Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

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Monoclonal antibody T311 specifically detects tyrosinase protein expression. Tyrosinase-derived peptides are recognized by CD8+ T-cells and applied in immunotherapy. We examined formalin-fixed paraffin-embedded tissue of 50 melanoma (primary n=31, metastatic n=19) and 41 control cases (junctional, dermal, compound, Spitz, Reed, balloon-cell nevi) by immunochemistry using the alkaline phosphatase-anti-alkaline phosphatase method after antigen retrieval. Staining with mAb T311 showed a sensitivity of 94% for melanoma with a very high specificity for melanocytic cells. Immunopositivity (94% of melanomas overall) correlated inversely with clinical stage: clinical stage I and stage II showed 100%, stage III and stage IV 86% immunoreactivity each. Staining changed from an exclusively homogeneous pattern in early stages to a more heterogeneous pattern in later stages. Melanocytic control tissue like nevi of different subtypes all showed weak to moderate, homogeneous immunoreactivity with polarity towards the epidermis. RT-PCR ELISA analysis of short-term melanoma cell cultures displayed mRNA expression in only half of the originally immunopositive tumors only, suggesting rapid mRNA expression loss in culture. mAb T311 allows detection of melanoma-associated tyrosinase protein expression and thus profiling of melanomas using routine archival tissue suited for immunotherapy approaches involving tyrosinase derived epitopes.

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Expression of HLA class I molecules is essential for the recognition of tumor cells by CD8+ T cells. In this study, 48 bioptic samples of 42 patients in all stages of melanoma were investigated after short-time cultivation of tumor cells. To confirm melanocytic origin of cultured cells, samples were screened for mRNA expression of melanoma markers gp100, tyrosinase, MAGE-3, MelanA, and MUC18 by reverse transcriptase-polymerase chain reaction. Surface expression of specific HLA-A and -B allospecificities on melanoma cells were analyzed with a standard microcytotoxicity assay after stimulation with interferon (IFN)-alpha and compared with the background found in peripheral blood mononuclear cells from the corresponding patients. Immunohistochemistry and flow cytometry confirmed specific losses in cases where the appropriate monoclonal antibodies were available. The level of expression of HLA-I, HLA-II, and intercellular adhesion molecule 1 antigens on melanoma cells cultured in the presence or absence of IFN-alpha and IFN-gamma was determined cytofluorometrically. All cell cultures tested were found to be positive for one or more melanocytic markers by reverse transcriptase-polymerase chain reaction. The specific HLA-I alleles on the cultured cells were detectable in 45 of 48 samples. In 11 cases a specific loss of one HLA-I allele was observed (2 x A2, B7, B8, B18, 4XB44, B47, B49). Ten of these samples were derived from locoregional lymphnode metastases or from distant metastatic tumors. Only one sample from a primary melanoma showed a specific loss of HLA-I (B47). IFN-alpha upregulated expression of HLA-I up to 4-fold. IFN-gamma enhanced the appearance of HLA-II up to 35-fold and the expression of intercellular adhesion molecule 1 up to 40-fold. Selective loss of HLA-I allospecificities might be a major step in tumor progression.

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IntroductionPrimary cutaneous lymphomas are the second most common group of extranodal non-Hodgkin lymphomas after primary gastrointestinal lymphomas. 1 They have highly characteristic clinical features and are considered prototype malignancies because they can be recognized clinically very early. In addition, because of their locations, they are well accessible for the study of immune and molecular biology. Most cutaneous lymphomas are low-grade T-cell lymphomas such as mycosis fungoides and Sézary syndrome (SS). 2 These neoplasms are not curable, even using aggressive chemotherapy and radiotherapy. 3 The malignant lymphocytes mostly express the T-cell markers CD2, CD3, CD4, and CD5. Often they lack CD7, and they display T-helper 2 (Th2) cytokines such as IL-5 and IL-10 and express the IFN-␥ receptor chain 2 (IFN␥R2). 4 IFN-␣, alone or in combination with retinoids or psoralen plus ultraviolet A irradiation, is helpful, especially in early disease. 5 Interferons are known for their pleiotropic effects. Besides the up-regulation of major histocompatibility complex molecules and the inhibition of cell proliferation and tumor growth, IFN can induce resistance to viral replication in all cells. 6 Interferons exert their effects by inducing overlapping sets of effector proteins through specific cell surface receptors (IFN␣R and IFN␥R). In this context, a cross-talk between IFN-␥ and IFN-␣/␤ signaling components has been reported recently. 7 It has been shown that the IFN␣R1 chain favors the association of the IFN␥R1 and the IFN␥R2 chains.Because of the beneficial clinical impact of IFN-␣ and the inhibitory potential of IFN-␥ on benign human and murine Th2 lymphocytes, 8 we studied the effects of IFNs on CTCL-derived tumor cells purified by FACS-sorting from the peripheral blood of 4 patients with SS. 4 This subtype of cutaneous T-cell lymphoma is characterized by an accumulation of malignant T-lymphocytes in skin and peripheral blood. 2 Furthermore, we evaluated the susceptibility of these SS cells to viral infections with vesicular stomatitis virus (VSV). Patients and methods PatientsSS was diagnosed using standard clinical criteria (erythroderma, lymphadenopathy), histologic criteria (subepidermal lymphoid infiltrate showing strong epidermotropism and expressing a T-helper cell phenotype), hematologic criteria (more than 1000 SS cells/mL), and an elevated CD4-CD8 ratio. 9-12 All patients were seronegative for human T-cell lymphocytic virus-I antibodies by enzyme-linked immunosorbent assay. Of the 4 patients whose T-cell clones were identified and investigated, 2 were in stage III (T 4 N 1 M 0) and 2 were in stage IVa (T 4 N 3 M 0 ) disease. 13 They did not receive any systemic therapy in the last 4 weeks before blood was taken. However, topical steroids had to be applied to control itching.Molecular biology studies for the T-cell receptor ␥ chain, 14 Southern blot analysis, or both documented the presence of a dominant T-cell clone in the peripheral blood of all patients. In all patients, a dominant CD4 ϩ V␤ ϩ (do...