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Journal of Virology, July 2003, p. 7411-7424, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7411-7424.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Activation of Tumor Antigen-Specific Cytotoxic T Lymphocytes (CTLs) by Human Dendritic Cells Infected with an Attenuated Influenza A Virus Expressing a CTL Epitope Derived from the HER-2/neu Proto-Oncogene

Clay L. Efferson,1 Jeanne Schickli,2 Byung Kyum Ko,1 Kouichiro Kawano,1 Sara Mouzi,1 Peter Palese,2 Adolfo García-Sastre,2* and Constantin G. Ioannides1,3

Departments of Gynecologic Oncology,1 Immunology, M.D. Anderson Cancer Center, Houston, Texas 77030,3 Department of Microbiology, Mt. Sinai School of Medicine, New York, New York 100292

Received 1 November 2002/ Accepted 31 March 2003


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ABSTRACT
 
The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-{gamma}) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75+ CD45RO+ cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-{alpha} in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.


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INTRODUCTION
 
Vaccination is increasingly used as an experimental approach for cancer therapy, and the rate of responses has substantially improved by current strategies (reviewed in reference 40). Stimulation of a potent and specific immune response against tumor cells will most likely result in tumor clearance. To achieve effective antitumor responses, recent emphasis has been placed on approaches that stimulate strong cellular responses. These responses are mediated by T cells and particularly by cytotoxic T lymphocytes (CTLs). The induction of a strong CTL effector and memory response was recently demonstrated to be dependent on the dose of antigen (Ag) and the number of Ag-presenting cells (APCs) presenting this Ag (26, 48). An increase in the concentration of the Ag can be achieved by using a live virus to deliver the Ag to APCs. Viral replication results in sustained high-level intracellular synthesis of viral proteins. An increase in the number of specific APCs can be achieved by using viruses which stimulate potent CTL responses, indicative of efficient viral Ag expression into macrophages and/or dendritic cells (DCs) with high efficiency. Influenza viruses efficiently infect DCs, resulting in the potent activation of CTL responses (5, 7, 29). Based on these considerations, insertion of a tumor Ag or of a part of a tumor Ag into an influenza virus protein should lead to high-level expression of the tumor Ag on DCs with consequent high-level expression of the CTL epitope in the major histocompatibility complex class I (MHC-I) molecules.

Since live viruses generally induce potent immune responses, vaccines based on live (replication-competent) viruses might be effective cancer vaccines. The development of such vaccines is complex due to the possible risk of disease associated with live virus immunization (39). Vaccines which use influenza virus as a vector are particularly attractive because influenza virus effectively infects immature DCs and monocytes (11, 36), which perform most of the Ag presentation in vivo. Thus, the use of influenza virus as a vector ensures that a large number of APCs present the tumor Ag. Second, the number of influenza virus-encoded proteins is relatively small compared with other currently used viruses, a fact which limits the diversity in Ag presentation (10). The influenza virus strain that we have used as the viral vector in this study is derived from the influenza A/PR8/34 virus. This virus strain, which is being utilized as a backbone for the growth of the currently used inactivated influenza virus vaccines, has been demonstrated to be nonpathogenic in humans (3, 4). However, this strain is pathogenic in mice. Recent studies demonstrated that recombinant influenza viruses (rIVs) containing a truncated nonstructural (NS) protein 1 (NS1) were highly attenuated and did not induce disease but elicited protective immunity in mice (15, 47, 49). Based on these findings, we constructed an rIV A/PR8/34 virus vector (KIF-NS virus) with a truncated NS protein expressing an immunodominant CTL epitope, E75 (KIF), derived from the human HER-2/neu proto-oncogene recognized by ovarian tumor-specific, HLA-A2 restricted, CTLs (16). This virus was constructed by using recently developed techniques to generate influenza virus from plasmid DNAs (17, 34). As additional attenuation markers, we used a mutant influenza A/PR8/34 virus backbone containing single-amino-acid mutations in three more viral genes: PB2 S490N, PA K358E, and NP E494D. These mutations were found to attenuate the virus in mice without affecting its ability to grow in tissue culture and embryonated eggs (J. Schickli, unpublished data). Immature DCs infected with KIF-NS virus activated and expanded T cells from healthy donors and cancer patients. A part of these T cells specifically recognized the immunodominant CTL epitope E75, HER-2 (amino acids 369 to 377). These CTLs also lysed tumor cells overexpressing HER-2. Lysis of tumor cells was inhibited by T2 cells pulsed with E75, suggesting that KIF-NS virus activated a population of E75-specific cytolytic effectors.


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MATERIALS AND METHODS
 
Cells. Peripheral blood mononuclear cells (PBMC) were collected from healthy individuals under institution-approved protocols. Similarly, ovarian ascites and breast pleural effusions were collected from discarded specimens under institution protocols. Ovarian and breast tumor-associated lymphocytes (OVA-TAL and BR-TAL) were generated as described previously (24, 25) and stored frozen before being used in the experiments. Cells of the myelomonocytic cell line THP-1 were maintained in culture in complete RPMI 1640 medium containing 10% fetal calf serum (FCS). The T2 cell line (HLA-A2+) was a gift from Peter Cresswell (Yale University) and was maintained in culture in Iscove's medium supplemented with 10% FCS. SKOV3.A2 tumor cells (HLA-A2+, HER-2hi) were generated in our laboratory (16) and were used as indicators of tumor recognition by CTL.

Plasmids and peptides. pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, and pCAGGS-NP are expression plasmids encoding the PB2, PB1, PA, and NP proteins, respectively, of influenza A/WSN/33 (WSN) virus under the control of a chicken ß-actin promoter. Expression plasmids for the viral RNAs of influenza A/PR/8/34 (PR8) virus (pPOLI-PB2/PR8, pPOLI-PB1/PR8, pPOLI-PA/PR8, pPOLI-HA/PR8, pPOLI-NP/PR8, pPOLI-NA/PR8, pPOLI-M/PR8, and pPOLI-NS/PR8) contain the corresponding cDNAs in a pUC18-based plasmid between a truncated human RNA polymerase (POL) I promoter and sequences of the hepatitis delta virus ribozyme (41). pPOL1-PB2-S490N/PR8, pPOL1-PA-K358E/PR8, and pPOL1-NP-E494D/PR8 are identical to pPOLI-PB2/PR8, pPOLI-PA/PR8, and pPOLI-NP/PR8, respectively, except for the indicated amino acid substitution in the corresponding viral sequence. pPOLI-NA/KIF expresses a mutated neuraminidase (NA) protein from the PR8 virus in which the amino acid residues 65 to 71 (TTSVILT) of the stalk domain of the NA were replaced by the nine-amino-acid sequence KIFGSLAFL, the CTL epitope E75 corresponding to amino acids 369 to 377 of the HER-2 gene product (16) (Fig. 1). pPOLI-NS/WSN-126 is a derivative of pPOLI-NS-RT (17). pPOLI-NS/WSN-126 expresses a mutated NS gene of WSN virus, which codes for a truncated NS1 protein of 126 amino acids due to the insertion of two stop codons at nucleotide 405 of the NS1 open reading frame. Synthetic peptides E75, derived from the HER-2 protein (amino acids 369 to 377), and M1 derived from the influenza A virus matrix protein (amino acids 58 to 66) were prepared by the Synthetic Antigen Laboratory of M.D. Anderson Cancer Center, purified by high-performance liquid chromatography, and stored frozen in a stock solution at 2.0 mg/ml in phosphate-buffered saline. The purity of peptides ranged between 95 and 97%.



