Affiliations of authors: J. W. Hodge, D. W. Grosenbach, H. Sabzevari, J. Schlom, Laboratory of Tumor Immunology and Biology, Division of Basic Sciences, National Cancer Institute, Bethesda, MD; A. N. Rad, Howard Hughes Medical Institute, Research Scholar's Program at the National Institutes of Health, Bethesda; A. Gómez Yafal, L. Gritz, Therion Biologics Corporation, Cambridge, MA.
Correspondence to: Jeffrey Schlom, Ph.D., National Institutes of Health, 10 Center Dr., Rm. 8B09, Bethesda, MD 20892-1750 (e-mail: js141c{at}nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Dendritic cells are highly specialized APCs that function as the principal activators of quiescent T cells and, thus, of cellular immune responses in vivo (7). Consequently, the unparalleled capacity of dendritic cells to induce antigen-specific T-cell responses has focused the attention of many investigators on the potential effectiveness of these cells in immunoprevention and immunotherapy. In experimental murine models, dendritic cells pulsed with peptides from tumor-associated antigens have been shown to induce antigen-specific antitumor responses in vivo (811), and fusion of dendritic cells with tumor cells has also been shown to enhance antitumor immunity (12). In addition, peptide-pulsed dendritic cells are being explored for the prevention and treatment of infectious diseases, such as acquired immunodeficiency syndrome (13,14), tuberculosis (15), chlamydia (16), and Epstein-Barr virus-implicated syndromes (17).
Since dendritic cells express high levels of histocompatibility molecules and most known costimulatory molecules, including B7-1, ICAM-1, and LFA-3, it is generally believed that the addition of a vector to express even higher levels of these costimulatory molecules would be of little, if any, advantage. On the other hand, one could theorize that hundreds of thousands of years of evolution would have placed the capacity of dendritic cells to activate T cells into a "median" state between (a) the ability to activate T cells to specific antigens such as those of microbial pathogens and (b) the induction of autoimmunity to self antigens of the host. Thus, we theorize that the natural expression levels of specific costimulatory molecules in either immature or mature dendritic cells would accommodate this median state of efficacy of dendritic cell populations. Poxvirus vectors were used to hyperexpress the triad of costimulatory molecules because (a) they can accommodate and express multiple transgenes and (b) they possess a high efficiency of infection of most cell types. Both replication-defective avipox (fowlpox; rF) and replication-competent vaccinia (rV) recombinant vectors were employed to hyperexpress the recombinant costimulatory molecules. The purpose of this study was to determine if the use of these vectors that can facilitate hyperexpression of these costimulatory molecules would enhance the capacity of dendritic cells to stimulate various T-cell responses.
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MATERIALS AND METHODS |
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Animal care. Animals were used in accordance with the guidelines of the National Cancer Institute's Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals.
Cell lines and cell cultures. The OVA-specific CD8+ effector/cytotoxic T-cell line was generated from C57BL/6 mice and recognizes the OVA peptide. This cell line was generated from splenocytes of mice vaccinated with OVA peptide in adjuvant; it was used in all subsequent assays involving OVA peptide as signal 1. The cytotoxic T-lymphocyte (CTL) line was maintained by weekly in vitro stimulation cycles with irradiated naive splenocytes (used as APCs) in complete medium (RPMI-1640 medium with fetal calf serum [10%], glutamine [2 mM], sodium pyruvate [1 mM], HEPES [7 mM], gentamicin [50 µg/mL], 2-mercaptoethanol [50 µM], and nonessential amino acids [0.1 mM]; Biofluids, Rockville, MD), supplemented with 1 µg/mL OVA peptide and 10 IU/mL murine interleukin 2 (IL-2) (Boehringer Mannheim Biochemicals, Indianapolis, IN). Before these cells were used as responders in antigen-specific proliferation assays, they were purified by centrifugation at 700g for 30 minutes over a FicollHypaque gradient (density = 1.119 g/mL; Sigma Chemical Co., St. Louis, MO) and replated in six-well culture plates (106 cells/mL, 5 mL/well) in complete medium supplemented with 10 U/mL murine IL-2 for 24 hours; these cells were considered to be "rested" OVA-specific T cells. For cytotoxicity assays, the target tumor cell line used was EL-4. EL-4 cells are T-cell lymphoma cells derived from C57BL/6 mice (H-2b class I allele-positive) and were obtained from the American Type Culture Collection (Manassas, VA), catalog No. TIB-39.
Preparation of dendritic cells.
Bone marrow was derived from 6- to 8-week-old female C57BL/6 mice (Taconic Farms, Germantown, NY). The procedure used in this study was a modified version of that described by Inaba et al. (20). Briefly, bone marrow was flushed from the long bones of the limbs and passed over a FicollHypaque gradient. Bone marrow cells were depleted of lymphocytes and Ia+ cells with the use of a cocktail of magnetic beads coated with monoclonal antibodies (MAbs) specific for CD4, CD8, and MHC class II antigens (MiniMACS; Miltenyi Biotec, Auburn, CA). The cells were plated in six-well culture plates (106 cells/mL, 5 mL/well) in complete medium supplemented with 10 ng/mL granulocytemacrophage colony-stimulating factor (GM-CSF) and 10 ng/mL interleukin 4 (IL-4) (R&D Systems, Minneapolis, MN). They were replated in fresh cytokine-supplemented medium on days 2 and 4. At 6 days of culture, the cells were harvested for infection, analysis, and vaccinations. Bone marrow-derived dendritic cells generated in the presence of GM-CSF and IL-4 exhibit an intermediate maturation stage with respect to phenotype and in vitro antigen-presenting capacity (21). For the indicated experiments, dendritic cells were further matured by treatment with 100 ng/mL murine tumor necrosis factor (TNF)- (Boehringer Mannheim Biochemicals), 0.1 µg/mL lipopolysaccharide (LPS) (Sigma Chemical Co.), or 5 µg/mL CD40-specific MAb (PharMingen, San Diego, CA) during the final 24 hours of culture.
