protein HLA-A2 epitope E75 (E75-TCR+) and T cells expressing TCR specific for an epitope derived from the product of the "Hairy" HES/AES-1/2 gene, which is associated with the Notch receptor (G60-TCR+). Figure 6A to E show that there were more E75-TCR+ cells than G60-TCR+ cells in both TR-NS1-SKBR3 and TR-NS1-SKOV3 cocultures. There were also more E75-TCR+ cells present in the CD8lo population that expresses low levels of CD8 (MFI of CD8, 101 to 102) than in the CD8hi (expressing high level of CD8) population (MFI of CD8, >102).



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  		FIG. 6. TR-NS1-SKBR3 and TR-NS1-SKOV3 cells stimulated HLA-A2+ CD8+ cells by cross-priming. (A to E) Expression of E75-TCR+ and G60-TCR+ cells in HLA-A2+ CD8+ cells. (A) For a negative isotype control, cells were stained with dNP, followed by phycoerythrin (PE)-conjugated anti-mouse IgG1 antibody, and by FITC-conjugated mouse IgG2a for an isotype control for CD8-specific Ab. (B and C) E75-TCR+ CD8lo and E75-TCR+ CD8hi cells in CD8+ cells stimulated with TR-NS1-SKBR3 and TR-NS1-SKOV3 cells. (D and E) G60-TCR+ CD8+ cells after stimulation with TR-NS1-SKBR3 and TR-NS1-SKOV3 cells. The upper left quadrant contains CD8lo cells with an MFI of CD8 of 101 to 102. The upper right quadrant contains CD8hi cells with an MFI of CD8 of 102 to 103. Cells were stained with dE75 (HLA-A2-IgG1 dimers) and dG60 (HLA-A2-IgG1 dimers) as described in Materials and Methods. The numbers in the top left and top right quadrants are the percentages of positive cells in the whole gated population. E75-TCR+ CD8+ cells in control cultures stimulated with uninfected SKOV3 and SKBR3 were less than 0.5% of all cells (data not shown). (F and G) TR-NS1-SKOV3 cells induce E75-specific CTL. Cold targeted inhibition of lysis is shown. The targets were SKOV3 cells (F) and SKOV3.A2 cells (G). Inhibitory targets were T2 cells not pulsed with peptide (T2-NP) or T2 cells pulsed with 25 µg/ml E75 overnight (T2-E75). The numbers in parentheses are the percentages of specific inhibition of lysis by T2-E75 compared with T2-NP. Cells were infected with 32 PFU of TR-NS1 virus per cell.

There were also more E75-TCR+ cells in TR-NS1-SKBR3-stimulated CD8+ cells than in TR-NS1-SKOV3-stimulated CD8+ cells, indicating that cross-priming by DC was not only effective but was also dependent on the amount of the precursor protein in the tumor cells. SKBR3 cells express larger amounts of HER-2 protein than SKOV3 cells do (not shown). Internal-control G60-TCR+ cells were present in smaller numbers than E75-TCR+ cells, both in the CD8lo cells and in the CD8hi cells (Fig. 6D and E).

To address whether CTL activated by TR-NS1-SKOV3 were Ag specific and functional, we determined the ability of E75-pulsed T2 cells (HLA-A2+) to inhibit lysis of SKOV3 and SKOV3.A2 cells. SKOV3.A2 cells express a transfected HLA-A2 molecule; therefore, in contrast with parental SKOV3 cells, they were HLA-A2 matched with the effectors and the inhibitors, T2 cells. Figure 6F and G show that lysis of SKOV3 cells mediated by effectors induced by TR-NS1-SKOV3 cells was not inhibited by T2 cells pulsed with E75. In contrast, CTL induced by TR-NS1-SKOV3 recognized E75 on SKOV3.A2 presented by HLA-A2, as indicated by inhibition of tumor lysis by T2-E75 cold targets and lack of inhibition of tumor lysis by control T2 cells not pulsed with peptide.

In conclusion, our results show that infection of tumor cell lines by TR-NS1 virus resulted in death of some of the tumor cells. The effects were dependent on the host tumor cells. However, tumor cells more resistant to inhibition of proliferation by TR-NS1 virus, together with IL-12, expanded and activated effectors from PBMC, including tumor Ag-specific CD8+ cells, which eliminated the virus-resistant tumor cells.


