Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Abstract

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.

FIGURE 1

DC phenotype before electrofusion. DCs from donor 4 were stained with FITC- (x-axis; FL1-H) and PE- (y-axis; FL2-H) conjugated mAbs as indicated and analyzed by FACS.

FIGURE 2

FACS analyses of DC-TC hybrid cells generated by electrofusion. 888 melanoma cells were labeled with CFSE (x-axis; FL1-H) and were fused with DCs from donor 4. After overnight culture, both adherent and nonadherent cell populations were stained with PE- (y-axis; FL2-H) conjugated mAbs as indicated.

FIGURE 3

Fluorescence micrographs of hybrid cells generated by electrofusion. A–C, 888 melanoma cells were labeled with CFSE (A) and were fused with DCs from donor 4. After overnight culture, the adherent cell population was stained with PE-conjugated anti-HLA-DR (B). Finally, a two-color overlay was prepared to enable visualization of double-positive cells (C). As estimated by FACS analysis, 29% of cells in the adherent fraction of DC-888mel fusion cells from donor 4 in this experiment were positive for both CFSE and HLA-DR. White arrows indicate melanoma cells that were only positive for CFSE. D–F, As a control, 888 melanoma cells were labeled with CFSE (D) and were fused in the absence of DCs. After overnight culture, the adherent cell population was stained with PE-conjugated anti-HLA-DR (E). Finally, a two-color overlay was prepared to enable visualization of double-positive cells (F).

FIGURE 4

Fluorescence micrographs of hybrid cells generated by electrofusion. A–C, 888 melanoma cells (without prior CFSE labeling) were fused with DCs from donor 7. After overnight culture, the adherent cell population was concomitantly stained with FITC-conjugated anti-HLA-DR (A) and anti-HLA-A24 that was indirectly coupled to PE (B). Finally, a two-color overlay was prepared to enable visualization of double-positive cells (C). The white arrow indicates an individual tumor cell that only expressed HLA-A24, and the blue arrow indicates a single DC that only expressed HLA-DR. D–F, As a control, 888 melanoma cells (without prior CFSE staining) were fused in the absence of DCs. After overnight culture, the adherent cell population was concomitantly stained with FITC-conjugated anti-HLA-DR (D) and anti-HLA-A24 that was indirectly coupled to PE (E). Finally, a two-color overlay was prepared to enable visualization of double-positive cells (F).