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  		FIG. 1. Schematic representation of wild-type NA and mutant NA/KIF proteins of rIVs used in our studies. The E75 amino acid residues (bold) in the chimeric NA/KIF protein replaced amino acid residues 65 to 71 in the NA stalk (italics). CT, cytoplasmic tail; TM, transmembrane domain.

Construction and characterization of recombinant influenza A viruses. MDCK and 293T cells were maintained in minimal essential medium (MEM; Gibco-BRL) and Dulbecco's MEM (Gibco-BRL), respectively, containing 10% FCS (Harlan) and penicillin-streptomycin (Gibco-BRL). Rescued viruses were first grown at 37°C in MDCK cells in MEM containing 3 µg of trypsin (Sigma)/ml, 0.3% bovine serum albumin (Sigma), and penicillin-streptomycin, and they were subsequently passaged in embryonated chicken eggs. Viruses were rescued by transfecting mixtures of approximately 106 293T and MDCK cells with a combination of 12 plasmids (0.5 µg of each) with Lipofectamine 2000 (Gibco-BRL) in OPTIMEM medium, as described previously (41). Twenty-four hours posttransfection, the medium was replaced by virus growth medium. Seventy-two hours posttransfection, supernatants were passaged into fresh MDCK monolayers and further amplified in embryonated eggs. rIV-PR8 was rescued by transfecting pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, pCAGGS-NP, pPOL1-PB2-S490N/PR8, pPOLI-PB1/PR8, pPOL1-PA-K358E/PR8, pPOLI-HA/PR8, pPOL1-NP-E494D/PR8, pPOLI-NA/PR8, pPOLI-M/PR8, and pPOLI-NS/PR8 plasmids. rIV-KIF (Fig. 1) was rescued by transfecting pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, pCAGGS-NP, pPOL1-PB2-S490N/PR8, pPOLI-PB1/PR8, pPOL1-PA-K358E/PR8, pPOLI-HA/PR8, pPOL1-NP-E494D/PR8, pPOLI-NA/KIF, pPOLI-M/PR8, and pPOLI-NS/PR8 plasmids. rIV-KIF-NS was rescued by transfecting pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, pCAGGS-NP, pPOL1-PB2-S490N/PR8, pPOLI-PB1/PR8, pPOL1-PA-K358E/PR8, pPOLI-HA/PR8,pPOL1-NP-E494D/PR8, pPOLI-NA/KIF, pPOLI-M/PR8, and pPOLI-NS/WSN-126 plasmids. The identity of the rescued viruses was confirmed by reverse transcription (RT)-PCR and restriction sequence analysis of the mutated gene segments.

Animal infections. Six-week-old BALB/c mice were anesthetized and infected intranasally with 50 µl of phosphate-buffered saline containing 103 or 104 PFU of the indicated influenza A virus. Animals were monitored daily and were euthanized when observed in extremis. All procedures were in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals.

Cytokine and CTL assays. Alpha interferon (IFN-{alpha}), IFN-{gamma}, interleukin-2 (IL-2), and IL-12 were detected as described previously (1, 28) by using the corresponding enzyme-linked immunosorbent assay kits obtained from Biosource International and R&D Systems. For Ag recognition, CTL assays were performed as described previously (16, 24, 25) by using as targets 51Cr-labeled T2 cells which were either pulsed with E75 (T2-E75) at various concentrations or were not pulsed with peptide (T2-NPP). The experiments were performed in triplicate. The percentage of E75-specific lysis was determined by subtracting the percentage of lysis of T2-NPP targets from the percentage of lysis of T2-E75 targets. For tumor lysis, indicator HER-2hi, HLA-A2+ SKOV3.A2 cells were used as targets. To determine the specificity of recognition of the endogenously presented E75 by the tumor we performed cold-target inhibition of tumor lysis experiments. T2-E75 and T2-NPP cells were added to wells containing 51Cr-labeled SKOV3.A2 cells followed by the effectors. Cells were incubated for 4 to 20 h. The percent specific inhibition of tumor lysis by T2-E75 was determined by subtracting the percentage of tumor lysis in the presence of T2-E75 from the percentage of tumor lysis in the presence of T2-NPP and dividing the number by the percent tumor lysis in the presence of T2-NPP (16, 24). P values were calculated by using the Student t test.

Flow cytometry. Determination of the number of cells expressing a T-cell receptor (TCR) specific for E75 bound to HLA-A2 (E75+-TCR cells) was performed by using E75 dimers (dE75). In brief, dE75 were prepared by incubating 10 µl of the empty HLA-A2-immunoglobulin G (IgG) dimers with 10 µM peptide E75 at 37°C for 18 h as described in the manufacturer's instructions. Empty HLA-A2-IgG dimers were obtained from BD Pharmingen, San Diego, Calif. Negative-control dimers (dNPP), which were empty HLA-A2-IgG dimers not pulsed with peptide, were prepared in parallel and tested in the same experiment. Positive-control influenza matrix peptide M1 (amino acids 58 to 66) dimers (dM1) were prepared simultaneously using the same amount of M1 peptide. Cells were incubated with 10 µl of dE75, 10 µl of dM1, and 10 µl of dNPP followed, after washing, by anti-mouse IgG1 phycoerythrin (PE)- or fluorescein (FITC)-conjugated secondary antibody. For multiple-color analysis, cells were incubated first with dE75, then with anti-mouse IgG1 PE- or FITC-conjugated secondary antibody, and then with anti-CCR7 (µ,{kappa}) antibody followed by biotinylated anti-mouse IgM or, alternatively, when dE75-PE was used, with anti-CCR7 allophycocyanin-conjugated antibody. For CD45RA and CD45RO expression, cells were then stained with the corresponding specific antibodies followed by FITC- or allophycocyanin-conjugated anti-mouse IgG2a secondary antibodies or anti-mouse IgG2b as described previously (12). Antibodies to leukocyte surface Ags CD1c, CD3, CD4, CD8, CD14, CD19, CD56, and CD83 were obtained from BD Pharmingen. Immune rabbit polyclonal serum against the influenza WSN virus containing antibodies to both H1 and N1 (NA) serotypes was generated in the laboratory of P. Palese. Monospecific goat sera against the H1 and N1 proteins of influenza virus were obtained from the National Institute of Allergy and Infectious Diseases, National Institutes of Health Reference Reagent Repository. DCs infected with the parental rIV-A/PR8/34 (PR8) and the rIV-KIF and rIV-KIF-NS viruses were first incubated with preimmune rabbit Igs (2 mg/ml) to diminish nonspecific binding of the serum to Fc receptors and then incubated with a 1:400 dilution of the anti-WSN serum or of the anti-N1 serum followed by FITC-conjugated rabbit anti-goat Igs (Sigma Chemical Co.). Both anti-WSN and anti-N1 antibodies were used to determine the expression of influenza proteins on the DC surface. Cells were analyzed in a flow cytometer (Becton-Dickinson) with the Cell Quest software.

