Supplementary Figure 1. Basal Expression of Surface Molecules on Various BCP-ALL Cell Types

Supplementary Figure 1. Basal expression of surface molecules on various BCP-ALL cell types.

Untreated BCP-ALL cells were isolated and analyzed for basal expression of various surface antigens. Bar graphs indicate isotype-corrected median fluorescence intensities (MFI) for costimulatory molecules CD40 and CD86, for the intercellular adhesion molecule CD54 and for the antigen-presenting molecules MHC class I and class II on the BCP-ALL cell lines RS4;11, KOPN-8, 018Z, MHH-CALL-2, Nalm-6 and Reh as well as on Xeno- and primary BCP-ALL cells. Error bars indicate SEM. Results are from at least 3 independent experiments for each cell type.

Supplementary Figure 2. IL-21 induces cell death in the BCP-ALL cell line RS4;11.

RS4;11 cells were incubated for 48 hours with CpG (2.5 µg/ml), IL-4 (400 U/ml), IL-21 (100 ng/ml) or CD40L (1 µg/ml) along with an enhancer for ligands (1 µg/ml), alone or in combinations as indicated. The percentage of viable BCP-ALL cells was determined by morphological criteria using FACS analysis according to morphological criteria (FSC/SSC). Bar graphs represent average of viable cells from at least 3 independent experiments, error bars indicate SEM, ** indicates p < 0.01.

Supplementary Figure 3. CpG, IL-4 and CD40L induce secretion of pro-inflammatory cytokines by BCP-ALL cells.

Primary BCP-ALL cells were incubated with CpG (2.5 µg/ml), IL-4 (400 U/ml), IL-21 (100 ng/ml) or CD40L (1 µg/ml) along with an enhancer for ligands (1 µg/ml), alone or in combinations as indicated. After 48 hours supernatants were harvested and analysed in a cytometric bead array. Bar graphs represent average concentrations of the indicated cytokines from four experiments. Error bars indicate SEM, * indicates p < 0.05.

Supplementary Figure 4. Pretreated BCP-ALL cells induce proliferation of co-cultured PBMC.

BCP-ALL cells (cell line KOPN-8 or RS4;11 or Xeno-BCP-ALL) were either left untreated or treated for 48 h with CpG B (2.5 µg/ml), IL-4 (400 U/ml) and CD40L (1 µg/ml) with enhancer for ligands (1 µg/ml), then served as stimulator cells for PBMC isolated from healthy donors at ALL:PBMC ratios of 1:5, 1:10, 1:100, 1:1000 for 5 days. 3H-Thymidine (5 µCi/ml) was added 18 h before analysis. Bar graphs represent PBMC proliferation as average of triplicates in counts per minute (CPM). Error bars indicate SEM. The data acquired with Xeno-BCP-ALL cells are representative for 3 individual experiments with similar results.

Supplementary Figure 5. Experimental design of autologous anti-leukemic CTL generation.

Leukemia cell samples obtained at diagnosis from three individual pediatric BCP-ALL patients were expanded using the NOD/SCID/huALL model. ALL cells were isolated from ALL-bearing recipients and cryopreserved. Meanwhile, ALL patients were successfully treated and achieved continuous remission. PBMC were isolated from peripheral blood obtained from these patients 6 months after the end of anti-leukemic treatment. Corresponding ALL samples were re-passaged in the NOD/SCID/huALL model, incubated with and without CpG, IL-4 and CD40L and used as stimulators in cytotoxicity assays. Cryoconserved untreated autologous ALL cells served as targets.