Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2 s -M2) alone or in combination with Tet-repressor (tTS-Kid)

Differentiation of CD8+ T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8+ T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8+ T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7+ CD45RA+/− cytotoxic factor− cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8+ T cell population toward CCR7− CD45RA−/+ perforin+ granzyme B+ differentiation stages. Distinct CD8+ T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7+ perforin− CD8+ T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7+CD45RA− cells, proliferation coupled to differentiation to the CCR7− perforin+ stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8+ T cells differentiated toward CCR7− cytotoxic factor+ stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8+ T cells.

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1-dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1 ؊ cells) nor upregulated (in APAF-1 ؉ cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1 ϩ and APAF-1 ؊ tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 -specific inhibitors in both APAF-1 ϩ and APAF-1 ؊ tumor cells. In response to 1 to 100 mol/L of cisplatin, camptothecin, or celecoxib, APAF-1 ؉ melanomas (n ‫؍‬ 12) did not show significantly increased levels of apoptosis compared with APAF-1 ؊ tumors (n ‫؍‬ 7), with the exception of enhanced apoptosis in response to a very high dose (100 mol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.

Neoplastic cells are thought to have defective expression of costimulatory molecules. However, in this study, we show that human melanoma cells express LIGHT/TNFSF14, a ligand of herpesvirus entry mediator on T cells and of lymphotoxin beta receptor on stromal cells. In vitro, melanoma cells stained for LIGHT in the intracellular compartment, with weak or negative cell surface expression. However, LIGHT was expressed on tumor-derived microvesicles released from melanoma cells. In vivo, LIGHT was found in metastatic lesions, and the extent of lymphotoxin beta receptor expression on the stromal cells was significantly associated with a "brisk" T-cell infiltrate in the neoplastic tissue. In the lesions with a brisk T-cell infiltrate, stromal cells surrounding the tumor also stained for the T-cell attractant chemokine CCL21. The intratumoral T lymphocytes frequently expressed herpesvirus entry mediator and were characterized by a differentiated phenotype. Coculture of lymphocytes with LIGHT(+) melanoma-derived microvesicles or even with LIGHT(+) melanoma cells in the presence of interleukin-2 costimulated LIGHT-dependent CD3(+)CD8(+) T-cell proliferation. However, lymphocyte coculture with LIGHT(+) microvesicles in the presence of interleukin-2 was also associated with an apoptotic response as documented by increased binding of Annexin V by CD3(+)CD8(+) T cells. These data suggest that LIGHT constitutively expressed in human melanoma cells and microvesicles may contribute to regulate T-cell responses to tumor cells.

The epithelial cell adhesion molecule, Ep-CAM, has been historically considered a target of passive immunotherapy using monoclonal antibodies, and more recently, of a first Pox-vector-based cancer vaccine Phase I trial in colorectal cancer patients. To shed further light on the use of this antigen, we isolated the mouse and rhesus homologues of human Ep-CAM and explored different genetic vaccination modalities based on the use of adenoviral vectors as well as DNA electroporation (DNA-EP). Immune responses to Ep-CAM were measured by IFN-c ELISPOT and intracellular staining assays using overlapping sets of peptides covering the entire coding regions. We found the most powerful vaccination regimen to be constituted by DNA-EP-prime/Adeno-boost mixedmodality protocols. Vaccination in rhesus macaques resulted in breakage of immunological tolerance in a minority of cases. Similarly, a low frequency of responders was observed with the mouse Ep-CAM vaccine in outbred CD1 mice. When immunized CD1 mice were analyzed for MHC haplotype and TCR expression levels, we observed that immune responders all had the same q/q MHC class I haplotype and showed higher expression levels of the TCRVb4 and TCRVb8 T cell receptors. Our results underscore the current limitations in our capacity to induce efficient cancer vaccines against self antigens like Ep-CAM, but also represent a first effort to identify predictive biomarkers of response.See accompanying commentary: http://dx