Article Figures & Data
Figures
- FIGURE 1.
Morphology of bone marrow-derived DCs (×200 magnification).
- FIGURE 2.
In vitro assessment of the functional activity of bone marrow-derived DCs. A, In a mixed lymphocyte reaction, increasing numbers of DCs derived from C57BL/6 bone marrow (1 × 102-1 × 104 DCs) were used to stimulate 2 × 105 allogeneic BALB/c T lymphocytes. Untransduced DCs, as well as DCs transduced with Ad vector encoding green fluorescent protein (Ad2/EGFP), were tested. The levels of proliferation induced were measured by tritiated thymidine incorporation after 5 days of culture. Results shown represent the mean cpm of triplicate wells ± SEM. The background proliferation of untransduced DCs and Ad2/EGFP-transduced DCs incubated alone was highest with 1 × 104 DCs, with cpm values of 2867 ± 681 and 3392 ± 367 cpm, respectively. Results from one representative experiment are shown. A decrease in the levels of proliferation induced by the highest concentration of untransduced DCs (1 × 104) was observed in two of three experiments, and the reason for this phenomenon is unclear. B, To assess primary Ag-specific proliferation, naive C57BL/6 T lymphocytes (2 × 105/well) were incubated with syngeneic untransduced DCs or with DCs transduced with wt Ad2 or Ad2 vectors expressing various transgenes (104 DCs/well). Proliferation levels were assessed after 5 days of culture. Background proliferation of transduced DCs incubated alone was as follows: wt Ad2, 5004 ± 42; Ad2/EGFP, 1198 ± 293; Ad2/β-gal-4, 3059 ± 1137; Ad2/mTRP-2, 920 ± 208; and untransduced DCs, 2867 ± 681 cpm. The induction of Ag-specific proliferation by transduced DCs was observed in three of three separate experiments.
- FIGURE 3.
Induction of CTL activity following immunization with Ad2/hugp100v1 vector or Ad2/hugp100v1-transduced DCs. Spleens from groups of three animals were collected 15 days after i.v. administration of vehicle (A), Ad2/hugp100v1-transduced DCs (B), or i.d. delivery of Ad2/hugp100v1 vector (C). Pooled spleen cells from each group were restimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugp100v1 and were tested for cytolytic activity after 6 days of culture. Targets consisted of B16 cells and SVB6KHA fibroblasts untransduced or transduced with Ad2/hugp100v1 or wt Ad2 deleted for E3 (SVB6KHA-Ad2Δ2.9). SD for mean percent lysis values was below 15%. Similar results were obtained in three separate studies.
- FIGURE 4.
Specificity and cross-reactivity of effector cells induced by Ad2/hugp100v2-transduced DCs. Spleen cells from mice immunized with DCs transduced with Ad2/hugp100v2 or Ad2/EV were tested in an ELISPOT assay. The number of IFN-γ-producing cells was counted after 48 h of stimulation with an MHC class I-restricted CTL peptide epitope from hugp100 (open bars), the homologous epitope from mgp100 (filled bars), or an irrelevant H-2b-binding CTL epitope from OVA (slashed bars). Results shown are the mean number of IFN-γ-producing spleen cells ± SEM of triplicate wells after subtracting the background values obtained with spleen cells incubated alone. ∗, p < 0.005, and ∗∗, p < 0.025, compared with OVA-stimulated spleen cells by Student’s t test.
- FIGURE 5.
Induction of CTL activity by transduced DCs delivered via the s.c. or i.v. route. Spleens from groups of three mice were collected 16 days after the s.c. (A and B) or i.v. (C and D) delivery of Ad2/hugp100v1-transduced DCs. Pooled spleen cells from each group were restimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugp100v1 and were tested for cytolytic activity after 6 days of culture. Target cells consisted of 51Cr-labeled YAC cells (NK cell target), C57BL/6-derived EL4 lymphoma cells, and B16 melanoma cells incubated with effector CTLs in the presence (B and D) or absence (A and C) of excess unlabeled “cold” YAC cells. SD for mean percent lysis values was below 15%.
- FIGURE 6.