Recombinant poxviruses. Two recombinant poxvirus vectors (rF and rV) were used in this study. The recombinant fowlpox viruses rF-B7-1 and rF-CEA/TRICOM have been described previously (6). The recombinant fowlpox virus rF-TRICOM was constructed by the insertion of foreign sequences into the BamHI J region of the genome of the POXVAC-TC (Schering Corp., Kenilworth, NJ) strain of fowlpox virus as described previously (22) and contains the murine B7-1 gene under the control of the SE/L promoter (23), the murine LFA-3 gene under the control of the I3 promoter (24), and the murine ICAM-1 gene under the control of the 7.5k promoter (25). The recombinant vaccinia viruses rV-B7-1 and rV-TRICOM have been described previously (6). Nonrecombinant wild-type fowlpox virus was designated FP-WT, whereas wild-type vaccinia virus (Wyeth strain) was designated V-WT. These wild-type viruses were used as controls for the TRICOM and B7-1 recombinant vectors to rule out the possibility that any effects in T-cell activation were due to vector alone.
Infection of dendritic cells. Dendritic cells were harvested on day 6 of culture and were washed with Opti-Mem (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). The cells (12 x 106/mL) were then either (a) mock-infected with Hanks' balanced salt solution, (b) infected with FP-WT, rF-B7-1, or rF-TRICOM at a multiplicity of infection (MOI) of 50 plaque-forming units (PFUs) per cell, or (c) infected with V-WT, rV-B7-1, or rV-TRICOM at 25 MOI in Opti-Mem for 5 hours. Complete medium (37 °C) was added after infection, and the cells were incubated at 37 °C overnight. After 18 hours, the cells were harvested for immunostaining, in vitro costimulation analysis, and in vivo administration.
Flow cytometry analysis. Cell-surface staining utilized three-color immunofluorescence. Staining was performed with primary fluorescein isothiocyanate-labeled antibodies CD11c, CD11b, H-2Kb, H-2Db, CD19, and Pan-NK as well as primary phycoerythrin-labeled antibodies directed against the MHC class II allele IAb, CD48 or murine LFA-3, CD86 (B7-2), CD3, and CD14 and the biotin-labeled antibodies CD80 (B7-1), CD57 (ICAM-1), and CD40. Biotin-labeled antibodies were subsequently labeled with streptavidinCy-chromeTM. All antibodies were purchased from PharMingen (Mountain View, CA). Cell fluorescence was analyzed and compared with that of the appropriate isotype-matched controls (PharMingen) with a FACScan cytometer using the Lysis II software (Becton Dickinson, Mountain View, CA).
In vitro costimulation analysis: pharmacologic signal 1. Female, 6- to 8-week-old C57BL/6 mice were obtained, and naive T cells were isolated as described previously (26). T cells were added at 105/well in 96-well, flat-bottomed plates (Costar, Cambridge, MA). Stimulator cells consisted of either uninfected dendritic cells, mock-infected dendritic cells, or dendritic cells infected with fowlpox vectors (FP-WT, rF-B7-1, or rF-TRICOM) or vaccinia vectors (V-WT, rV-B7-1, or rV-TRICOM) for 5 hours. After 18 hours, the dendritic cells were irradiated to achieve 20 Gy and added at 104/well. The cells in all wells were cultured in a total volume of 200 µL of complete medium in the presence of several concentrations (2.50.3125 µg/mL) of concanavalin A (Con A) (Sigma Chemical Co.) for 2 days. The cells were labeled for the final 1218 hours of the incubation with 1 µCi/well [3H]thymidine (New England Nuclear, Wilmington, DE) and harvested with a Tomtec cell harvester (Wallac Inc., Gaithersburg, MD). The incorporated radioactivity was measured by liquid scintillation counting (Wallac 1205 Betaplate; Wallac, Inc.). The results of the triplicate wells were averaged and are reported as mean counts per minute (CPM) with 95% confidence intervals (CIs).
Mixed-lymphocyte reaction: (H-2d versus H-2b). The mixed lymphocyte reaction was used to assess the stimulatory function of dendritic cells for allogeneic (H-2d) and syngeneic (H-2b) naive T cells. T cells were isolated from BALB/c (H-2d) or C57BL/6 (H-2b) mice as described previously (6). Stimulator cells consisted of dendritic cells (H-2b) that were either uninfected, mock infected, or infected with FP-WT, rF-B7-1, rF-TRICOM, V-WT, rV-B7-1, or rV-TRICOM for 5 hours. Complete medium was then added, and the cells were incubated at 37 °C for 18 hours. The cells were then irradiated to achieve 20 Gy and washed. T cells (5 x 104/well) were cocultured with graded numbers (5 x 103 to 6.25 x 102) of stimulator cells in complete medium in flat-bottomed, 96-well culture plates, then incubated at 37 °C in 5% CO2 for 4 days, labeled for the final 1218 hours of the incubation with 1 µCi/well [3H]thymidine, harvested, and analyzed as described in the previous paragraph.
