Evaluation of dendritic cells loaded with apoptotic cancer cells or expressing tumour mRNA as potential cancer vaccines against leukemia

Abstract

Background: Leukemia is a clonal disorder characterized by uncontrolled proliferation of haematopoietic cells, and represents the most common form of cancer in children. Advances in therapy for childhood leukemia have relied increasingly on the use of high-dose chemotherapy often combined with stem-cell transplantation. Despite a high success rate and intensification of therapy, children still suffer from relapse and progressive disease resistant to further therapy. Thus, novel forms of therapy are required.

Methods: This study focuses on dendritic cell (DC) vaccination of childhood leukemia and evaluates the in vitro efficacy of different strategies for antigen loading of professional antigen-presenting cells. We have compared DCs either loaded with apoptotic leukemia cells or transfected with mRNA from the same leukemia cell line, Jurkat E6, for their capacity to induce specific CD4+ and CD8+ T-cell responses. Monocyte-derived DCs from healthy donors were loaded with tumor antigen, matured and co-cultured with autologous T cells. After one week, T-cell responses against antigen-loaded DCs were measured by enzyme-linked immunosorbent spot (ELISPOT) assay.

Results: DCs loaded with apoptotic Jurkat E6 cells or transfected with Jurkat E6-cell mRNA were both able to elicit specific T-cell responses in vitro. IFNgamma-secreting T cells were observed in both the CD4+ and CD8+ subsets.

Conclusion: The results indicate that loading of DCs with apoptotic leukemia cells or transfection with tumour mRNA represent promising strategies for development of cancer vaccines for treatment of childhood leukemia.

Figure 1

Phenotype of generated DCs. Expression of antigens were determined by flow cytometry (a) Before loading with tumor antigens (b) After loading with apoptotic Jurkat E6-cells and following maturation with TNFα, IL1β, IL6 and PgE2 for 24 h and (c) After transfection with Jurkat E6-cell mRNA and maturation for 24 h. The histograms show staining with the appropriate mAb.

Figure 2

Staurosporin-induced apoptosis of Jurkat E6-cells. T cells were cultured for 3 h with 1 μM staurosporin and examined by Annexin V-FLUOS staining and flow cytometry.

Figure 3

Phagocytosis of apoptotic Jurkat E6 cells by immature DCs. At day 5 of culture, DCs were co-cultivated with apoptotic Jurkat E6 cells, previously labelled with green fluorescent dye. (a) Flow cytometry of immature DCs stained with anti CD1a-PE and apoptotic Jurkat E6 cells stained with PKH-67 green. (b) Confocal microscopy analysis shows the presence of intracellular apoptotic cells labelled with PKH-26 red within DCs labelled with PKH-67 green (yellow cell, arrow) or in the process of being phagocytized (yellow and red cell, arrow).

Figure 4

mRNA transfectation of DCs. (a) Flow cytometric analysis of DCs after transfection with EGFP/pCIpA₁₀₂ mRNA (10 μg/400 μl) by square-wave electroporation and maturation for 24 h in medium with maturation cocktail. Control cells were mock electroporated without mRNA. (b) Fluorescence microscopy of DCs 24 h after transfection with EGFP/pCIpA₁₀₂ mRNA.

Figure 5

ELISPOT analysis of T-cell activation. T cells were stimulated with DCs loaded with tumour antigen (mRNA or apoptotic cells) or control DCs as indicated. (a) Mean number of IFN-γ positive spots obtained with the indicated numbers of un-fractionated T cells, and CD4+ and CD8+ T-cell subsets from individual donors. (b) Frequency of reactive T cells. The data represent mean values of all donors calculated by the formula: [(spots loaded DC - spots control DC)/10⁵ T cells].