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DISCUSSION
 
In this paper we report several novel findings regarding the ability of a recombinant attenuated IAV, TR-NS1 virus, to activate cellular immune responses against tumors resulting in tumor elimination. These findings were obtained with prostate tumor cells of the line LNCap, which is a poor host for TR-NS1 virus-mediated oncolysis, and were confirmed with breast and ovarian tumor cells, which are good hosts for replication of IAV variants expressing a truncated NS1 protein (TR-NS1) or lacking the NS1 protein ({Delta}NS1). {Delta}NS1 and other oncolytic viruses have been reported to induce irreversible inhibition of growth of tumors that express constitutively activated Ras->MAPK pathways and/or have defects in the IFN-{alpha}/ß response systems (8, 24, 32). We found that TR-NS1 virus infection activated ERK-1 in LNCap tumor cells. TR-NS1 virus infection also transiently inhibited LNCap cell proliferation and induced apoptosis in LNCap cells within 24 h. Cytokines, such as IFN-{gamma} and IL-12, also partially eliminated tumor cells. Human PBMC together with IL-12 completely eliminated TR-NS1-LNCap cells. Our results indicate that LNCap cells were eliminated as a result of synergistic action of the virus, cytokines, and cellular effectors.

CD8+ cells and NK cells activated by TR-NS1-LNCap and TR-NS1-SKOV3 cells mediated tumor lysis, indicating that infection of tumor cells by TR-NS1 virus was needed for proliferation and activation of Ag-specific CD8+ CTL. CD8+ T-cell activation was at least partly mediated by cross-priming by APC and not only directly by the tumor (alloreactivity), since CD8+ cells generated by stimulation with TR-NS1-SKOV3 mediated Ag-specific HLA-A2-dependent lysis of the uninfected HLA-A2+ tumor cells.

We found that both wild-type PR8 and TR-NS1 viruses were able to infect the majority of the tumor LNCap cells. PR8 virus replicated better in LNCap cells as indicated by the higher level of expression of HA (Fig. 1F and G). Although both wild-type and TR-NS1 viruses are oncolytic, an attenuated virus, such as TR-NS1 virus (46), is preferred as an oncolytic agent to reduce the potential side effects of wild-type virus infection in nontransformed cells. Interestingly, TR-NS1 virus is more attenuated in normal cells than in tumor cells, and it is known to have preferential specificity for replication in transformed cells (32). In addition, TR-NS1 virus was more oncolytic than the parental PR8 virus (Fig. 1C and D). This effect may be due to the truncated NS1 protein, which might have an impaired function in apoptosis inhibition (50). Consistent with this, TR-NS1 virus infection induced higher levels of apoptosis in LNCap cells than PR8 virus did (Fig. 2). Further studies are needed to address this point.

Our results have revealed antitumor effector pathways activated in virus-infected tumor cells that lack constitutively activated ERK (Fig. 7). We propose that TR-NS1 virus activated ERK-1 in these cells, more likely as a consequence of activation of PKC at the time of infection (41). ERK activation might allow for viral replication and induction of apoptosis in a proportion of tumor cells. APC, such as macrophages (M{phi}) and DC, were attracted to the tumor cells and most likely phagocytosed dying TR-NS1 virus-infected tumor cells. Peptides from tumor proteins presented by APC to T cells induced CTL expansion and differentiation, which resulted in tumor lysis.



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  		FIG. 7. Schematic representation of the proposed activation of anticancer pathways by TR-NS1 virus. The apoptosis induction and cytolytic pathways are labeled 1 and 2, respectively.

How tumor cells were selected for death by virus remains unclear. Cell cycle analysis did not indicate that TR-NS1 virus targeted cells in a particular phase of the cell cycle. We found a proportionally higher decrease in the number of cells in the S phase than in the G1 phase; this effect was stronger at higher TR-NS1 virus MOIs. It is also possible that some tumor cells in G1 phase were more sensitive to death by TR-NS1 virus than, for example, cells in G2/M phase.