In vitro costimulation analysis: activation of memory/effector T cells.
Rested OVA-specific T cells (responders) were added at 5 x 104/well to 96-well, flat-bottomed plates. Stimulator cells consisted of dendritic cells that were uninfected or infected with either FP-WT, rF-B7-1, rF-TRICOM, V-WT, rV-B7-1, or rV-TRICOM for 5 hours. After 18 hours, the stimulator cells were irradiated to achieve 20 Gy. The cells in all wells were cultured in a total volume of 200 µL of complete medium. The costimulation assay was carried out with the use of the following sets of conditions to quantitate the strength of the TRICOM vectors to enhance T-cell activation: (a) a 10 : 1 fixed ratio of responder-to-stimulator cells that were cultured in the presence of several concentrations of OVA peptide or control peptide VSVN or (b) a fixed concentration of OVA peptide or VSVN peptide cultured at responder-to-stimulator cell ratios of 10 : 1 to 320 : 1. The cells were cultured for 72 hours, labeled for the final 1218 hours of incubation with 1 µCi/well [3H]thymidine, harvested, and analyzed as before. Stimulated T cells were also analyzed for the expression of cytokine genes and apoptosis-related genes by reverse transcription (RT)multiplex polymerase chain reaction (MPCR). Defined cytokine primer sets for murine interferon gamma (IFN ), IL-2, IL-4, and interleukin 10 (IL-10) as well as defined apoptosis-related gene primer sets for murine lymphocyte interleukin 1-converting enzyme (LICE) (a prototype of the caspase family), bcl-2, Bax, bcl-xS, and bcl-xL were purchased commercially (Cytoexpress Kit; Biosource International, Camarillo, CA). Assays were performed according to the manufacturer's instructions. Polymerase chain reaction products contained in bands on agarose gels were quantified with the use of a Kodak DC 120 Digital Camera and Kodak Digital Science Software (Eastman Kodak, Rochester, NY). The quantity of DNA for a given band was calculated by a comparison with a known quantity and expressed as a percent of the housekeeping gene transcript glyceraldehyde phosphate dehydrogenase. In other experiments, naive CD4+ and CD8+ T-cell populations were prepared with the use of magnetic beads coated with MAbs specific for CD4 and CD8 (MiniMACS) and stimulated with the dendritic cell populations described above in the presence of 2.5 g/mL Con A for 48 hours. Supernatants were collected and analyzed for murine IL-2, IFN
, IL-10, and IL-4 by capture enzyme-linked immunosorbent assay (ELISA) as described previously (27). Apoptosis of T cells was assessed with the use of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick-end labeling (TUNEL) assay, as described previously (28) (Fluorescein FragEL DNA Fragmentation Detection Kit; Oncogene Research Products, Cambridge, MA).
CTL induction in vivo and cytotoxicity analysis. Uninfected dendritic cells (1 x 106) or dendritic cells that were infected with either FP-WT, rF-B7-1, rF-TRICOM, V-WT, rV-B7-1, or rV-TRICOM were washed twice in Opti-Mem and resuspended in 1 mL of the same medium containing 10 µM of OVA peptide. After 2 hours of incubation at 37 °C, the cells were washed twice in Hanks' balanced salt solution and resuspended in this solution for injections. Peptide-pulsed dendritic cells (1 x 105 cells/mouse), with and without all vectors, were injected once intravenously. Other mice were vaccinated subcutaneously with 100 µg OVA peptide in Ribi/Detox adjuvant (Ribi ImmunoChem Research, Hamilton, MT) to a final volume of 100 µL to compare the potency of this vaccination strategy with that of dendritic cell vaccination strategies. Fourteen days after the vaccination, spleens from three animals per group were removed, dispersed into single-cell suspensions, pooled, and coincubated with 10 µg/mL of OVA peptide for 6 days. The majority of lymphocytes were recovered by centrifugation at 700g for 20 minutes through a density gradient (LSM; Organon Teknika, West Chester, PA). EL-4 cells were prepared for use as targets in a standard cytolytic assay using 111In, as described previously (29). Target cells were then pulsed with 10 µM OVA peptide for 1 hour at 37 °C, while a second group of target cells was pulsed with VSVN peptide. Lymphocytes and peptide-pulsed targets (5 x 103 cells/well) were suspended in complete medium, combined at effector-to-target cell ratios ranging from 80 : 1 to 10 : 1 in 96-well, U-bottomed plates (Costar), and incubated for 5 hours at 37 °C with 5% CO2. After incubation, supernatants were collected with the use of a Supernatant Collection System (Skatron, Sterling, VA), and radioactivity was quantitated with the use of a gamma counter (Cobra Autogamma; Packard, Downers Grove, IL). The percentage of specific release of 111In was determined by the standard equation: % specific lysis = [(experimental spontaneous)/(maximum spontaneous)] x 100. Where indicated, CTL activity was converted to lytic units (LU) with the use of the following formula: LU18% = [(1 x 106)/(5000 x number of effector cells to reach 18% lysis)], as described by Wunderlich and Shearer (30).
