Inhibition of melanoma growth after treatment with dendritic cells in a Tyr-SV40E murine model requires CD4+ T cells but not CD8+ T cells.

Melanomas are promising targets for immunotherapy, as they express a number of tissue-specific antigens against which immune responses can be elicited. We have previously described transgenic mice in which malignant cutaneous melanomas are produced. The 1042 melanoma cell line, derived from a primary melanoma in one of these mice, was used here to generate tumours by subcutaneous inoculation in syngeneic animals. All mice injected with 1 x 10(6) cells of the 1042 cell line developed a tumour. CD4+ T cells, CD8+ T cells and macrophages infiltrated the tumours. Treatment with dendritic cells pulsed with peptides from melanogenic proteins slowed tumour growth and resulted in increased numbers of infiltrating lymphocytes and macrophages, expansion of CD4+ T cells specific for 1042 cell antigens, and increased levels of 1042-specific immunoglobulin G1 (IgG1) and IgM in serum. The frequency of cytotoxic T lymphocytes (CTLs) specific for the MART-1 melanocytic antigen did not increase after dendritic cell treatment. Indeed, the presence of CD8+ T cells was apparently not required for the anti-tumour effects: slowing of tumour growth was not abrogated in animals depleted of CD8+ T cells using antibodies, or in syngeneic CD8-/- animals. In contrast, treatment with dendritic cells + peptides was ineffective after depletion of CD4+ T cells and in syngeneic CD4-/- mice. This experimental system therefore provides an opportunity to investigate CD4-dependent anti-tumour effector mechanisms, and for studies designed to activate the quiescent CTLs which infiltrate melanomas.

Nuclear cloning of embryonal carcinoma cells.

Embryonal carcinoma (EC) cells have served as a model to study the relationship between cancer and cellular differentiation given their potential to produce tumors and, to varying degrees, participate in embryonic development. Here, nuclear transplantation was used to assess the extent to which the tumorigenic and developmental potential of EC cells is governed by epigenetic as opposed to genetic alterations. Nuclei from three independent mouse EC cell lines (F9, P19, and METT-1) with differing developmental and tumorigenic potentials all were able to direct early embryo development, producing morphologically normal blastocysts that gave rise to nuclear transfer (NT)-derived embryonic stem (ES) cell lines at a high efficiency. However, when tested for tumor or chimera formation, the resulting NT ES cells displayed an identical potential as their respective donor EC cells, in stark contrast to previously reported NT ES cells derived from transfer of untransformed cells. Consistent with this finding, comparative genomic hybridization identified previously undescribed genetic lesions in the EC cell lines. Therefore, nonreprogrammable genetic modifications within EC nuclei define the developmental and tumorigenic potential of resulting NT ES cells. Our findings support the notion that cancer results from the deregulation of stem cells and further suggest that the genetics of ECs will reveal genes involved in stem cell self-renewal and pluripotency.

Freezing and thawing of bone marrow-derived murine dendritic cells with subsequent retention of immunophenotype and of antigen processing and presentation characteristics.

Murine dendritic cells (DCs) are widely used for experimental vaccinations in mouse models. A high-yield method for freezing and thawing batches of these cells, if compatible with retention of cell immunophenotype, would reduce the time required for repeated preparations from DC precursors in bone marrow (BM), as well as variability among lots. Following depletion of specific lineages, murine bone marrow cells from C57BL/6 inbred-strain mice were grown in medium containing 10% fetal calf serum (FCS) and granulocyte/macrophage colony-stimulating factor (GM-CSF); after 6 days, large numbers of immature DCs were obtained. The immature cells were frozen in complete medium with GM-CSF and 10% DMSO, at a cell density of 5x10(6) DCs/ml. After thawing, 80% of DCs survived; they were induced to mature by addition of lipopolysaccharide (LPS). In comparison with fresh DCs, the thawed DCs had similar morphology, purity, and expression of class I (H-2D(b) and H-2K(b)) and class II major histocompatibility complex (MHC) proteins, as well as CD11b, CD11c, CD40, CD80, and CD86 molecules. Freeze-thawing did not affect trafficking to T cell areas of spleen, nor reduce the capacity to stimulate an alloresponse. Frozen-thawed cells were also proficient at uptake, processing, and presentation of native or denatured ovalbumin (OVA) protein to a peptide-specific T cell hybridoma, and were able to induce T cell responses in vivo after being loaded with denatured OVA protein. The ability to freeze and thaw DCs, and to obtain high yields without altering their essential properties, will facilitate future immunotherapy experiments in laboratory mouse models.