Transduction and differentiation of dendritic cells from human hematopoietic progenitor cells

Dendritic cells (DC) are highly potent antigen presenting cells, and may be able to stimulate effective anti-tumor immunity. Therefore, the ability to constitutively express tumor antigen genes in DC might be a powerful method to uncover new tumor antigens in vitro and to actively immunize in vivo. However, the growth of large numbers of DC has been difficult, and such cells have not been receptive to gene transfer. Since DC can differentiate from hematopoietic progenitor cells (HPC), we are retrovirally transducing human HPC with tumor antigen genes, followed by in vitro differentiation into DC. In initial experiments, human CD34⁺ HPC were transduced with marker genes (β-galactosidase or murine B7-1), and differentiated in vitro into DC using GM-CSF, TNFa, and stem cell factor. The resulting cell population contained 20-30% DC by morphological and phenotypic analyses, and was functionally far superior to PBMC in allogeneic MLR. By FACS analysis, >5% of cells expressed both the transduced marker gene and the DC phenotype (HB7-2⁺. class IP, CDla⁺, hB7-l⁺. The DC population expanded 20 fold during differentiation. Using this method, the melanoma tumor antigen gene MART-1 was introduced into DC. The MART-1 transduced DC stimulated high levels of cytokine release by MART-1 specific tumor infiltrating lymphocytes (2816 pg/mt IFNy vs 193 pg/ml for controls transduced with an irrelevant gene). This indicates that they were able to express, process, and present a MART-1 epitope on MHC class I molecules. This expression and presentation was stable, persisting beyond two weeks without selection. We are currently trying to raise specific anti-MART-1 CTL by in vitro stimulation of autologous PBL with the MART-1 transduced DC. In addition, we are evaluating the ability of transduced DC to present endogenously expressed antigens on MHC class II molecules.