Statistical analysis. The results from triplicate wells of all in vitro T-cell proliferation assays were averaged and are reported as mean CPM with 95% CIs. Statistical comparisons between groups were carried out by use of analysis of variance (Statview 4.1; Abacus Concepts Inc., Berkeley, CA). All tests were two-sided. P values were calculated at 95%. The error bars representing 95% CIs are depicted for all values in all graphs. In some cases, the variation was such that the error bars were obscured by the plot symbol.
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RESULTS |
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To determine the efficiency of poxvirus infection of dendritic cells, we infected the cells with either an rV virus encoding B7-1, ICAM-1, and LFA-3 (designated rV-TRICOM) or an rF virus encoding B7-1, ICAM-1, LFA-3, and human carcinoembryonic antigen (CEA) (designated rF-CEA/TRICOM). In the latter case, CEA was used as a reporter gene, since the majority of fowlpox structural proteins are under the control of late fowlpox promoters and, thus, are not expressed in infected cells. After 18 hours, the cells were analyzed for their expression of cell-surface markers associated with the particular viral infection (Fig. 1, AD). Uninfected control dendritic cells expressed CD11b (97%98%) and were equivalent to background levels (1%) for the expression of CEA (Fig 1
, A) or vaccinia proteins (Fig. 1
, C). After infection with rF-CEA/TRICOM, 87% of the dendritic cells coexpressed both CD11b and CEA (Fig. 1
, B). These dendritic cells failed to express fowlpox proteins as detected by polyclonal rabbit anti-fowlpox sera (data not shown), which is in agreement with a report (31) stating that fowlpox does not replicate in mammalian cells. Ninety-four percent of the dendritic cells infected with rV-TRICOM coexpressed both CD11b and vaccinia proteins (Fig. 1
, D). These data demonstrate that dendritic cells are efficiently infected by both rF- and rV-TRICOM vectors.
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An in vitro model was used to analyze how increased levels of B7-1, ICAM-1, and LFA-3 expression affect the induction of naive T-cell stimulation. In this model, the first signal for T-cell activation was delivered via a pharmacologic reagent (Con A), and the additional, or costimulatory, signal(s) were delivered to the T cell via dendritic cells or dendritic cells expressing higher levels of the three costimulatory molecules as a consequence of recombinant TRICOM vector infection. In these and all subsequent studies reported here, FP-WT and V-WT were also used to rule out effects due to the vector alone. As shown in Fig. 1 (panels E and F), both uninfected and mock-infected dendritic cells induced proliferation of T cells, which was dependent on the concentration of signal 1 (Con A). Dendritic cells infected with FP-WT (designated DC/FP-WT) induced T-cell proliferation similar to that of uninfected dendritic cells. Higher levels of B7-1 expression in dendritic cells infected with rF-B7-1 (designated DC/rF-B7-1) significantly increased proliferation of T cells compared with uninfected dendritic cells (P = .002 at highest dose of Con A). Moreover, dendritic cells infected with rF-TRICOM (designated DC/rF-TRICOM) induced even further increases in T-cell proliferation at all concentrations of Con A (P<.001). In addition, when T cells were stimulated with DC/rF-TRICOM, 17-fold less signal 1 (Con A) was needed to induce proliferation to levels comparable to those of uninfected dendritic cells. These experiments were then repeated with the use of wild-type and recombinant vaccinia vectors. Dendritic cells infected with rV-TRICOM (designated DC/rV-TRICOM) induced increases in T-cell proliferation at all Con A concentrations compared with uninfected control dendritic cells or dendritic cells infected with rV-B7-1 or V-WT. As with rF-TRICOM, when T cells were stimulated with DC/rV-TRICOM, 23-fold less Con A was needed to induce proliferation to levels comparable to those of uninfected dendritic cells (Fig. 1
, F). It should be noted that dendritic cells infected with V-WT actually showed a decrease in their ability to act as APCs compared with uninfected dendritic cells, which is most probably due to the slight decrease in the expression levels of MHC class I and class II as a result of V-WT infection (Table 1
). However, as shown in Table 1
and Fig. 1
, F, the increases in expression levels of B7-1, ICAM-1, and LFA-3 as a result of rV-TRICOM infection more than compensated for this vaccinia effect and made these dendritic cells more potent APCs, compared with control uninfected dendritic cells, at all concentrations of signal 1. These experiments were repeated five times with similar results.
Enhanced Allostimulatory Activity by Dendritic Cells Infected With TRICOM Vectors
The effects of both rF-TRICOM (Fig. 2, A, C, and E) or rV-TRICOM (Fig. 2
, B, D, and F) infection on the stimulatory capacity of dendritic cells was assessed by allospecific mixed lymphocyte reaction. Both uninfected dendritic cells and mock-infected dendritic cell populations (H-2b, C57BL/6) induced a strong proliferation (78 000 CPM) of naive allogeneic (H-2d, BALB/c) T cells (Fig. 2
, A and B). The stimulatory capacity of dendritic cells was increased slightly after infection with rF-B7-1 (Fig. 2
, C). Infection of dendritic cells with rF-TRICOM statistically significantly increased the stimulatory capacity over dendritic cells and DC/rF-B7-1 at all dendritic cell-to-responder T-cell ratios (P<.001; Fig. 2
, C). Importantly, dendritic cell populations infected with either FP-WT or rF-TRICOM vectors failed to stimulate syngeneic T cells (Fig. 2
, E). When these experiments were repeated with the use of vaccinia vectors (Fig. 2
, B and D), DC/rV-TRICOM induced marked increases in allogeneic T-cell proliferation when compared with uninfected dendritic cells and dendritic cells infected with rV-B7-1 (designated DC/rV-B7-1) at all dendritic cell-to-responder T-cell ratios (P<.001). DC/rV-TRICOM, DC/rV-B7-1, or dendritic cells infected with V-WT (designated DC/V-WT), however, failed to stimulate syngeneic (H-2b, C57BL/6) T cells (Fig. 2
, F). These experiments were repeated four times with similar results.
