Tumor-derived CRCL activates DCs. (A) 12B1-derived chaperone proteins stimulate DCs to express MHC-II and costimulatory molecules. Day 6 bone marrow–derived DCs, grown in GM-CSF and IL-4, were exposed to 50 μg/mL 12B1 HSP70 (□), CRCL (▪), or unfractionated lysate (▨) for 24 hours, harvested, and analyzed by flow cytometry for expression of the cell-surface markers indicated. MFI of CD40, CD80, CD86, and MHC-II (I-Ad) on CD11c⁺ gated nontreated DCs was considered as one. The numbers in the ordinate represent the fold increase in MFI of treated DCs when compared with nontreated DCs. Means and SEM from 3 experiments are shown. (B) DCs pulsed with CRCL have increased IL-12 production. ELISPOT assays were performed to measure IL-12 production by DCs. At day 6 of culture, 3 × 10⁵ DCs were cultured with 50 μg/mL 12B1-derived lysate, HSP70, or CRCL in the presence of 10 ng/mL GM-CSF and IL-4 each for 24 hours (DC + CRCL or DC + HSP70 versus DC or DC + lysate, P < .05; DC + CRCL versus DC or DC + HSP70, P < .05; representative data from 3 experiments are shown). (C) 12B1-derived CRCL increases DC capacity to stimulate allogeneic splenocyte proliferation. DCs were cultured as in “Materials and methods,” harvested, treated with Mitomycin C, and washed. Splenocytes from C57BL6 mice were added (10⁵ per well) and cultured with the indicated ratios of pretreated BALB/c DCs for 4 days. [³H]thymidine was added and the cells were cultured for an additional 18 hours before the incorporated radioactivity was counted (DC + CRCL versus DC, DC + lysate, or DC + HSP70; P < .05; representative data from 2 experiments are shown). cpm indicates counts per minute.