Stimulation of T cells by influenza viruses. Monocyte-derived DCs were used as APCs for these experiments. Immature DCs were generated from adherent PBMCs, as described previously, by culture in granulocyte-macrophage colony-stimulating factor and IL-4 for 4 to 6 days (1, 28). To induce DC maturation, 5- to 6-day-old DCs were treated either with lipopolysaccharide (LPS; Sigma) at a 20-ng/ml final concentration or with tumor necrosis factor alpha (TNF-{alpha}, 100-IU/ml final concentration) for 48 h as described previously (27). Afterwards, DCs were washed, resuspended in L-15 medium without FCS, and infected with recombinant PR8, KIF, and KIF-NS viruses for 1 h. DCs were then washed three times in medium with 10% FCS and plated in 24- and 48-well plates. Responders used in these experiments were plastic nonadherent PBMCs from three HLA-A2+ donors and the OVA-TAL and BR-TAL T-cell lines. OVA-TAL and BR-TAL were >95% CD3+. Responders were added to DCs 1 to 2 h later to allow initiation of viral replication and expression of influenza virus proteins in DCs. In some experiments, IL-12 was added at priming at a concentration of 100 to 300 pg/ml (3 IU/ml) 1 h after the addition of responders as described previously (1, 28). The addition of IL-12 was considered necessary in the initial experiments since it was unclear whether the amount of IL-12 induced in DC by KIF and KIF-NS was sufficient to activate IFN-{gamma} induction in T lymphocytes. IL-2 was added 48 h later at a final concentration of 300 IU/ml and maintained in the wells through the entire culture period. Restimulations were performed under the same conditions, by using PR8, KIF, and KIF-NS DCs from the same donor as APCs. Responders were first rested by culture in complete RPMI medium without IL-2 for 48 h. DCs were infected with the same amount of virus (PFU equivalents) used at priming. The responder-to-stimulator ratio was 25:1. IL-12 was not added in restimulation experiments.


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RESULTS
 
Construction of recombinant KIF and KIF-NS influenza virus vectors. KIF and KIF-NS rIVs expressing the E75 epitope as part of their NA gene (Fig. 1) were generated by plasmid transfections (17, 41). These two viruses share 7 genes derived from PR8 virus. The PB2, PA, and NP genes of these viruses contain single-amino-acid mutations (PB2-S490N, PA-K358E, and NP-E494D) responsible for an increase in the PR8 virus 50% lethal dose for mice of more than 2 log (J. Schickli, unpublished observations). Table 1 shows survival rates in mice infected with wild-type PR8 virus and with recombinant PR8 virus containing these three point mutations. Both KIF and KIF-NS viruses contain these three point mutations. The KIF and KIF-NS viruses differ in their NS1 genes, which in the case of the KIF-NS virus has been mutated to encode a truncated NS1 protein of 126 amino acids. Previous observations have demonstrated that influenza viruses expressing carboxy-terminally truncated NS1 proteins were attenuated but retained immunogenicity in mice (15, 47). KIF and KIF-NS viruses grew to titers of approximately 107 PFU/ml in 10-day-old embryonated chicken eggs. The identity of the rIV was confirmed by RT-PCR and sequencing of their NA and NS genes. When indicated, recombinant PR8 virus containing the PB2-S490N, PA-K358E, and NP-E494D mutations was used as control virus (not expressing E75).


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  		TABLE 1. Recombinant PR8 virus containing amino acid substitutions PB2-S490N, PA-K358E, and NP-E494D is attenuated in mice

Influenza KIF-NS virus infection induced expression of NA on the surface of immature human DCs. The ability of KIF-NS virus containing E75 in its NA to infect immature DCs and express its NA on the surface was determined. DCs treated with LPS were used as mature DC controls. The purity of monocyte-derived DCs was more than 95%, as indicated by positive staining with anti-CD1c monoclonal antibody (MAb) and negative staining for monocyte and T- and B-cell markers (CD14, CD3, and CD19) (Fig. 2A). In separate experiments, monocyte-derived DCs stained positive with anti-CD13 MAb (data not shown). Figure 2B shows that, 20 h after infection with KIF-NS virus at 2 PFU/cell, both immature and LPS-treated DCs expressed NA on their surfaces. These experiments demonstrated that the recombinant KIF-NS virus is infectious not only for LPS-treated DCs but also for immature DCs.




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  		FIG. 2. (A) Surface marker expression on immature monocyte-derived DCs. The intensity of staining with the indicated antibodies is shown by solid lines. Grey lines represent isotype control staining. Percentages of positive cells (M1) are shown in the right corner. (B) Expression of NA on the surface of DCs infected with KIF-NS virus. (a) Negative-control DCs stained with preimmune goat Igs; (b) control immature DCs stained with anti-N1 goat Igs; (c) control mature DCs treated with LPS and stained with anti-N1 goat Igs; (d) DCs as in panel b, infected with KIF-NS virus; (e) DCs as in panel c infected with KIF-NS virus. Numbers in the corners indicate percentages of NA-positive cells. One representative experiment from three independently performed experiments with donor 1 DCs is shown. (C) (a, b, c, and d) CD83 expression on immature monocyte-derived DCs; (a and b) mock-infected DCs; (c and d) DCs infected for 18 h with 0.8 PFU/cell of KIF (c) or KIF-NS (d) virus. Cells were double-stained with isotype control PE (a), anti-CD83 PE (b, c, and d), preimmune goat serum FITC (a), or anti-NA goat Ig FITC (b, c, and d). Only CD83 staining is shown. Numbers in the right corners indicate percentages of CD83+ cells (top number) and mean fluorescence intensities (bottom number). (e, f, g, and h) Dot plots of NA+ cells in the gated CD83- population (upper left quadrants shown in the dot plots represented in panels a, b, c, and d. Numbers in the right corner represent percentages of NA+ cells. (D) CD83 and NA expression on TNF-{alpha}-treated DCs. (a and b) Mock-infected DCs; (c and d) DCs infected with 0.8 PFU of KIF-NS virus/cell; (a and c) isotype controls; (b and d) anti-CD83 PE plus anti-NA FITC-specific antibodies.