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Studies were undertaken to determine if the T-cell stimulatory capacity of peptide-pulsed dendritic cells could be enhanced by infecting these cells with rF-B7-1 and rF-TRICOM. To that end, the H-2Kb-restricted OVA peptide and an OVA-specific CD8+ effector T-cell line were used. Dendritic cells were exposed to different concentrations of OVA peptide in vitro and were incubated in the presence of the OVA T-cell line (Fig. 3). The conventional (i.e., uninfected) dendritic cells induced a strong proliferation of OVA-specific T cells when incubated with the OVA peptide (Fig. 3
, A; open squares). These dendritic cells did not induce proliferation of OVA-specific T cells when incubated with the control peptide VSVN (Fig. 3
, A; open diamonds). DC/rF-B7-1 increased the overall peptide-specific proliferation of these cells twofold at 1 µM concentration of peptide. In addition, DC/rF-B7-1 induced proliferation similar to that of uninfected or mock-infected dendritic cells in the presence of fourfold less peptide (0.25 µM). DC/rF-TRICOM, however, markedly and significantly (P<.001) increased the overall proliferation of these T cells and, in the presence of 13-fold less OVA peptide (0.077 µM; Fig. 3
, A [closed circles]), induced proliferation comparable to that of uninfected dendritic cells (Fig. 3
, A). For further evaluation of the capacity of fowlpox-infected dendritic cells to present peptide, dendritic cells were pulsed with a single concentration of OVA peptide (1 µM) and incubated in the presence of T cells at several different ratios (Fig. 3
, B). On a per-cell basis, twofold less DC/rF-B7-1 was required to induce proliferation levels comparable to those of dendritic cells (Fig. 3
, B; closed triangles versus open squares). The greatest stimulatory effect was that of DC/rF-TRICOM, which induced T-cell proliferation levels comparable to those of uninfected dendritic cells with eightfold fewer cells (i.e., ratios of T cells to dendritic cells of 10 : 1 for uninfected dendritic cells versus 80 : 1 for DC/rF-TRICOM) (Fig. 3
, B; closed circles versus open squares). Experiments were also carried out with the use of dendritic cells infected with vaccinia vectors (Fig. 3
, C and D). As before, these dendritic cells did not induce proliferation of OVA-specific T cells when incubated with the control peptide VSVN (Fig. 3
, C; open diamonds). DC/rV-B7-1 increased the overall peptide-specific proliferation of these cells twofold (Fig. 3
, C) and induced proliferation similar to that of uninfected or mock-infected dendritic cells in the presence of 2.5-fold less peptide (1 µM versus 0.4 µM). However, DC/rV-TRICOM statistically significantly (P<.001) increased the overall proliferation of these T cells, as compared with uninfected dendritic cells, and induced proliferation comparable to that of uninfected dendritic cells in the presence of 33-fold less OVA peptide (0.03 µM versus 1 µM) (Fig. 3
, C). When these dendritic cell populations were pulsed with a single concentration of OVA peptide and incubated in the presence of several ratios of T cells (Fig. 3
, D), fourfold fewer DC/rF-B7-1 cells were required to induce proliferation levels comparable to those of uninfected dendritic cells (closed triangles versus open squares). Among these vectors, the greatest stimulatory effect was that of DC/rV-TRICOM, which induced proliferation levels comparable to those of dendritic cells with 32-fold fewer cells (10 : 1 versus 320 : 1) (closed circles versus open squares). These experiments were repeated five additional times with the same results.
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It has been reported that B7-1 costimulation prolongs T-cell IL-2 messenger RNA (mRNA) half-life and increases levels of IL-2 transcription, resulting in production of considerable amounts of secreted IL-2 (32,33). In addition, T-cell costimulation with LFA-3 has been reported to have an effect on a variety of cytokines, notably IL-2 and IFN (2). Cytokine expression in OVA-specific CD8+ effector T cells stimulated for 48 hours with OVA peptide-pulsed dendritic cells or OVA peptide-pulsed dendritic cells infected with rF-B7-1 or rF-TRICOM was analyzed at the RNA level for expression of IL-2, IFN
, IL-10, and IL-4 message with the use of RTMPCR. Incubation of OVA-specific CD8+ T cells with OVA peptide-pulsed dendritic cells or peptide-pulsed dendritic cells infected with FP-WT induced IL-2 message (Fig. 4
, A), whereas pulsed DC/rF-B7-1 induced 32% more IL-2 message than did dendritic cells. However, T cells stimulated with peptide-pulsed DC/rF-TRICOM induced 60% more IL-2 message than conventional pulsed dendritic cells. Incubation of OVA-specific CD8+ T cells with either dendritic cells alone or control peptide VSVN-pulsed dendritic cells induced no detectable IL-2 message (data not shown).
