SW1-P2 cells express less ATF-2 in the nucleus and more in the cytoplasm than SW1-C cells, and this is correlated with decreased malignancy and increased ability to undergo apoptosis after exposure to radiation or certain chemicals (6). As we now show, neither SW1-C nor SW1-P2 cells became immunogenic after transfection to express CD83. SW1-P2 cells became rejectable after expression of CD83 or anti-CD137 scFv. However, outgrowth of cells from either line was delayed in mice immunized against M2-CD83 or M2-1D8 cells, and immunization with a mixture of mitomycin C-treated cells from M2-CD83 plus M2-1D8 prevented tumor formation by SW1-P2 cells in five of five and by SW1-C cells in three of five mice. We conclude that M2 cells expressing CD83 can induce a tumor-destructive immune response also against SW1 cells and that this response can be made more effective by combining them with M2 cells expressing anti-CD137 scFv. A similar approach may be therapeutically beneficial against certain human cancers. Keywords: tumor vaccines, immunotherapy, antigen presentation, activating transcription factor 2 CD83 is a marker of activated human dendritic cells (1). In humans, CD83 ligands are primarily expressed on resting monocytes and a subpopulation of activated T cells (2). Transfection of cells from the human B cell line T51 to express CD83 increases their ability to stimulate proliferation of allogeneic peripheral blood mononuclear cells, including CD8+ cytotoxic T lymphocytes, and this effect is abolished in the presence of a soluble CD83 fusion protein with an Ig tail (3). In mice, the ligands for CD83 are primarily expressed on B cells (4), and fusion proteins that express the extracellular part of mouse CD83 (mCD83) inhibit antigen-specific WF 11899A T cell proliferation and IL-2 secretion in cultures of mouse spleen (4). Injection of a CD83 mouse Ig fusion protein can facilitate the outgrowth of transplanted cells from the P815 mastocytoma and decrease a tumor-directed cytotoxic T lymphocyte response (3). Furthermore, cells from the M2 clone of mouse melanoma K1735 cells transfected to express human CD83 were shown to be rejected by syngeneic mice, some of which also rejected a subsequent transplant of nontransfected (WT) cells from the M2 clone (3). Although mCD83 and human CD83 share 63% amino acid identity (5), an immune response to human antigens may contribute to the immunogenicity of mouse tumor cells expressing human CD83. Therefore, we have now immunized mice with M2 cells expressing mCD83. Furthermore, we have used mitomycin C (MMC)-treated rather than live tumor cells as immunogen in many of our experiments to make them more relevant as models for potential human application. This method also facilitates comparisons between immunizing tumor cells that express CD83 or an irrelevant molecule. To make our tumor model more demanding, we extended our studies to a different K1735 clone, SW1-C, and the SW1-P2 line, which had been obtained by transfecting SW1 cells to express a 50-aa peptide derived from the NH2-terminal domain of activating transcription factor type 2 (ATF-2) (6, 7). Expression of ATF-2, which belongs to the ATF/CREB (cAMP-response element-binding protein) family, within the nucleus rather than the cytoplasm is associated WF 11899A with poor prognosis of melanoma (8). SW1-P2 cells express less ATF-2 in the nucleus and more in the cytoplasm than SW1-C cells, and this is correlated with decreased malignancy and increased ability to undergo apoptosis after exposure to radiation or certain chemicals (6). As we now show, neither SW1-C nor SW1-P2 cells became immunogenic after transfection to express CD83. Neither did transfection to express anti-CD137 single-chain variable region fragments (scFv) cause SW1-C or SW1-P2 cells to be rejected, whereas similarly transfected M2 cells are efficacious as therapeutic vaccines against established WT M2 tumors (9). However, outgrowth of both SW1-C and SW1-P2 was delayed in mice immunized with MMC-treated M2 cells that expressed either CD83 or anti-CD137 scFv, and it could be prevented in most mice concomitantly immunized with both M2-CD83 and M2-1D8 cells, probably because these cells engaged the immune system in different ways. Materials and Methods Tumor Lines. K1735 is a melanoma of C3H/HeN origin from which clones M2 and SW1 were isolated (10, 11). SW1-P2 was derived by transfecting SW1 cells to WF 11899A express an ATF-2-derived peptide (7). The SW1-C and SW1-P2 cell lines were chosen because pilot tests revealed that they were not rejected by immunocompetent mice after transfection to express anti-CD137 scFv derived from hybridoma 1D8 (data not shown), a procedure that makes cells from the M2 clone highly immunogenic (9). Ag104 is a spontaneous sarcoma of C3H/HeN originally obtained from H. Schreiber (University of Chicago, Chicago) and has low immunogenicity (12). Tumor cells transplanted to MF1 mice were WF 11899A derived from cultures, and suspensions comprising >90% live tumor cells were prepared by exposing cultures.