Because we used immature DCs in most stimulation experiments, we determined the ability of KIF and KIF-NS viruses at lower multiplicities of infection (0.8 PFU/cell) to induce rapid expression of CD83 as an indicator of maturation. Figure 2C (panels c and d versus b) shows that, compared with mock-infected DCs, 18 h after infection with KIF and KIF-NS viruses of immature DCs, the numbers of CD83+ cells increased by 100% (KIF) and 70% (KIF-NS). The mean fluorescence intensity for CD83 did not increase. NA+ DCs were 70% of CD83- cells (Fig. 2C, panels g and h versus f) in both KIF and KIF-NS virus-infected DCs. Only a few CD83+ DCs expressed NA on their surface. Thus, CD83 induction may be more efficient in cells expressing low (not detectable) levels of viral NA Ag. Alternatively, CD83 induction might be due to extracellular factors released by infected DCs and not due to the endogenous replicating virus. In a second experiment with even lower multiplicities of infection (0.3 PFU/cell), we found that DC incubation with KIF and KIF-NS viruses for 1 h followed by culture for 18 h increased the numbers of CD83+ cells by 30% and increased the mean fluorescence intensity for CD83 by 30% compared with DCs which were mock infected (data not shown). The NA+ cells were much lower (2.7% for KIF and 7.1% for KIF-NS viruses), but again, NA was detected on CD83- cells. It has been reported that monocytes require 10 to 12 days of culture to differentiate in CD83+ CD14- cells or at least 2 to 3 days of culture with TNF-{alpha} to differentiate in CD83+ cells. Thus, the low levels of CD83 within 18 h of incubation with virus may not indicate its later up-regulation (56). To address whether CD83 expression on DCs is enhanced by TNF-{alpha} and KIF-NS virus, we determined the numbers of CD83+ cells and the levels of expression of CD83 and NA on KIF-NS (0.8 PFU/cell) virus-infected DCs which were cultured for 48 h in TNF-{alpha}. KIF-NS virus induced a twofold increase in CD83+ DCs compared with TNF-{alpha} alone (27 versus 14%) (Fig. 2D, panel d versus b). Thirty percent of the NA+ DCs expressed CD83 while 87% of DCs expressed NA (Fig. 2D, panel d). Therefore, KIF-NS virus induced expression of CD83 in DCs productively infected, but this required previous culture of DCs in TNF-{alpha}. As shown previously for PR8 virus (11, 27), KIF-NS virus enhanced expression of CD86 and of MHC-I on DCs (data not shown).

rIV KIF-NS induced high levels of IFN-{alpha} in DCs. Since the NS1 protein was implicated in subverting the host innate immune responses to influenza virus (20, 21, 31, 46, 50), we investigated whether the truncation of NS1 in KIF-NS virus affected DC activation by measuring the induction of IFN-{alpha} and IL-12 in LPS-activated DCs. The results in Fig. 3A show that DCs infected with 2 PFU of KIF-NS virus/cell secreted high levels of IFN-{alpha} (>1 ng/ml) within 24 h compared with uninfected DCs or DCs infected with recombinant PR8 virus. The levels of IFN-{alpha} secreted by KIF-NS DCs increased during the following 24 h. The levels of IFN-{alpha} in DCs infected with recombinant PR8 virus did not increase. Similar results were obtained with immature DCs (data not shown). It is known that PR8 virus is a weak inducer of IL-12 in immature DCs (11). To address whether PR8 and KIF-NS viruses have a synergistic effect with LPS in augmenting IL-12 levels, DCs were first treated with LPS for 20 h then infected with either recombinant PR8 or KIF-NS viruses. The levels of IL-12 were determined 24 and 48 h later (Fig. 3B). In LPS-activated DCs, which secreted IL-12, PR8 virus inhibited IL-12 production by 30% during the first 24 h after infection. In contrast, KIF-NS virus inhibited IL-12 production by 10% during the same interval. Both PR8 and KIF-NS viruses enhanced IL-12 production by 30 to 40% above the levels induced by LPS alone during the next 48 h. Thus, infection with KIF-NS virus rapidly induced IFN-{alpha} in both immature and LPS activated DCs, but it did not have a strong potentiating effect in the induction of IL-12. Induction of IFN-{gamma} did not require exogenous IL-12 as a cofactor. High levels of IFN-{gamma} were obtained in the presence or absence of exogenous IL-12. Truncation of the NS1 protein did not interfere with the ability of the virus to express viral Ags in DCs.



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  		FIG. 3. Induction of IFN-{alpha} (A) and IL-12 (B) in DCs after infection with KIF-NS virus. LPS-stimulated DCs from a healthy donor were either left uninfected (UN) or infected with recombinant PR8 or KIF-NS virus. Viruses were used at concentrations of 2 PFU/cell. Supernatants were collected 24 and 48 h later and used to determine cytokine concentrations in duplicate determinations. IFN-{alpha}- and IL-12-specific enzyme-linked immunosorbent assay results indicate picograms of cytokine secreted by 105 DCs per milliliter. Values represent means ± standard deviations of triplicate determinations. In panel A, numbers at the tops of the columns represent the stimulation (n-fold) of IFN-{alpha} secretion in KIF-NS versus PR8 virus-infected DCs. Results from one experiment representative of two independently performed experiments are shown.

Stimulation of PBMCs and ovarian with KIF-NS virus-infected DCs induced high levels of IFN-{gamma}. One of the best-characterized immediate effects of stimulation of cellular immunity with influenza virus is the induction of IFN-{gamma} in leukocytes. The IFN-{gamma} response is mediated by both T cells and NK cells. PBMCs contain both T cells and NK cells, whereas long-term-cultured TALs are T-cell lines containing only CD8+ and CD4+ cells. To determine whether truncation of the NS1 protein diminished the ability of KIF-NS virus-infected DCs to induce IFN-{gamma}, nonadherent PBMCs from healthy donors and TALs from ovarian and breast cancer patients were stimulated in the same experiment with DCs infected with 1 PFU of KIF virus/cell (expressing E75 and a full-length NS1 protein) or with 1 PFU of KIF-NS virus/cell. IL-12 was not added to these cultures. Figure 4 shows that KIF and KIF-NS DCs showed similar potency in inducing IFN-{gamma} from either PBMCs (Fig. 4A) or BR-TAL-1 (Fig. 4C), and it was significantly higher than that of E75 peptide. The ability of KIF and KIF-NS DCs to induce IFN-{gamma} in PBMC was similar at restimulation (Fig. 4B). These results indicated that truncation of the NS1 protein did not affect the ability of the virus to induce IFN-{gamma} in this system.



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  		FIG. 4. Induction of IFN-{gamma} secretion in PBMCs from a healthy donor (donor 1) and BR-TAL-1 at priming with DCs infected with KIF and KIF-NS viruses. (A) Donor 1 priming; (B) donor 1 restimulation; (C) BR-TAL-1 priming. PBMCs or TALs (3 x 106) were incubated with 0.15 x 106 DCs infected for 1 h with 1 PFU of KIF or KIF-NS virus/cell. The responder-to-stimulator ratio was 20:1. Supernatants were collected every 24 h. Values represent means ± standard deviations of triplicate determinations. Results from one experiment representative of two independently performed experiments are shown. Open circles indicate the IFN-{gamma} levels secreted by responders in the presence of control noninfected DCs.

Stimulation of E75-specific responses by KIF-NS virus-infected DCs. Stimulation of the human T-cell population with influenza virus is expected to activate CD4+ and CD8+ cells specific to the vector, i.e., influenza epitopes and CD4+ and CD8+ cells cross-reacting with influenza virus proteins. To determine whether a part of the KIF-NS virus-induced IFN-{gamma} response is E75 specific, BR-TAL-1 primed with KIF-NS virus-infected immature DCs were restimulated with DCs pulsed either with peptide E75 or with the peptide corresponding to the influenza matrix CTL epitope M1 (amino acids 58 to 66) (positive control) or with DCs not pulsed with peptide (DC-NPP, negative control) in the presence of a low concentration (1 U/ml) of IL-12. Figure 5A shows that at restimulation, BR-TAL-1 rapidly secreted four-times-higher levels of IFN-{gamma} in response to the same DCs pulsed with peptide E75 within 40 h than it did in response to DC-NPP, indicating that the response was E75 specific. Breast TAL-1 that were stimulated by DCs infected with recombinant PR8 virus did not respond to stimulation with DC-E75 by IFN-{gamma} secretion. Control BR-TAL-1 which were incubated with DCs infected with KIF-NS virus did not secrete IFN-{gamma} in response to E75, suggesting that the E75 precursors were either too small in numbers or that they were not activated. Similar differences were obtained when IL-12 was absent in the cocultures, but the levels of IFN-{gamma} were lower (data not shown).



