Influence of interferon gamma on modulation of Fas expression by human colon carcinoma cells and their subsequent sensitivity to antigen-specific CD8+ cytotoxic T lymphocyte attack.
The inability of certain neoplastic populations to undergo Fas-mediated death by immune effector mechanisms may confer a selective survival advantage, which may contribute to tumor escape. In this study, we examined the role of Fas-mediated lysis in a human-antigen (Ag)-specific cytotoxic T lymphocyte (CTL)/colon carcinoma cell model, and the regulation of the lytic phenotype by interferon gamma (IFNgamma). Previously, we have identified mutated ras peptides reflecting the valine-for-glycine substitution at position 12 as unique HLA-A2-restricted, CD8+ CTL neo-epitopes. Peptide-specific CTL, established from both normal and carcinoma-bearing individuals, lysed in vitro a HLA-A2+ primary colon adenocarcinoma cell line, SW480, harboring the naturally occurring ras mutation. Pretreatment of SW480 cells with IFN-gamma was necessary to promote efficient Ag-specific CTL killing, although the mechanisms by which IFNgamma influenced the lytic outcome remains to be elucidated. Here, we show, by phenotypic analysis of SW480 cells, a significant up-regulation of HLA-A2, ICAM-1 and Fas molecules after IFNgamma pretreatment, which paralleled their sensitivity to lysis with anti-Fas stimuli. Moreover, nearly half of the lytic response to IFNgamma-treated SW480 cells was inhibited by neutralizing anti-Fas or anti-Fasligand (FasL) mAb, revealing for the first time an important functional role for Fas/FasL interactions in carcinoma cell killing by human Ag-specific CTL. mAb against HLA-A2, ICAM-1, the alpha T cell receptor (TCR) and Fas molecules inhibited lysis; however, if these CTL were preactivated to express functional FasL and then used as effectors, only anti-Fas mAb efficiently blocked lysis. IFNgamma also increased pro-caspase-3 protein expression and its subsequent activation in SW480 cells following Ag-specific CTL attack. Peptide-based caspase inhibitors blocked both caspase-3 activation and CTL-mediated lysis. Overall, these data suggested that IFNgamma (a) facilitated both Ag-dependent and Ag-independent events as a prerequisite for efficient CTL/target interactions, FasL up-regulation and triggering of Fas-dependent, as well as Fas-independent lysis (perforin); and (b) enhanced or restored a Fas-sensitive phenotype in SW480 cells, reflecting modulation of cell-surface and intracellular elements of the Fas pathway. Thus, IFNgamma may play an important role in the regulation of a human neoplastic cell death phenotype, which may have implications for our understanding of the processes of both tumor evasion and tumor regression following Ag-specific CTL attack.
Bergmann-Leitner
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Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1402, USA.
Abrams
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D015153:Blotting, Western; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis; D002467:Cell Nucleus / Q000201:enzymology; D003110:Colonic Neoplasms / Q000276:immunology* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000096:biosynthesis; D005434:Flow Cytometry; D017930:Genes, Reporter; D006801:Humans; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000494:pharmacology*; D019169:Jurkat Cells; D010455:Peptides / Q000276:immunology; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects; D019014:fas Receptor / Q000096:biosynthesis*; D018631:ras Proteins / Q000378:metabolism
D004792:Enzyme Precursors; D010455:Peptides; D019014:fas Receptor; D007371:Interferon-gamma; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D018631:ras Proteins