In the absence of exogenous rhuIL-15, only WT1 mRNA-electroporated CD34⁺ HPC-derived LCs stimulate primary WT1-specific CTLs in vitro that kill both immortalized and primary tumor cells. Cytokine-generated, CD34⁺ HPC-derived LCs (A-B) and moDCs (C-D) from the same healthy, HLA-A*0201⁺ donors were electroporated with WT1 mRNA, matured by a combination of inflammatory cytokines, and then added in serial doses to triplicate microwells containing 1 × 10⁵ autologous T cells. Exogenous rhuIL-15 (10 ng/mL) was added to the primary cultures stimulated by moDCs only in panel D. A total of 5 × 10³⁵¹Cr-labeled target cells were added directly to the primary culture microwells after only 7 days stimulation. ⁵¹Cr release was measured in the supernatants collected after 6 hours in panels A, C, and D. WT1⁺ primary AML blasts from patients expressing HLA-A*0201 were labeled with PKH-26 and evaluated as targets of CTLs stimulated by LCs in the absence of exogenous rhuIL-15 in panel B. These response assessments used a flow cytometry-based assay whereby lysed targets took up an otherwise membrane impermeable DNA stain, TO-PRO-3. Specific lysis was based on the frequency of PKH-26⁺ TO-PRO3⁺ relative to PKH-26⁺ TO-PRO3⁻ events. With either ⁵¹Cr or colorimetric labeling, specific lysis has been plotted against the y-axes with respect to the conditions of primary stimulation shown along the x-axes. The cytolytic activity generated per primary culture condition could then be compared between LC (A) and moDC stimulators, the latter without (C) or with (D) exogenous rhuIL-15 supplementation. (A,C-D) Data points are the averages ± SEM of triplicate means from each of 3 independent experiments using 3 different healthy donors. (A,C-D) Target cells were 697 cells (□, HLA-A*0201⁺, WT1⁺ cell line); SKLY-16 cells pulsed with HLA-A*0201-restricted WT1 peptide (▿; HLA-A*0201⁺, WT1⁻ cell line); unpulsed SKLY-16 cells (O; HLA-A*0201⁺, WT1⁻ cell line); and LCL721.221 cells (*; MHC class I-negative, NK cell sensitive cell line). (B) One representative experiment of 3 independent experiments performed where 697 cells were targeted by T cells stimulated by LCs, blocked (▵) or not (□) by anti–IL-15R-α during T-cell priming. Primary AML blasts (♢; HLA-A*0201⁺, WT1⁺) proved susceptible, whereas NK cell-sensitive LCL721.221 cells (*) proved resistant to CTLs stimulated by LCs in the absence of blocking anti–IL-15R-α. *P < .01; **P < .001 for pairwise comparisons between either 697 or SKLY-16 WT1-pulsed cells versus LCL721.221 at the top 2 stimulator doses (A,D). **P < .001 for pairwise comparisons between 697 cells targeted by T cells primed by LCs blocked or not with anti–IL-15R-α, and for AML blasts versus LCL721.221, also at the top 2 stimulator doses (B).