Impact of tumor on conditioned BM cells, and impact of tumor-pulsed DCs upon subsequent T-cell cocultures. (A) Following step 1 conditioning treatments listed in far left column, each group was replated at 4 million cells/well in fresh medium with only GMCSF added at the beginning of step 2 culture. Twenty-four hours later, individual wells were also exposed to 3 million viable irradiated MCA-205 tumor cells. FACS analyses of cells were performed at 44 hours (20 hours after addition of tumor). Histograms display expression of MHC class II, CD40, and B7.2 for each conditioning treatment, with or without exposure to tumor. The far right column displays supernatant content of IL12p70 heterodimer 20 hours after tumor exposure when rmIFN-γ was also added to culture. Supernatant IL12p70 content was below detection (< 31 pg/24 hr) following exposure to either rmIFN-γ or to tumor alone (not shown). Data are representative of 3 full comparison experiments. (B) Photos of individual T-cell cultures after 6-day coculture with tumor-pulsed DCs or with anti-CD3. Labels denote step 1 DC conditioning treatments; step 2 DC cultures were performed in rGMCSF + rIL-4, with irradiated MCA-203 cells added at 24 hours and CpG + LPS added at 44 hours. Four hours later, DCs were harvested for coculture with L-selectin^low T cells freshly harvested from MCA-203 tumor-bearing mice. T cells were cocultured with DCs at a 8:1 ratio or were activated with immobilized anti-CD3. Cocultures shown also received exogenous IL-2 + IL-7 + IL-15. Data are representative of 3 full comparison experiments, using an Olympus IX50 inverted microscope, an Olympus CPlan 10×/0.25 PhC objective lens, a Sony DSC-S85 Cybershot, and Adobe Photoshop (Adobe Systems, Mountain View, CA) for compilation.