Purified hybrid cells from dendritic cell and tumor cell fusions are superior activators of antitumor immunity.

The use of fusions between dendritic cells (DCs) and tumor cells as vaccines has been proved very effective in stimulating antitumor immune responses, both in animal studies and in early human clinical trials. Because of the difficulty of purifying the hybrid cells from the fusion, fusion mixtures were used in these studies. Recently, we developed a technique using fluorescent-dye staining and fluorescence-activated cell sorting that enabled the hybrid cells to be instantly purified from the fusion mixture. In the present study, the hybrid cells were purified from a fusion between mouse DCs and B16F0 melanoma tumor cells using the new technique. The purified cells, named instant dendritomas (IDs) were then compared with fusion mixtures in stimulating antitumor immune responses. The results from cytotoxicity assays, interferon-gamma production and in vivo lung tumor metastasis demonstrated that IDs are more effective than fusion mixture in stimulating antitumor immunity. Meanwhile, there was no significant difference in the antitumor immunities activated by IDs from allogenic fusion or IDs from syngenic fusion.

Murine tyrosinase expressed by a T7 vector in bone marrow-derived dendritic progenitors effectively prevents and eradicates melanoma tumors in mice.

Dendritic cell (DC)-mediated cancer immunotherapy is a very promising alternative approach to cancer treatment. In a previous study, we successfully transfected bone marrow-derived dendritic progenitors (BMDDPs) with a T7 vector--a nonviral, cytoplasmic-based autogene expression system--encoding a model tumor antigen, firefly luciferase, and subsequently stimulated the transfected cells to differentiate into DCs. When injected into experimental mice, those DCs generated a strong immune response against tumor cells bearing luciferase, which not only prevented occurrence of metastasis but also eradicated existing tumors. In the present study, we constructed a T7 vector encoding mouse tyrosinase, a well--known melanoma associated tumor antigen, and used it to transfect BMDDPs. Reverse transcriptase polymerase chain reaction and Western analysis confirmed the expression of tyrosinase by DCs differentiated from transfected BMDDPs. Two immunizations of these DCs at a dose of 2 x 10(6) of each successfully prevented tumor growth. More importantly, one injection of 2 x 10(6) of these DCs into mice followed by five doses of recombinant human interleukin-2 administration effectively eradicated existing tumors as indicated by pulmonary metastasis assay.

Rapid generation and flow cytometric analysis of stable GFP-expressing cells.

Expression of green fluorescent protein (GFP) represents a unique method for the fluorescent labeling of viable mammalian cells, with many potential applications. The studies detailed in this report examine the detection of GFP expression in murine cells using flow cytometry. Direct comparison of NIH 3T3 cells transiently expressing GFP or GFPS65T, a mutant form of GFP, showed that GFPS65T fluorescence (using 488 nm excitation) was detected more readily than fluorescence from wildtype GFP. Efficient generation of cell lines that stably expression GFPS65T was achieved using a plasmid vector that encoded a hygromycin phosphotransferase/GFPS65T fusion protein. Flow cytometric detection of NIH 3T3 cells expressing this fusion protein was improved using a 510/20 band pass filter instead of the standard filter setup for fluorescein detection. Additionally, staining the surface of these cells with phycoerythrin, RED 670, or allophycocyanin did not interfere with the detection of GFPS65T fluorescence, indicating that multiparameter analyses using GFPS65T fluorescence are possible. Importantly, we also observed that GFPS65T expression could be detected in NIH 3T3, BW5147, or freshly cultured Thy1lo CD3- murine bone marrow cells transduced with a retroviral vector encoding the fusion protein, suggesting that the potential applications of this system may be quite broad.

Reliable method for the simultaneous detection of cytoplasmic and surface CD3 epsilon expression by murine lymphoid cells.

During their development, T-cell precursors (pre-T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T-cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER-Golgi). In order to study the expression of the T-cell receptor TCR/CD3 complex during pre-thymic T-cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3 epsilon) and cytoplasmic CD3 epsilon (cCD3 epsilon). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti-CD3 epsilon in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X-100 was shown to remove surface-bound anti-CD3 epsilon. The present technique permitted the detection of discernible sCD3 epsilon and cCD3 epsilon double and single positive lymphocytes and may prove useful in defining bone marrow-resident pre-T cells.

Cysteine proteinase changes during macrocyst formation in Dictyostelium mucoroides.

Cysteine proteinases were detected in vegetative myxamoebae of Dictyostelium mucoroides DM7 using chromogenic substrates and by electrophoretic analysis (gelatin-SDS-PAGE) which revealed three enzymes, dmCP30, dmCP35 and dmCP46 (a minor form). During the initial stages of macrocyst formation the cysteine proteinaes were secreted and disappeared almost completely from the cells. High extracellular levels of activity towards N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide and of dmCP30 persisted throughout macrocyst development. Three new intracellular proteinases, dmCP31, dmCP36 and dmCP40, were produced as macrocysts formed but their activity was only detected by gelatin-SDS-PAGE. Their appearance was specific to the developmental pathway leading to macrocyst formation. This is the first direct evidence for the accumulation of cysteine proteinases during a developmental process in a cellular slime mould.