Background: For tumor vaccine-based immunotherapy of cancer, the expansion of tumor antigen-specific cytotoxic T lymphocytes (CTL) in the patients by blocking induced regulatory T (Treg) cells is the most important objective now. Fludarabine (FLU), a known anticancer drug, has been shown to downregulate Treg cells in vivo in chronic leukemia patients. Melanoma tumor antigen Mart-127–35-specific CD8+ CTLs generated in vitrowith total peripheral blood lymphocytes (PBL) lose their activity within 14–21 days with concomitant expansion of Treg cells. When CD4+ cells are removed from PBL and CTL are generated with purified CD8+ cells, the CTL survive and maintain their activity for a significantly longer period. Methods: We used a low dose of FLU in the cultures in Mart-1-specific CTL generation assays with total PBL. Blood samples were taken from HLA-A2-positive melanoma patients and normal donors. Autologous matured dendritic cells pulsed with Mart-127–35 peptide were used to generate CTL responses using purified CD8+ cells or total PBL. Results: The presence of FLU in the cultures with PBL helped to generate a significantly higher number of antigen-specific CTLs as detected by Mart-1 HLA-A2 tetramer staining. Specificity of such CTLs was determined by IFN-γ secretion or by cytotoxicity against the target cells bearing the specific antigen. The presence of FLU stopped the expansion of IL-10-producing CD4+ Treg cells in the cultures with PBL. Analyses of expanded CD4+ cells isolated from PBL in vitro cocultures with FLU showed a Th1 type of function. Those cells secreted higher amounts of IFN-γ and very low levels of IL-10, or no IL-10 at all, upon restimulation. Conclusion: The observations of the study are as important for adaptive immunotherapy of cancer as they are for vaccine-based approaches.

Keywords:
Cytotoxic T lymphocytes, Cytokine, Melanoma, Peripheral blood lymphocytes
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© 2008 S. Karger AG, Basel
2008
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