How PKC->ERK and MEK-ERK activation connected to death pathways in TR-NS1 virus-infected tumor cells is unknown. The mitogenic or death effects of ERK were reported to be dependent on the agonist (growth factor, tetradecanoyl phorbol acetate, or virus) and the duration and intensity of ERK activation (11, 44). It is also possible that IAV variants activate additional signaling pathways mediated by other kinases or MAPKs other than ERK (28). In separate experiments using low doses of TR-NS1 virus, which did not activate ERK-1/2, we noted that AKT was constitutively activated in LNCap cells, but its level of phosphorylation was not affected by TR-NS1 virus, while p38MAPK remained inactive (lacked phosphorylation) before and after TR-NS1 virus infection (S. Sellapan and C. G. Ioannides, unpublished observations). A recent study indicates that a related virus, reovirus, required activated Ras->p38MAPK pathway to infect nonpermissive fibroblasts (33). We do not know at this time whether these conclusions could be extended to TR-NS1 virus infection of LNCap tumor cells. Another recent study demonstrated strong tropism of IAV for tumors transformed by activated Raf (34). Since LNCap cells express high levels of HER-2, it is possible that the HER-2->Ras->Raf pathway may be activated and act synergistically with IAV.

Our results indicate that the virus-initiated tumor cell elimination was amplified by activation of NK cells and Ag-specific CD8+ cells (Fig. 7). Ag-specific CD8+ cells were activated by APC by cross-priming, i.e., presentation of peptide fragments from the apoptotic proteins by specialized APC. Activation of APC could have been mediated by TLR-dependent and -independent pathways. An impaired NS1 function might have been resulted in higher activation of DC. In fact, a recombinant IAV without NS1 induced higher activation of DC than wild-type virus did (27). However, further experimentation is required to prove this hypothesis. Activation and expansion of CD8+ T cells was enhanced by the polarizing cytokine IL-12. IAV-infected tumor cells were weak inducers of IL-12. APC, such as DC, produce IL-12 if stimulated properly; however, the level of IL-12 produced by differentiated, mature DC is much higher than that produced by immature DC. In our studies, for APC, we used either unseparated PBMC or immature MDC. An explanation for the observed effects is that in these systems, APC did not completely differentiate within 16 to 20 h and that exogenous IL-12 compensated for the lack of endogenous IL-12.

The CTL priming effects mediated by TR-NS1-LNCap were confirmed using SKBR3 breast and SKOV3 ovarian tumor cell lines. Both cell lines express constitutively activated HER-2 kinase and activated ERK. Compared to LNCap cells, both SKBR3 and SKBOV3 cells were more likely to die after TR-NS1 virus infection. Death induced by TR-NS1 virus was prevented mainly by the MEK->ERK inhibitor U1026, but not by the ERK inhibitor PD08909, indicating an event upstream of ERK was required. Inhibitors of p38 MAPK, Jun N-terminal protein kinase, and phosphatidylinositol 3-kinase had weak and insignificant protective effects. However, it is possible that at higher concentrations these inhibitors can be protective, although the functional significance of this protection needs to be determined.

The finding that attenuated IAV, such as TR-NS1 virus with weak oncolytic properties, can induce, with help from IL-12, tumor-lytic CTL after infection of nonpermissive tumors is novel for the human tumor system. Multiple attenuation markers were inserted in this recombinant virus, including single-amino-acid changes in the PB2 and PA polymerase genes and the nucleoprotein gene and a truncation in the NS1 gene that resulted in loss of virulence in mice (14, 46). Similar NS1 truncations result in attenuation of primary isolates of influenza virus (36, 43). Thus, the use of TR-NS1 virus in humans is likely to be safe.

In our experiments, we used EGF at concentrations 100- to 200-fold higher than the concentrations of IL-12. Complete inhibition of antitumor cell proliferation effects of TR-NS1 virus by EGF was not observed. A new generation of RNA vectors can direct the cellular responses toward CTL of the desired specificity, e.g., prostate tumor Ag (3), by expressing the protein in one vector and IL-12 from another vector (37). Integration of the growth-inhibitory and T-cell-activating pathways in one vector might amplify the anticancer effects of the virus. Such studies are ongoing in our laboratory.


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ACKNOWLEDGMENTS
 
This work was supported in part by DOD grant 01-10299, a grant from the W. M. Keck Foundation and grant P50 CA93459 (MDACC-melanoma SPORE) to C.G.I., and grants from the NIH to A.G.-S. Peptide synthesis was supported in part by Core grant 16672 to the M. D. Anderson Cancer Center.

We thank Peter Palese for critical discussions and Richard Cádagan for excellent technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029. Phone: (212) 241-7769. Fax: (212) 534-1684. E-mail: adolfo.garcia-sastre{at}mssm.edu. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back

{ddagger} These two authors contributed equally to this work. Back


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Journal of Virology, January 2006, p. 383-394, Vol. 80, No. 1
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.1.383-394.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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