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  		FIG. 5. (A and B) BR-TAL-1 primed by KIF-NS virus-infected DCs recognized E75 at restimulation with DCs pulsed with E75 peptide. BR-TAL-1 were primed with control mock-infected DCs (DC), DCs infected with recombinant PR8 virus (PR8-DC), and DCs infected with KIF-NS virus (KIF-NS DC). BR-TAL-1 were then restimulated either with DCs not pulsed with peptide (DC + NPP) or with DCs pulsed with E75 peptide (DC + E75) or M1 (amino acids 58 to 66) peptide (DC + M1). (A) IFN-{gamma} induction. Supernatants were collected 24 h later. (B) IL-2 induction. IL-2 was added at a concentration of 100 pg/ml 6 h later, and supernatants were collected 72 h later. Values represent mean picograms of cytokine/milliliter ± standard deviations of duplicate determinations. Results from one experiment representative of two independently performed experiments are shown. (C and D) In vitro expansion of PBMC from two healthy donors at priming with PR8-DC, KIF-DC, and KIF-NS DC (C) or priming and restimulation with KIF-NS DC (D). Initial, starting number of PBMC in each culture.

To determine whether KIF-NS virus-infected DCs activated E75-specific IL-2 secretion, we determined in parallel the ability of BR-TAL-1 stimulated with KIF-NS virus-infected DCs to secrete IL-2 at recall with DCs pulsed with E75 peptide. In many instances, the amount of IL-2 produced by CD8+ cells at Ag stimulation is low and IL-2 is consumed. Due to this fact, we added exogenous IL-2 during restimulation and we determined total IL-2 levels recovered from cells that were primed with mock-infected DCs or primed with KIF-NS virus-infected DCs after restimulation of each group with DC-NPP and DCs pulsed with M1 (amino acids 58 to 66) and DC-E75 peptides. In these peptide restimulation experiments, exogenously added IL-12 was omitted. The results in Fig. 5B show higher levels of IL-2 from BR-TAL-1 primed with KIF-NS DCs and restimulated with DCs pulsed with peptide E75 or the positive-control peptide M1 (amino acids 58 to 66), compared with BR-TAL-1 which were primed with mock-infected DCs (negative control) and restimulated with DCs pulsed with peptides E75 and M1. These results indicate that KIF-NS virus-infected DCs activated not only M1-specific (vector specific) T cells but also E75-specific T cells from BR-TAL. Thus, the E75 epitope in the selected position is presented by KIF-NS virus-infected DCs to T cells.

Stimulation of lymphocytes by virus-infected DCs also resulted in cell proliferation. Figures 5C and D show the levels of cell proliferation after stimulation with influenza virus-infected DCs of PBMCs from two healthy donors. For example, for donor 1, the starting numbers were 2 x 106 cells in all cultures. Ten days after stimulation with DC-NPP, the total number of cells was 3.7 x 106. The resulting cell numbers in cultures stimulated with DCs pulsed with E75 were 3 x 106 cells while those in cultures stimulated with DCs infected with KIF-NS virus were 7.8 x 106 cells. Those results indicate that cells stimulated by KIF-NS virus infection expanded better than cells stimulated by peptide and that the KIF and KIF-NS viruses did not kill leukocytes in PBMCs.

Stimulation of ovarian and breast TAL with HLA-A2-matched DCs infected with KIF-NS activated cytotoxic function of tumor-reactive CTLs. To address whether DCs infected with KIF-NS virus activated tumor-specific CTLs, OVA- and BR-TAL were stimulated in vitro with KIF-NS virus-infected HLA-A2-matched DCs. OVA- and BR-TALs were isolated from ascites and from pleural effusions, respectively, from HLA-A2+ patients (1). In all instances, tumor cells present were HER-2hi. HLA-A2-matched DCs from healthy donors were used as APCs. Both the ovarian and the two breast TALs used in these experiments showed weak cytolytic activity prior to stimulation and did not specifically recognize E75 after coculture with uninfected DCs (Fig. 6A). OVA-TAL-1 primed with KIF-NS virus-infected DCs specifically lysed E75-pulsed T2 cells (Fig. 6A).



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  		FIG. 6. Stimulation of OVA-TAL-1 with KIF-NS virus-infected DCs (KIF-NS DC) induces a subpopulation of E75-specific tumor-lytic CTL. (A) Concentration-dependent recognition of E75 by KIF-NS DC-stimulated OVA-TAL-1 effectors; controls using uninfected DCs as stimulators (no virus) are also shown. (B) Kinetics of cold-target inhibition of lysis of SKOV3.A2 cells by KIF-NS DC-primed OVA-TAL-1 by nonpulsed T2 cells (T2-NPP) and T2 cells pulsed with E75 peptide (T2-E75). E:T ratio, 20:1. The specific percentage of cold-target inhibition by T2-E75 it is not significant compared with inhibition by T2-NPP (P < 0.1). (C) Cold-target inhibition of tumor lysis. OVA-TAL-1 was stimulated two times with KIF-NS DC followed by a third stimulation with either DCs pulsed with E75 peptide (E75-ST) or KIF-NS DC (KIF-NS-ST) in a 4-h CTL assay. The numbers above the columns indicate the percent inhibition of lysis by T2-E75 in comparison with that of T2-NPP. E:T ratio, 4:1. Cold-target inhibition by T2-E75 is significant compared with inhibition by T2-NPP. *, P = 0.02; **, P = 0.01. Values represent mean percentages of specific lysis ± standard deviations of triplicate experiments.

Although most CTLs induced by peptides or tumor Ag expressed in viral vectors recognize the peptide presented on indicator cells, the main question is whether such CTLs can recognize the Ag endogenously presented by the tumor. To address this question, we performed tumor lysis experiments by using as a target the indicator tumor cell line SKOV3.A2 (HLA-A2+, HER-2hi) in the absence or in the presence of cold target inhibitors (unlabeled T2 cells pulsed with E75). Figure 6B shows that within the OVA-TAL-1 cells primed with KIF-NS DCs there was a population that lysed SKOV3.A2 tumor cells. Part of these CTLs appeared to be E75 specific because cold (unlabeled) T2 cells pulsed with E75 peptide partly inhibited cell lysis. However, this was only evident in the 18-h CTL assay, and the levels of lysis in the presence of E75-pulsed T2 cells were not significantly different from the levels of lysis in the presence of T2-NPP. This suggests that priming with KIF-NS DCs induced only a small number of tumor-reactive E75-specific CTL. To determine whether recognition of endogenously presented E75 is enhanced by restimulation with KIF-NS DCs, OVA-TAL-1 were stimulated for two additional times with KIF-NS DCs. To compare the stimulating ability of KIF-NS virus with that of peptide E75, OVA-TAL-1 cells were then restimulated in parallel either with DCs pulsed with E75 (DC-E75) or with KIF-NS virus-infected DCs. After CD4+ cell depletion, effectors generated by each method (>85% CD8+) were tested in cold-target inhibition of lysis of SKOV3.A2 tumor cells. The results in Fig. 6C show that SKOV3.A2 cell lysis by effectors primed and restimulated with KIF-NS virus-infected DCs or with DC-E75 was inhibited by 48 and 30%, respectively. These results suggested that tumor-reactive E75-specific CTLs expanded at restimulation with KIF-NS virus-infected or peptide E75-pulsed DCs. Thus, for OVA-TAL-1, KIF-NS virus appears to have similar effectiveness with the peptide in the expansion and activation of the cytolytic function of responders.

