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Background: Tumor elimination and the success of cancer immunotherapy depend on the proper expression of HLA class I complex (HLA-I) required for the presentation of tumor-associated peptides to cytotoxic T-lymphocytes. Tumors escape immune attack by losing HLA-I expression, often due to irreversible genetic/chromosomal alterations, including mutations in beta2-microglobulin (B2M) or lack of HLA-A2 allele due to a haplotype loss. The introduction of these genes and re-expression of the missing HLA-I specificity on the tumor cell surface is an attractive strategy to induce tumor rejection by T-lymphocytes. Methods: Using genomic HLA-I typing and gene sequencing we determined HLA-I phenotypes and alterations in different human tumor cell lines previously characterized in our laboratory. We used adeno- and adeno-associated viruses to reconstitute/up-regulate HLA-A2 or/and B2M expression in these cells in vitro. Using flow cytometry and immunocytochemistry we evaluated levels and patterns of HLA-I expression in these cells. Methods: Using genomic HLA-I typing and gene sequencing we determined HLA-I phenotypes and alterations in different human tumor cell lines previously characterized in our laboratory. We used adeno- and adeno-associated viruses to reconstitute/up-regulate HLA-A2 or/and B2M expression in these cells in vitro. Using flow cytometry and immunocytochemistry we evaluated levels and patterns of HLA-I expression in these cells. Conclusion: Using gene therapy, it is possible to recover normal B2M and HLA-A2 gene expression caused by structural “hard” alteration and to induce co-expression of both genes in cells naturally lacking HLA-A2 allele. In addition, we demonstrated that transfected tumor cells ae able to express seven HLA-I alleles. The recovery of the missing HLA-I molecules frequently observed in tumor cells using adeno and adeno-associated viruses can be a useful strategy to circumvent cancer immune escape and increase tumor rejection.

Background The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-γ-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-γ treatment in human melanoma cell lines., Methods Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan® Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-γ-treated cells., Results Altered IFN-γ mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-α led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-γ treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-α treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-γ in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-γ signaling pathway., Conclusion We observed two distinct mechanisms of loss of IFN-γ inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-γ signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-γ-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation., This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS), Red Genomica del Cancer (C03/10), Plan Andaluz de Investigacion, Servicio Andaluz de Salud (SAS) in Spain, from the ESTDAB project (contract no. QLRI-CT-2001-01325), from the European Network for the identification and validation of Antigens and biomarkers in Cancer and their application in clinical Tumor immunology (ENACT, contract No. 503306), and from the European Cancer Immunotherapy project (OJ2004/C158, 518234).