Fig. 1. Fusion of autologous DC with breast or ovarian carcinoma cells. (A) Cytocentrifuge preparations of DC (left panel), OVCa cells (middle panel), and DC/OVCa fusions generated from patient with OVCa. Cells were stained with TU36 (anti-HLA-DR, blue color) and OC-125 (anti-CA-125, red color) mAb and by immunohistochemical staining (magnification ×60). (B and C) Surface structure of cells examined by scanning electron microscope (SEM, ×4800); (B) DC, BRCa cells, and DC/BRCa fusions; (C) DC, OVCa cells, and DC/OVCa fusions.

Fig. 2. Intracellular structure of a 5-day cultured DC fused to breast carcinoma (BRCa) cells examined by TEM (×4800): (A) DC, (B) BRCa cell, and (C) DC/BRCa fusion cell. The lower panel shows the integration of DC and tumor cells (arrow). DC-N, DC nucleus; Tu-N, tumor nucleus.

Fig. 3. Colocalization of MUC1 and HLA class II molecules in DC/BRCa fusion cells. (A) DC/BRCa fusion cells were cultured for 5 days, prepared and examined by TEM (×7250). Arrow points to DC and tumor cell membrane where fusion has taken place. Corner panel is enlarged view of fusion line. (B) DC/BRCa fusion cell was prepared, stained with preembedded immunogold-labeled anti-MUC1 mAb (5-nm particles) and anti-HLA-DR mAb (10-nm particles), and examined by JEOL-100-CX immunoelectron microscopy (×47,500). Arrow points the fusion line between DC and tumor cells. Lower panels are enlarged view of membrane of DC side (✪) and tumor side () showing colocalization of MUC1 and HLA-DR.

Fig. 4. Activation of CD4 and CD8 T cells by autologous DC/OVCa fusion cells. T cells from ovarian cancer patient (HLA-A*0201 positive) were cocultured with autologous DC/OVCa fusion cells (A), DC mixed with OVCa cells (B), or OVCa cells alone (C) for 15 days and their phenotype assessed. On Days 0, 8, and 15, the T cells were collected, purified by passage through nylon wool, stained with indicated antibodies, and analyzed by flow cytometry. The intracellular IFN-γ secretion was assessed using a human IFN-γ secretion assay kit and antigen-specific T cells were detected by HLA-A*0201-MUC1 tetramer assay.

Fig. 5. Induction of tumor-specific and HLA class I-restricted CTL by DC/tumor fusion cells. (A) T cells were stimulated with autologous DC (□), BRCa cells (), DC/BRCa fusion cells (○), or DC mixed with BRCa cells () for 10 days and then incubated with ⁵¹Cr-labeled autologous breast carcinoma cells at indicated E/T ratios. (B) DC/BRCa-stimulated T cells from HLA-A*0201-negative patient were incubated with autologous BRCa cells (○), autologous monocytes (MC, □), MCF-7 breast cancer cells (, HLA-A*0201 positive), or K562 cells (, sensitive for NK cells) at indicated E/T ratios (left panel). HLA class I blocking assay in which autologous BRCa (●), autologous MC (▪), MCF-7 (), or K562 (♦) were preincubated with anti-HLA class I antibody (W6/32: 1:100 dilution) for 1 h, labeled with ⁵¹Cr, and then incubated with effector cells at indicated E/T ratios (right panel). (C) T cells from two ovarian carcinoma patients were stimulated by autologous DC (), OVCa cells (), DC/OVCa fusion cells (●), DC mixed with irradiated OVCa cells (▪), or irradiated DC mixed with OVCa cells (□) for 7 days and then incubated with ⁵¹Cr-labeled autologous OVCa at indicated E/T ratios. (D) T cells from a patient with ovarian cancer were cocultured with autologous DC/OVCa fusion cells, collected on Days 10 and 25, and incubated with autologous OVCa cells (gray bar) or OVCa cells preincubated with anti-HLA class I antibody (W6/32: 1:100 dilution, hatch bar) at a 30:1 ratio. Results are expressed as mean ± SD of three replicates.