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To determine if stimulation of OVA-specific T cells with peptide-pulsed dendritic cells infected with rF-B7-1 or rF-TRICOM would lead to increased cell survival or programmed cell death, the mRNA levels for selected apoptosis-related proteins were analyzed after 48 hours. The proteins bcl-2 and bcl-xL, which have been reported to suppress apoptosis (34), and Bax and bcl-xS, which reportedly increase cell susceptibility to apoptosis (34,35), were examined. As depicted in Fig. 4, C, T cells activated by peptide-pulsed dendritic cells expressed moderate quantities of bcl-2 message, whereas T cells stimulated with peptide-pulsed dendritic cells infected with rF-B7-1 or rF-TRICOM expressed 31% and 75% higher levels of bcl-2 message, respectively, than those stimulated with peptide-pulsed control dendritic cells. This observation is in agreement with that of Boussiotis et al. (36), who observed that bcl-2 was induced following CD28 costimulation. The expression level of bcl-2 in T cells was decreased by 65% following stimulation with DC/FP-WT, which was most likely a result of the interaction of the T cell with a dendritic cell expressing decreased costimulatory molecules due to wild-type virus infection more than 60 hours before message analysis. T cells stimulated with peptide-pulsed dendritic cells infected with any construct failed to modulate expression levels of Bax or of the caspase family LICE. Although it has been reported that CD28 costimulation augmented the expression of bcl-xL in activated T cells (34), stimulation of these OVA-specific CD8+ T cells with peptide-pulsed dendritic cells infected with any construct failed to induce detectable levels of bcl-xL or bcl-xS. These findings were confirmed with the use of TUNEL analysis of naive CD4+ and CD8+ T cells stimulated with the dendritic populations noted above for 48 hours in the presence of Con A as signal 1.
Effect of rV-TRICOM Infection on Dendritic Cells Matured With TNF-, LPS, or CD40
The functional maturation of dendritic cells has been shown previously to be associated with increased levels of certain T-cell costimulatory molecules (21,37,38). Bone marrow-derived dendritic cells generated in the presence of GM-CSF and IL-4 exhibit an intermediate maturation stage with respect to phenotype and in vitro antigen-presenting capacity (21). Experiments were conducted to examine the effect of rF-TRICOM or rV-TRICOM infection on dendritic cells that had been further "matured" by coculture with either TNF-, LPS, or CD40-specific MAb. Treatment of dendritic cells with TNF-
or CD40-specific MAb during the final 24 hours of culture resulted in some increased levels of MHC class II, B7-2, and ICAM-1, as determined by flow cytometry analysis (Table 2
); the increased level of these markers is in agreement with previous observations (21,37). Treatment with LPS resulted in increased levels of MHC class II, B7-2, ICAM-1, and CD40; this too is in agreement with previously reported observations (21,37,38). None of the other phenotypic markers were altered by TNF-
, LPS, or CD40-specific MAb (Table 2
). Functionally, treatment of dendritic cells with TNF-
, LPS, or CD40-specific MAb culminated in an 18%20% increase in peptide-specific proliferation over that of unmanipulated dendritic cells (Fig. 5
, A and C) at the highest level of peptide. These maturation-mediated increases in stimulatory capacity are also similar to those reported by other investigators (21,39). Infection of dendritic cells of intermediate maturity (grown in the presence of GM-CSF and IL-4) with rF-TRICOM (Fig. 5
, B) or rV-TRICOM (Fig. 5
, D) resulted in their ability to induce a substantial increase in T-cell proliferation. The use of more mature dendritic cells (matured with the use of TNF-
, LPS, or CD40-specific MAb) conferred only a slight increase in stimulatory capacity (Fig. 5
, A and C). These more mature dendritic cells, however, were markedly enhanced in their T-cell stimulatory capacity when they were infected with rF-TRICOM (Fig. 5
, B) or rV-TRICOM (Fig. 5
, D). These experiments were repeated four additional times with similar results.
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Experiments were conducted to determine if the enhanced T-cell stimulatory capacity of dendritic cells infected with TRICOM vectors noted in vitro with the use of Con A (Fig. 1, E and F), mixed lymphocyte reactions (Fig. 2
), and peptide-specific effector T cells (Fig. 3
) would translate to enhanced efficacy in priming naive T-cell responses in vivo. To that end, dendritic cells, DC/FP-WT, DC/rF-B7-1, and rF-TRICOM were pulsed with 10 µM OVA peptide and administered intravenously to C57BL/6 mice. The mice were also vaccinated with OVA peptide in Ribi/Detox adjuvant subcutaneously. Splenocytes were harvested 14 days after vaccination, restimulated in vitro for 6 days with irradiated splenocytes as APCs and 10 µg/mL OVA peptide, and assessed for their peptide-specific lytic ability against OVA-pulsed EL-4 cells. EL-4 cells pulsed with VSVN peptide were used as control target cells.
