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Adoptive T-cell immunotherapy may provide complementary therapy for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this study, we have analyzed the functional characteristics of anti-BCP-ALL effector T cells generated by co-culturing T lymphocytes and dendritic cells (DC) from allogeneic human stem cell transplantation (HSCT) donors. After 21-day co-culture with DC pulsed with CD40L+ apoptotic BCP-ALL blasts, T cells presented with both effector and central memory phenotype, and showed high and specific cytotoxic activity against leukemic cells (average lysis = 77%), mostly mediated by CD8+ T cells. Noticeably, growth of CD4 T cells was maintained (45% of total cells), which actively produced Th1 cytokines (IFN-gamma, TNF-alpha, IL-2), but not IL-4, IL-5 and IL-10. Anti-BCP-ALL T cells expressed CD49d and CXCR4 (implicated in the recruitment to bone marrow), and CD62L and CCR7 (involved in the migration to lymphoid organs). In accordance with this profile, T cells significantly migrated in response to the chemokines CXCL12 and CCL19. In conclusion, stimulation of T cells with CD40L+BCP-ALL cells-loaded DC not only elicited the generation of potent and specific anti-leukemic cytotoxic effectors, but also the differentiation of specific and functional Th-1 CD4 lymphocytes. These effectors are fully equipped to reach leukemia-infiltrated tissues and have characteristics to support and orchestrate the anti-tumor immune-response.

Three different second-generation lentiviral self-inactivating vectors containing CMV, EF1alpha and PGK promoter, respectively, and all carrying the exogenous GFP gene, were compared for expression in human B-cell precursor ALL blasts. At a comparable percentage of transduction and vector DNA copy number, CMV clearly showed better efficiency of transcription. Human bone marrow stromal cells were favored compared to the MRC-5 cell line, as support for cell viability during infection. Cells were infected and analyzed after variable culture times ranging from 4 to 12 days, to reduce the possibility of pseudotransduction. In 10/ 14 samples, we detected more than 20% GFP-positive cells after exposure to high-titer viral supernatants. We then tested a similar vector carrying the human CD40L cDNA and, in similar infection conditions, obtained more than 20% transduction in 6/6 samples. The levels of transduction obtained were sufficient to induce the upregulation of CD83 molecule in cocultured immature dendritic cells.