To confirm the ability of KIF-NS DCs to activate tumor-lytic E75-specific CTLs from TALs, the experiments were repeated by using breast TAL (BR-TAL) from two patients as responders. The results in Fig. 7A showed that priming of BR-TAL-1 with HLA-A2-matched DCs infected with KIF-NS virus led to CTLs which lysed the indicator SKOV3.A2 cells. The results also revealed that lysis by BR-TAL-1 was significantly inhibited (19%) by E75-pulsed T2 cells compared with T2-NPP. Inhibition of lysis reached 40% when the assay was continued for 20 h, indicating that for BR-TAL-1, one stimulation with KIF-NS virus-infected DCs was sufficient to activate a subpopulation of tumor-reactive effectors.



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  		FIG. 7. Stimulation of BR-TAL with KIF-NS virus-infected DCs induced E75-specific CTL. (A) BR-TAL-1; (B and C) BR-TAL-2. Cold-target inhibition of tumor lysis experiments. (A) BR-TAL-1 primed with KIF-NS virus-infected DCs; (B) BR-TAL-2 primed with KIF virus-infected DCs followed by restimulation in parallel with PR8 (panel PR8) and KIF-NS (panel KIF-NS) virus-infected DCs were used in a 4-h cold-target CTL inhibition assay. (C) The same experiment as in panel B, with 20-h cold-target inhibition assays. Values represent means ± standard deviations of triplicate determinations. Numbers above the columns indicate percentages of inhibition by T2-E75. *, significant difference (P = 0.05) between lysis of SKOV3.A2 in the presence of T2 cells not pulsed with peptide (T2-NPP) and T2 cells pulsed with E75 peptide (T2-E75).

A different schedule of stimulation was used for BR-TAL-2 with the aim to address whether the vector expressing E75 was needed to maintain the E75-specific activity. BR-TAL-2 were primed with KIF-DCs (containing E75 and wild-type NS1) and expanded in culture for 1 week. These BR-TAL-2 were restimulated in parallel with either recombinant PR8 virus-infected DCs (as a control) or with KIF-NS DCs. One week later, the responders from each stimulation group (KIF->PR8) and (KIF->KIF-NS), were tested for lysis of SKOV3.A2 tumor cells and inhibition of lysis by T2-NPP- and E75-pulsed T2 cells. The results (Fig. 7B and C) show that T2-E75 inhibition of lysis of SKOV3.A2 cells by effectors generated by the approach KIF->PR8 was marginal even in the 20-h assay. In contrast, tumor lysis by effectors generated by the approach KIF->KIF-NS was significantly inhibited by E75-pulsed T2 cells (>40% inhibition). Inhibition was evident in the 20-h CTL assay but not in the 4-h assay. These results confirmed that KIF-NS DCs could activate a subpopulation of tumor-reactive E75-specific CTLs from three of three TALs, although there were responder-related differences in the magnitude of responses.

Stimulation of PBMCs from healthy donors by KIF-NS virus-infected DCs induced E75-specific CTLs. In contrast with tumor infiltrating/associated lymphocytes (TIL/TALs) which contain ex vivo activated memory CTLs, PBMCs from healthy donors contain mainly naive cells. The number of precursors for tumor Ag specific cells is low (0.5 to 0.05%). We tested the ability of KIF-NS DCs to activate E75-specific CTLs by using HLA-A2+ DCs from two healthy donors as APCs and autologous PBMCs as responders. Donor 1 PBMCs primed with autologous DCs infected with KIF and KIF-NS virus showed weak specific recognition of E75 in a CTL assay with E75-pulsed T2 cells as targets. The percentages of lysis by KIF-DC-induced CTLs at an effector-to-target cell ratio of 30:1 were 48.4% ± 2.9% and 59.4% ± 3.5% against nonspecific T2-NPP and T2-E75 target cells, respectively. The percentages of lysis induced by KIF-NS virus-infected DCs against the same targets were 9.3% ± 1.1% and 20.5% ± 1.1%, respectively (data not shown). Since KIF virus infection appeared to be more efficient than KIF-NS in activating E75-specific CTLs, we confirmed the immunogenicity of KIF-NS DCs in experiments with a second donor.

Donor 2 PBMCs primed with KIF-NS DCs recognized E75-pulsed T2 cells, but the levels of lysis of T2-E75 and T2-NPP were similar. We rationalized that a significant part of the lytic activity was due to NK cells activated by the influenza virus. CD56 defines a population of CD8+ T cells with NK cells markers but with nonvariant CDR3 regions (44). CD56+ but not CD57+ NK-type T cells are potently cytotoxic for both NK-resistant and NK-sensitive targets. The experiment was then performed in the presence of MAb to CD56. Figure 8A shows that in the presence of anti-CD56 MAb, KIF-NS DC-primed CTLs recognized E75-pulsed T2 cells significantly better than control T2-NPP cells. These results indicated that E75-specific CTLs were induced upon the priming of PBMCs with KIF-NS DCs. The results in Fig. 8B show that after three in vitro stimulations with KIF-NS DCs, the resulting CTLs specifically recognized E75-pulsed cells at an effector-to-target cell ratio as low as 4:1 and the lysis was E75 concentration dependent. To determine the ability of these PBMC-derived CTLs to recognize tumor cells, we determined the inhibition of lysis of SKOV3.A2 cells by E75-pulsed cold target T2 cells. The results in Fig. 8C show that donor 2 CTLs stimulated with KIF-NS virus-infected DCs recognized E75 on SKOV3.A2 cells (20% inhibition by T2-E75). Tumor-reactive E75-specific CTLs were a small population in the effectors since significant inhibition of lysis was evident only after 18 h of incubation with tumor and cold targets.



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  		FIG. 8. Stimulation of PBMCs from donor 2 with KIF-NS virus-infected DCs (KIF-NS DC) induce E75-specific CTL. (A) Kinetics of recognition of E75 by PBMCs from donor 2 8 days after priming with KIF-NS DC and expansion in IL-2. The E:T ratio was 30:1. Targets were T2-E75 ({triangleup}, {blacktriangleup}) and T2-NPP ({circ}, •). +anti-CD56, effectors were incubated with anti-CD56 MAb prior to addition to the assay. (B) Concentration-dependent recognition of E75 by CTLs from donor 2 after three in vitro stimulations with KIF-NS DC ({blacksquare}) or with uninfected autologous DCs ({square}). The target was T2-E75. The E:T ratio was 4:1. (C) Kinetics of cold-target inhibition of tumor lysis. Effectors were donor 2 CTLs after three in vitro stimulations with KIF-NS DC. The E:T ratio was 20:1. Cold targets were T2-NPP ({blacksquare}) and T2-E75 ({square}). Results represent mean percent specific lysis values ± standard deviations of triplicate determinations. Numbers above the columns indicate percent inhibition by T2-E75. *, significant difference (P = 0.05) between lysis of SKOV3.A2 in the presence of T2-NPP and T2-E75.