As seen in Fig. 6, A, CTLs generated from mice vaccinated with peptide/adjuvant exhibited modest levels of CTL activity. Mice vaccinated with peptide-pulsed, uninfected dendritic cells exhibited a greater peptide-specific CTL response (Fig. 6
, B) than mice vaccinated with peptide in adjuvant; infection of peptide-pulsed dendritic cells with FP-WT vector yielded results similar to those seen with peptide-pulsed, uninfected dendritic cells (Fig. 6
, C versus B). Mice vaccinated with peptide-pulsed DC/rF-B7-1 (Fig. 6
, D) exhibited a CTL response that was statistically significantly stronger than that of mice vaccinated with peptide-pulsed dendritic cells (Fig. 6
, B) (LU = 16.1 and 3.2, respectively; P = .001). Mice vaccinated with peptide-pulsed dendritic cells that had been infected with rF-TRICOM exhibited T cells with even more potent lytic capacity (>20 LU) than mice vaccinated with peptide-pulsed dendritic cells (P = .001) or peptide-pulsed dendritic cells infected with rF-B7-1 (P = .006). Again, there was no in vivo effect of the vector alone. Similar experiments were then conducted with the use of the recombinant vaccinia vectors (Fig. 6
, FJ). The induced CTL response was somewhat blunted in mice vaccinated with peptide-pulsed DC/V-WT (Fig. 6
, H) versus those vaccinated with peptide-pulsed dendritic cells (Fig. 6
, G). In contrast, mice vaccinated with peptide-pulsed DC/rV-TRICOM (Fig. 6
, J) exhibited a CTL response that was significantly stronger than that of mice vaccinated with uninfected or vector control-infected, peptide-pulsed dendritic cells (LU = 14.3; P = .001).
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DISCUSSION |
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Recently, the role of multiple combinations of costimulatory molecules in the activation of T cells was examined (6). Poxvirus (fowlpox and vaccinia) vectors were used because of their ability to efficiently express multiple genes. Murine tumor cells provided with signal 1 and infected with either rF- or rV-TRICOM vectors induced the activation of both CD4+ and CD8+ T-cell populations to far greater levels, compared with the use of either one or two costimulatory molecules (6). Results employing the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell-to-T-cell ratios. In vivo vaccination experiments using a recombinant human CEA/TRICOM vector in CEA transgenic mice also indicated that TRICOM recombinants could enhance antigen-specific T-cell responses. Because functional maturation of dendritic cells is believed to be associated with increased surface expression of T-cell costimulatory molecules (43,44), we focused our efforts on infecting dendritic cells with viral vectors (i.e., rF-TRICOM or rV-TRICOM) that enable the cells to hyperexpress this triad of costimulatory molecules and on analyzing the consequences of this infection in vitro and in vivo.
The poxviral vectors examined in these studies were shown to infect dendritic cells with high efficiency (Fig. 1). Phenotypically, substantial differences were noted in the levels of B7-1, ICAM-1, and LFA-3 expression between dendritic cells, DC/rF-B7-1 or DC/rV-B7-1, and DC/rF-TRICOM or DC/rV-TRICOM (Table 1
). The expression levels of certain surface markers (CD11b and B7-2) decreased after infection with any of the vaccinia constructs and could be attributed to virus-mediated inhibition of the host-cell protein-synthesis machinery. The decrease in these markers was not noted after infection with fowlpox vectors. Infection of dendritic cells with rF-B7-1 induced a substantial increase in the surface expression of B7-1, whereas infection with rF-TRICOM substantially increased the expression of B7-1, ICAM-1, and LFA-3. Similar results were noted after infection of dendritic cells with vaccinia vectors. When these dendritic cell populations were assessed functionally by measurement of either stimulation of naive T cells in the presence of Con A as signal 1 (Fig. 1
, E and F), stimulation in a primary allogeneic culture (mixed lymphocyte reaction) (Fig. 2
), or stimulation of a peptide-specific effector T-cell line (Fig. 3
), stimulation with DC/rF-B7-1 or DC/rV-B7-1 was superior to that of dendritic cells alone or dendritic cells infected with control vectors, whereas DC/rF-TRICOM and DC/rV-TRICOM clearly induced proliferation superior to that of DC/rF-B7-1, DC/rV-B7-1, or conventional uninfected dendritic cells. On a per-cell basis, DC/rV-TRICOM induced levels of proliferation comparable to those of dendritic cells with 12.5- to 32-fold fewer cells (Fig. 3
, C and D). Taken together, these data suggest that a much lower dose of DC/rF-TRICOM or DC/rV-TRICOM is required to stimulate T cells, which is an important consideration for clinical applications.
Infection with TRICOM constructs led to increased levels of IL-2 and IFN production from CD8+ effector T cells (Fig 4
, A and B), while no detectable levels of type 2 cytokines (IL-10 and IL-4) were produced (data not shown). One question raised concerns about the potential of overstimulated T cells, thus resulting in apoptosis. The expression of bcl-2 has been shown to block apoptosis in many experimental systems (45). The results shown in Fig. 4
, C, demonstrate expression of bcl-2 in T cells stimulated with dendritic cells. However, the expression level of bcl-2 in T cells was increased 31% and 76% following stimulation with DC/rF-B7-1 or DC/rF-TRICOM, respectively. These results are in agreement with those of previous studies, which found that costimulation through the CD28 receptor appears to play an important role in enhancing the resistance of activated T cells to undergo apoptosis in culture (34). This could be attributed to augmentation of cytokine production by these cells (Fig. 4
, A and B) and to potential increased levels of survival genes. There were no substantial changes in any T-cell group in the expression levels of Bax, which has been reported to increase cell susceptibility to apoptosis (35), or LICE, a member of the caspase gene family responsible for the cascade of catalytic steps toward apoptosis (46).
