Article Figures & Data
Figures
- Fig. 1.
Intravenously administered reovirus can reduce lymph node metastatic melanoma burden, prime antitumor immunity, and induce proinflammatory cytokines. C57BL/6 mice were seeded with subcutaneous B16-tk tumors (5 × 105 cells). Ten days later, mice were treated intravenously with 5 × 108 pfu reovirus or with PBS. Ten days after that, tumor draining lymph node and spleen were explanted, and lymph nodes were dissociated and plated overnight in culture. A, total genomic DNA from 106 lymph node cells was screened with primers specific for the HSV-tk gene. Equal loading of DNA was confirmed using primers specific for a genomic fragment in the tyrosinase gene promoter. B, 106 cells from the dissociated lymph node cultures were seeded in medium containing 1.25 μg/mL puromycin to select for viable B16-tk cells, which were present in the lymph node at the time of resection. Within 5 to 10 days, individual puror colonies appear, which were counted. C, splenocytes recovered at day 10 were pulsed, in triplicates of 750,000, with the synthetic TRP-2180-188 SVYDFFVWL peptide (i) or with the irrelevant H-2Kb-restricted OVA SIINFEKL peptide (ii). Forty-eight hours later, supernatants were assayed by ELISA for IFN-γ. D, explanted tumor draining lymph node were dissociated and plated overnight in culture. Supernatants were assayed for IL-12. Representative of one of two independent experiments.
- Fig. 2.
Analysis of human dendritic cell phenotype, cytokine secretion, and viability after coculture with reovirus-infected Mel888 cells. Dendritic cells were incubated with lipopolysaccharide (250 ng/mL), uninfected Mel888 cells, or Mel888 cells infected with 0.1, 1, and 10 pfu reovirus/Mel888 cell at a 1:3 ratio for 24 h. A, surface expression of dendritic cell phenotypic markers, CD86, CD80, CD83, CD40, and MHC class II, was examined by flow cytometry. Median fluorescence intensity is shown in each plot. Contact dependence of phenotypic changes was examined using a 0.4 μm Transwell. Representative of at least four independent experiments. B, levels of IL-12p70, IL-6, and TNF-α in supernatant were determined by ELISA. Representative of six independent experiments. C, proportion of nonviable dendritic cells was examined by flow cytometry, gating on dendritic cells labeled with CellTracker Green, and stained with propidium iodide. Representative of two independent experiments.
- Fig. 3.
Dendritic cells phagocytose uninfected and reovirus-infected Mel888 cells equally efficiently. Dendritic cells labeled with CellTracker Green were cocultured at 4°C and 37°C at a 1:3 ratio with Mel888 cells or Mel888 cells infected with 0.1, 1, or 10 pfu reovirus and labeled with CellTracker red. A, uptake of Mel888 by dendritic cells was examined by flow cytometry after coculture for 40 min or 4 h. The percentage of dendritic cells double-labeling red and green was assessed. Representative of six independent experiments. B, uptake of Mel888 tumor material by dendritic cells after 4 h at 37°C was confirmed by fluorescence microscopy of living cultures. At 4°C, no Mel888-derived material (red) was observed within dendritic cells (green), whereas at 37°C Mel888 material was observed to be internalized within dendritic cells (arrow). Bar, 40 μm. For zoomed image: bar, 10 μm. C, Mel888-derived material internalized within dendritic cells localized to late endosomal/lysosomal structures as shown by colocalization of red-dyed material with Lysotracker blue-labeled intracellular compartments within green-labeled dendritic cells (arrow). Bar, 10 μm. Representative of data from two independent donors. Similar images were obtained following 4 h coculture of dendritic cells with uninfected or reovirus-infected Mel888 cells.
- Fig. 4.
Naive priming of human tumor-specific cytotoxic lymphocytes by dendritic cells loaded with reovirus-infected Mel888 cells. PBMC were incubated with autologous dendritic cells loaded overnight with Mel888 cells or Mel888 cells infected with 0.1 pfu reovirus per Mel888 cell (at 1:3 ratio), restimulated 7 d later, and assayed at 14 d. A, lymphocyte proliferation was determined by trypan blue exclusion. B, cytotoxicity of lymphocytes primed in the presence or absence of reovirus was determined by 51Cr release assay using Mel888 cells and a range of melanoma and nonmelanoma cell lines as targets. Two experiments representative of at least six independent donors are shown in A and B. C, specificity of the activity of lymphocytes primed by dendritic cells loaded with reovirus-infected Mel888 cells toward Mel888 cells was further tested using the CD107 lymphocyte degranulation assay toward a larger panel of melanoma and nonmelanoma cell lines. D, a pan-MHC class I blocking antibody and isotype control was used to test the MHC class I restriction of degranulation, measured by the CD107 assay, toward Mel888 cells. The percentage of CD8 T cells degranulating is shown. Representative of at least four independent experiments.
- Fig. 5.
Cytokine production in CTL cultures. A, levels of IFN-γ in CTL cultures were determined by ELISA each week. Data from one donor representative of nine.
- Fig. 6.
Cross-priming of MART-1-specific CTLs by dendritic cells loaded with reovirus-infected Mel888 cells. HLA-A2+ PBMCs were incubated with autologous dendritic cells loaded overnight with uninfected Mel888 cells (a HLA-A2− cell line) or reovirus-infected Mel888 cells as in Fig. 5. A total of three stimulations were done at 7-d intervals, with analysis on day 21. Initial PBMC and the lymphocyte populations generated after 2 and 3 wk in culture were examined by flow cytometry after labeling with a MART-1-specific pentamer and a negative control (which labeled no detectable cells; data not shown). Percentage of CD8+ T cells labeled with the MART-1 pentamer. Representative of five independent experiments.
Volume 14, Issue 22