Expansion and differentiation of E75+ TCR cells from PBMCs at priming with KIF-NS virus-infected DCs. Our experiments indicate that after stimulation with KIF-NS virus-infected DCs, E75 TCR+ cells expanded. To address whether KIF-NS DCs also expanded CD8+ cells specific for CTL epitopes on viral proteins, PBMCs from three distinct donors were primed and restimulated with KIF-NS DCs. The resulting cultures contained variable proportions of E75+ TCR cells and influenza matrix (M1)+ TCR cells. (Table 2). These results show that both E75-specific and M1-specific T cells expanded after KIF-NS DC stimulation. There was a significant increase in cell numbers in cultures stimulated with KIF-NS DC compared with cultures stimulated with peptide E75-pulsed DCs (Fig. 5C and D).


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  		TABLE 2. E75 TCR+ and M1 TCR+ cells in PBMC cultures stimulated with KIF-NS DCs

We wanted to know whether priming with KIF-NS DCs followed by culture in IL-2 led to the expansion of effector CTLs (characterized by the presence of CD45RO and the lack of CCR7 markers) and of central memory CTL (characterized by the presence of both CD45RO and CCR7 markers) (9, 12). To address these questions, PBMCs from donor 2 were stimulated with immature DCs infected with KIF-NS virus, IL-2 was added, and the number of E75+ TCR cells was determined 10 days later by dE75 staining. Control PBMCs from the same donor were stimulated with uninfected DCs and cultured under the same conditions. Analysis of the presence and numbers of E75+ TCR cells was performed in at least six independent experiments, with various gate settings for negative controls, i.e., cells stained with dNPP. In the same experiments, we performed analysis of the expression of the chemokine receptor CCR7 and of CD45RO, which are present in activated T cells. One analysis is summarized in Fig. 9, where gates for the E75+ TCR cells in the control samples were set at or around 0.1%.



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  		FIG. 9. Priming with KIF-NS virus-infected DCs (KIF-NS DC) induced expansion of E75+ TCR cells. (A and B) Expansion of E75+ TCR cells from PBMCs of donor 2 primed with autologous DCs and isotype control FITC (x axis) versus control dimer NPP-PE (y axis) (A) or anti-CD8 FITC (x axis) versus dimer E75-PE (y axis) (B). (C and D) Expansion of E75+ TCR cells in PBMCs from donor 2 primed with KIF-NS DC. (C) Isotype control FITC-conjugated (x axis); negative-control dNPP-PE (y axis); (D) anti-CD45RO FITC-conjugated (x axis) versus dE75 (y axis). Results were obtained after analysis of at least 20,000 cells. One representative experiment of two performed in parallel is shown. These results are representative of two independently performed determinations with cells from the same donor and are representative of donors 1 and 2 (Fig. 8) stimulated in parallel. (E and F) Increase in CD8+ cells in PBMCs from donor 2 after priming with KIF-NS DC. (E) Forward scatter (x axis) versus isotype control (y axis); (F) forward scatter (x axis) versus CD8+ cells (y axis). More than 15,000 cells were analyzed. (G and H) CCR7 expression in E75+ TCR cells from donor 2 primed with KIF-NS DC. (G) Negative-control IgM APC (x axis) versus dNPP-PE (y axis); (H) CCR7-APC (x axis) versus dE75-PE (y axis). More than 32,000 cells were analyzed.

In PBMCs stimulated with autologous DCs and expanded in IL-2, the isotype control MAb-stained dNPP-positive cells were 0.26% while dNPP+ isotype control negative cells were 0.07% (Fig. 9A). E75+ TCR, CD8+ cells were 0.39% while E75+ TCR, CD8- cells were 0.28% (Fig. 9B). Thus, after subtracting the negative control values, E75+-TCR-specific CD8+ cells were 0.13% while E75+-TCR-specific CD8- cells were 0.21%. Stimulation with KIF-NS DCs led to a substantial increase in the numbers of E75+ TCR cells (Fig. 9C and D, isotype controls). This increase was as high as 10-fold when the results shown in Fig. 9D are compared with those shown in Fig. 9B. Most of E75+ TCR cells (3.3%) expressed the CD45RO marker, indicating that they were activated and not naive T cells (Fig. 9D). Priming with KIF-NS DC increased the numbers of CD8+ cells from 35.6% (Fig. 9B, lower right quadrant) to 83.14% (Fig. 9F, upper right quadrant). Figure 9D shows the cells stained by dE75, with the mean fluorescence intensity ranging between 102 and 104 (medium plus high affinity). In the same experiment, M1+ TCR cells were 3.8% (data not shown).

Since, in donor 2, CD8+ cells represented 83% of the population, cells were not stained with anti-CD8 but were stained with anti-CD45RO and anti-CCR7 MAb in parallel with dE75. Analysis of the cells stained with dE75-PE and anti-CCR7-APC showed that E75+ TCR, CCR7- cells were 0.60% while E75+ TCR CCR7+ cells were 2.77% (Fig. 9G, isotype controls, and H, specific antibodies). These results are in agreement with results obtained from analysis of CCR7 expression in the gated E75+ TCR, CD45RO+ population (Fig. 9D). Of the E75+ TCR CD45RO+ cells, 18.3% were CCR7- while 81.7% were CCR7+ (data not shown). This suggested that priming of PBMCs with KIF-NS DCs induced expansion of E75+ TCR cells, but the majority of these cells had a central memory and not effector phenotype.

We also estimated the number of E75+ TCR effectors in the CTL assay (Fig. 8A). Since 6,000 targets were used and the effector-to-target cell ratio was 30:1, then the total number of effectors was 180,000/well. Of these, 1,086 were E75+ TCR, CCR7- while 4,986 were E75+ TCR, CCR7+. Thus, the ratio of E75 TCR+ effectors to targets was 0.18 for CCR7- cells and 0.83 for CCR7+ cells. Assuming that NK cells were only 4% of the total effectors, this means 7,200 NK cells/well, or a ratio of NK cells to targets of 1:2. This NK-to-target ratio is higher than the ratio of E75+ TCR cells to targets. The presence of a low number of E75+-TCR-specific effectors in the wells of CTLs may explain the nonspecific lysis in the absence of anti-CD56 MAb. The presence of E75+ TCR, CCR7- cells at a ratio of 0.18 may explain why high levels of E75-specific tumor lysis were detectable mainly in 18-h CTL assays.