The maturation stage of murine dendritic cells is usually defined by the phenotypic profile and, at times, by functional characteristics; however, there is considerable variability in these definitions in the literature (21,37,47). We have described the dendritic cells used in these studies (cultured with GM-CSF and IL-4) as dendritic cells of intermediate maturity; however, based on the phenotypic definitions by some investigators, these dendritic cells would be termed "mature." The addition of TNF-, LPS, or CD40-specific MAb to the dendritic cells used in these studies for "maturation" resulted in slight increases in MHC class II, B7-2, and ICAM-1 molecules on the cell surface (Table 2
) and in a modest increase in the stimulatory capacity of dendritic cells (Fig. 5
). These findings confirm those of Morse et al. (39), who reported that CD40 ligand treatment of dendritic cells resulted in a 17% improvement in alloreactive proliferation over that of untreated dendritic cells. In addition, using a T-cell line specific for OVA323339, Labeur et al. (21) showed that CD40 or LPS treatment of dendritic cells resulted in an 8.5% enhancement in T-cell proliferation, whereas TNF-
treatment of dendritic cells resulted in no enhancement. Treatment of dendritic cells with TRICOM vectors, however, resulted in a dendritic cell with far greater stimulatory capacity than dendritic cells treated with either LPS, CD40-specific MAb, or TNF-
(Fig. 5
). Dendritic cells pretreated with TNF-
, LPS, or CD40 displayed a substantial increase in stimulatory capacity when they were infected with rV-TRICOM (Fig. 5
, B and D). Thus, the enhanced stimulatory capacity induced by infection of dendritic cells with TRICOM vectors was noted with both mature dendritic cells and dendritic cells of intermediate maturity.
Since the in vitro findings demonstrated that infection with TRICOM vectors endowed dendritic cells with an enhanced ability to stimulate T cells, these reagents were also examined in vivo. The model peptide OVA was chosen to demonstrate the broad application of dendritic cells infected with rF-TRICOM or rV-TRICOM. As shown in Fig. 6, OVA-pulsed dendritic cells were able to induce OVA-specific CTL activity (3.25.2 LU) following a single vaccination of 1 x 105 cells. This level of CTL induction was similar to that seen by Porgador et al. (48) when using this system. Vaccination with the same dose of OVA-pulsed DC/rF-TRICOM induced markedly and statistically greater levels of CTL activity (>20 LU) than did vaccination with OVA peptide-pulsed dendritic cells (Fig. 6
). Similar results were demonstrated when peptide-pulsed dendritic cell populations were infected with rV-TRICOM (Fig. 6
).
Recently, it was reported that, under certain circumstances, vaccination with dendritic cells could result in the induction of autoimmune responses. Ludewig et al. (49) noted that dendritic cells pulsed with a model tumor antigen also expressed on pancreatic ß islet cells resulted not only in the mice rejecting model antigen transduced tumors, but also in the mice developing autoimmune diabetes. Roskrow et al. (50) reported that dendritic cells pulsed with acid-eluted peptides from A20 tumor cells protected mice challenged with A20 tumors; however, the mice went on to develop a fatal autoimmune disorder. It should be noted, however, that several preclinical (812,51) as well as clinical (10,5256) studies have now demonstrated the efficacy of dendritic cell therapy in inducing specific T-cell responses and antitumor responses without any evidence of autoimmunity. It is, thus, likely that the tissue distribution, immunogenicity of the antigen used, and the dose and scheduling of the dendritic cell therapy may be factors that need to be considered.
Several groups have been exploring the introduction of exogenous model genes into dendritic cells by retrovirus (7,5759), adenovirus (6062), or poxvirus (17,60,62) vectors containing a single transgene. The advantage of using recombinant poxviruses to introduce genes into dendritic cells is the efficient level of infection (Fig. 1, Tables 1 and 2
) and the ability of the poxvirus to accommodate large amounts of foreign DNA and to express multiple transgenes. To date, as many as seven genes have been inserted into the vaccinia virus genome (63).
The avipox viruses represent potentially attractive vectors for use in dendritic cell vaccines because avipox viruses, such as fowlpox and ALVAC (canarypox), can be administered numerous times to enhance immunogenicity (64). Since avipox viruses are replication defective, the consequences of any host immune responses should be minimal. Avipox viruses are also distinct from vaccinia virus in that the inserted transgene is expressed in infected cells for 1421 days before death. In a vaccinia-infected cell, the transgene is expressed for 12 days until cell lysis. A potential concern inherent in using a dendritic cell immunogen containing a vaccinia vector is that vaccinia immunity might inhibit the effectiveness of subsequent vaccinations. Indeed, a single dose of dendritic cell populations infected with any of the recombinant vaccinia viruses reported here induced anti-vaccinia antibody titers ranging from 1 : 4000 to 1 : 9000. However, these antibody titers had no effect on the efficacy of the subsequent dendritic cell vaccinations in the generation of CTLs (data not shown). One potential explanation for this is that vaccinia virus does not replicate in dendritic cells (65,66) and, thus, is protected from any potentially neutralizing antibodies because of its intracellular location. Again, there would be less concern about inducing neutralizing antibodies if nonreplicating poxviral vector systems, such as fowlpox, canarypox, or Modified Vaccinia Ankara (MVA), were used (67).
Dendritic cell immunogens, either peptide-loaded, apoptotic body-loaded, or transfected with tumor RNA, among other methodologies, are currently being evaluated for clinical use. Infection of these dendritic cell immunogens with rF-TRICOM or rV-TRICOM could well improve the efficacy of these reagents in priming specific immune responses. These studies, thus, have implications in vaccine strategies for a range of human cancers and infectious diseases.
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Manuscript received January 14, 2000; revised May 25, 2000; accepted May 31, 2000.
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