Induction of long-term antitumor protection by Ad2/hugp100v1-transduced DCs. Groups of five C57BL/6 mice were injected i.v. with vehicle (A), 5 × 105 untransduced DCs (B), or 5 × 105 Ad2/hugp100v1-transduced DCs (C). The animals were challenged 15 days later with a s.c. injection of 2 × 104 B16 melanoma cells. Results shown depict tumor growth in individual animals over time. All animals that were still tumor-free 50 days after B16 challenge received a second injection of B16 cells to test for immunological memory. Results are representative of six separate studies using five to eight mice per group.
- FIGURE 7.
Factors involved in the effectiveness of immunization with Ad vector-transduced DCs. A, Nature of the Ag: groups of five C57BL/6 mice were injected i.v. with 5 × 105 DCs that were either untransduced or transduced with Ad2/hugp100v1, Ad2/mgp100, or Ad2/mTRP-2 vector. The animals were challenged 15 days later with a s.c. injection of 2 × 104 B16 melanoma cells. B, Involvement of CD4+ T cells: groups of eight wt or 10 CD4 KO C57BL/6 mice were immunized s.c. with 5 × 105 DCs transduced with Ad2/mTRP-2, or Ad2/EV as a negative control. The animals were challenged 14 days later with a s.c. injection of 2 × 104 B16 melanoma cells. C, Dose dependence: groups of eight C57BL/6 mice were immunized s.c. with increasing doses (5 × 103-5 × 106) of Ad2/mTRP-2-transduced DCs. One group received vehicle as a negative control. Animals were challenged with 2 × 104 B16 cells s.c. 15 and 64 days later. All results are shown as the percentage of tumor-free mice in each group over time.
- FIGURE 8.
Immunization with Ad vector-transduced DCs in animals with preexisting immunity against Ad. Groups of 12 naive or 12 Ad-immune mice were immunized s.c. with 5 × 105 Ad2/mTRP-2-transduced DCs. In parallel, groups of 8 naive or 8 Ad-immune mice received the same number of DCs transduced with Ad2/EV as a negative control. The ELISA titers of Ad-specific Abs present in immune serum collected the day before DC immunization ranged from 12,800 to 51,200. All mice were challenged s.c. with 2 × 104 B16 cells 15 days after administration of DCs. The kinetics of tumor growth are depicted for each individual animal.
- FIGURE 9.
A, Active treatment of established B16 tumor cells with Ad vector-transduced DCs. Twenty C57BL/6 mice were injected s.c. with 1.5 × 104 B16 cells on day 0. Four days later, the animals were divided randomly into groups of five and were treated s.c. with 5 × 105 DCs that were untransduced or transduced with Ad2/EV, Ad2/hugp100v1, or Ad2/mTRP-2 vector. Results shown represent the percentage of tumor-free mice in each group over time. Note that several animals had already developed tumors when tumor measurement was initiated on day 16. Results are representative of six separate studies using five to eight mice per group. B, Active treatment of established B16 tumor cells using transduced DCs presenting two vs one MAA. Forty-six C57BL/6 mice were injected s.c. with 2 × 104 B16 cells on day 0. Four days later, the animals were divided randomly into six groups, which were treated with a s.c. injection of 5 × 105 DCs transduced with either Ad2/EV (•), Ad2/hugp100v2 (▪) or Ad2/mTRP-2 (▴). Two groups received a mixture of DCs transduced separately with Ad2/hugp100v2 or Ad2/mTRP-2 in the amount of 2.5 × 105 (♦) or 5 × 105 (○) of each DC population. An additional group was treated with vehicle (□) as a negative control. All groups contained eight animals, except for the vehicle control group, which was limited to six mice. Results are presented as the mean tumor size over time. Similar results were obtained in two separate studies.
Tables
DC Sample B7.1 B7.2 MHC I MHC II ICAM-I CD11c CD13 Untransduced 80 77 41 70 96 81 80 Transduced 84 83 85 79 94 71 74 -
a Results shown are representative of seven separate experiments and are expressed as the percentage of bone marrow-derived DCs staining positive for each marker. DCs were untransduced or transduced with Ad2/β-gal-4.
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