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DISCUSSION
 
In this study, we demonstrated that DCs infected with an rIV with a truncated NS1 protein containing a HER-2, HLA-A2-restricted CTL epitope encoded in the NA are capable of stimulating CTL responses in vitro in T cells from both ovarian and breast TAL as well as from HLA-A2+ randomly selected healthy donors. PBMC priming was associated with expansion of E75+ TCR cells (more than 10-fold) and induction of E75+ TCR cells of both central memory (CD45RO+ CCR7+) and effector (CD45RO+ CCR7-) phenotypes. The induction of IFN-{alpha} by KIF-NS virus in DCs is in agreement with studies on the IFN-antagonistic functions of the NS1 protein of influenza A virus. Deletion of the carboxy-terminal domain of the NS1 protein leads to an enhanced IFN-{alpha}/ß response in influenza virus-infected cells (21) due to a decrease in NS1 dimerization that partly impairs the ability of the NS1 to prevent IFN-{alpha}/ß synthesis in infected cells (49). Taking into consideration the recently reported effects of IFN-{alpha} on the induction of survival and maturation of CTLs (22, 37), the use of vectors based on rIVs with truncated NS1 proteins may be a promising strategy for human cancer vaccination. CTLs induced by KIF-NS DCs specifically recognized the E75 peptide presented by HLA-A2+ cells in cytokine assays. Furthermore, a part of these KIF-NS virus-induced CTLs recognized E75 on indicator tumor cells. OVA-TAL-1 stimulated three times by KIF-NS DCs recognized tumor in the brief (4 h) CTL assay. KIF-NS virus-stimulated breast TALs recognized E75 presented by SKOV3.A2 tumor cells in CTL assays. Since all stimulations were performed under the same conditions with DCs from the same healthy donor as the APCs, the differences in lysis may reflect differences in the number of CTL precursors, their state of differentiation, or their ability to expand following virus stimulation.

Demonstration of activation of Ag-specific tumor-lytic function and of Ag-specific IFN-{gamma} and IL-2 induction by human tumor Ag expressed in PR8-based vectors is novel for human tumor systems. Several possibilities are currently considered for the potentiation of the stimulatory ability of PR8 NS-based vectors: (i) expression levels of the epitope may need to be increased (52) and (ii) the Ag could be optimized by sequence changes for the induction of complete CTL functional differentiation (33, 42). Interestingly, induction of CTLs by influenza virus is less dependent of help (53), and in fact, influenza virus leads to declining CD4+ cells but not CD8+ memory cells (23). Recent studies started to investigate the use of RNA viruses for Ag presentation and experimental cancer vaccination (19, 38, 54, 55). Interestingly, activation of cytokine secretion by tumor-specific CD8+ CTLs by using DCs infected with an avian rIV expressing the melanoma-derived tumor-associated Ag MAGE-3 as an additional gene has been demonstrated (43). However, this strategy of expression may result in variable levels of Ag expression from virus batch to virus batch.

In this study we have used an attenuated influenza virus vector of human origin to stimulate tumor-specific CTLs. Stimulating CTLs by using rIV with truncated NS1 proteins may have several advantages over other strategies: unlike synthetic tumor peptides who have weak binding affinity to MHC-I and require high concentrations of peptide for exogenous loading, influenza virus delivers and expresses the epitopes endogenously. The increase in precursor protein concentration should lead to an increase in the amount of the CTL epitope delivered and presented by APCs. Based on the stable NA expression on DCs, the presentation of the epitope may be stable for at least 24 to 48 h. This allows the engagement of a significantly larger number of cells than when DCs are pulsed with peptide (2). Influenza virus also has the ability to induce a proinflammatory type 1 environment in the absence of adjuvant (35), similar to the one induced by poly(I-C) or double-stranded RNA (10, 11). This ability is probably enhanced by truncation of the NS1 protein (30), which is an antagonist of double-stranded RNA-activated pathways (3, 18). In contrast with other vectors which mediate their effects through mechanisms which depend on the death of the host, and representation of Ag by DCs, the influenza virus-mediated effects are direct, in that they infect DCs which mediate Ag presentation, and controllable by the virus dose (4). The positions of a large number of influenza CTL epitopes associated with various MHC-I molecules are known. This allows precise insertion of tumor CTL epitopes at defined sites. Longer sequences of 20 to 50 amino acids from tumor Ag can be inserted in favorable positions for expression of hybrid influenza virus-tumor Ag proteins (5). Although our studies have been focused on in vitro stimulation of tumor-specific CTL responses, it may also be possible to use influenza virus vectors expressing tumor Ags in vivo. This may have the advantage of resulting in tumor cell killing not only due to CTLs induced against the tumor cells but also due to direct virus-mediated oncolysis. In this regard, it is noteworthy that NS1-modified influenza viruses have specific oncolytic properties (6).

The ability of influenza virus to infect DCs in vitro is in general considered proof of the principle that influenza virus infects DCs in vivo. Recent results suggest that influenza virus is also able to infect DCs in vivo (29). One concern regarding the use of influenza vectors for cancer vaccination is that APC presentation can be blocked by anti-influenza virus antibodies. Recent evidence indicates that a brief Ag contact of a minimum of 1 h is sufficient to activate effector and memory CTLs (26, 48). Thus, T-cell activation should precede activation of Ig synthesis. Furthermore, the presence of protective anti-influenza antibodies may be helpful in controlling virus spread from the site of inoculation. However, the antiviral antibodies can neutralize the infectious virus in vivo, reducing the infectious dose. Alternatively, the influenza virus vector can be customized to contain viral antigenic determinants from a virus strain not recognized by anti-influenza virus antibodies present in the patient. It should also be possible to use an influenza virus vector to induce a population of Ag-specific CTLs and to maintain the response focused by boosting with peptides or with a second viral vector (32). On the other hand, anti-influenza antibodies bound to the infectious virus might facilitate delivery of the virus to DCs through FcR-mediated uptake endocytosis. The effects of antiviral antibodies in CTL induction are still unclear.

While influenza viruses with truncated NS1 proteins are attenuated in mice, and in our engineered vector we have included additional attenuation markers in three more viral genes, there is a concern as to the use of rIV in cancer patients with weakened organisms (8, 13). One possibility is to further attenuate the influenza virus by creating temperature-sensitive variants by mutations in NA (2) or by mutating its M2 gene (45, 51). Another is to use recombinant viruses expressing even shorter NS1 proteins, which results in a more profound viral attenuation (14, 47). Such questions need to be carefully addressed in animal and clinical studies.

In conclusion, the studies presented above demonstrated the feasibility of stimulating HER-2-specific CTL responses by using a novel vector consisting of a recombinant influenza A virus with a truncated NS1 protein. Ongoing studies in our laboratory are determining the effects of the vector and of epitope variants in the induction of tumor-specific central memory CTL and effector CTL differentiation. Our results suggest that influenza vectors may be efficient therapeutic tools to induce the differentiation of CTL responses to tumor Ag.


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ACKNOWLEDGMENTS
 
This work was supported in part by grant 17-97-I-7098 and NIH grants to A.G.-S. and P.P. Peptide synthesis was supported in part by the Core Grant CA 16672.

We thank James Reuben and Hui Gao for assistance with flow cytometry.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Box 1124, Mount Sinai School of Medicine, 1 Gustave L. Levy Pl., New York, NY 10029. Phone: (212) 241-7769. Fax: (212) 534-1684. E-mail: adolfo.garcia-sastre{at}mssm.edu. Back


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Journal of Virology, July 2003, p. 7411-7424, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7411-7424.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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