Stem cells conditioned in Flt3L + IL-6 are licensed for DC1-polarization, spontaneous maturation, and resistance to tumor-associated immunosuppressive factors. (A) Production of IL-12 by Flt3L + IL-6–conditioned BM cells following step 2 exposure to CpG plus LPS. Culture was performed as in Figure 1D, comparing outcomes with or without CpG plus LPS treatment. FACS dot plots show results of intracellular cytokine assays for IL12p40 production. Percentages shown in upper right quadrants are those of total BM cells specifically staining dually positive for MHC class II and intracellular IL-12 at the end of culture. Numbers in far right column show ELISA content of IL12p70 heterodimer from culture supernatants run in parallel without monensin. Data are representative of 8 experiments. (B) Step 2 culture as in Figure 1E, except following step 1 conditioning each group was replated in fresh medium for 72 hours solely with rGMCSF (no IL-4 or TLR agonists), after which FACS analyses were performed (selected groups shown in panel B; all groups shown in Figure S3a). Number within each histogram is the calculated mean fluorescence specificity index at 72 hours. Results shown are representative of 3 comprehensive comparisons. A similar but even more pronounced pattern of spontaneous maturation was observed during 48 to 72 hours step 2 culture in GMCSF + IL-4 (Figure S3B). (C) Step 2 cultures performed as in Figure 1E except that tumor-associated immunosuppressive factors were also added at 0 hour as listed in far left column. Data are representative of 3 experiments. (D) Same as in panel A, except listed immunosuppressive factors were added at 0 hour, 1 hour, 2 hours, or 4 hours of step 2 culture, with supernatants analyzed by ELISA in triplicate for IL12p70 content at 44 hours of step 2 culture. Error bars indicate SD. Data are representative of 3 experiments.