ABSTRACT
S100A4 (metastasin 1) belongs to the S100 family of Ca(2+) binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-gamma ELISPOT and cytotoxicity assays. In addition, IFN-gamma responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1(+) fibroblasts in comparison to HLA-A1(-) fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.
Subject(s)
Epitopes/immunology , HLA-A1 Antigen/immunology , Melanoma/immunology , Peptide Fragments/immunology , S100 Proteins/immunology , Skin Neoplasms/immunology , Amino Acid Sequence , Cell Line, Tumor , Cells, Cultured , Epitopes/metabolism , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Melanoma/pathology , Molecular Sequence Data , S100 Calcium-Binding Protein A4 , Sequence Alignment , Skin Neoplasms/pathology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
In an attempt to trigger increased mucosal secretory immune responses against bacterial surface antigens, we constructed an optimized human interleukin (hIL)-6-secreting Salmonella typhimurium strain (X4064(pCH1A+pYL3E)), utilizing the hemolysin (Hly) exporter for secretory delivery of a functional hIL-6-hemolysin fusion protein (hIL-6-HlyA(s)). Through stable introduction of a second hIL-6-HlyA(s) expression plasmid (pYL3E) in the previously described X4064(pCH1A) strain, hIL-6-HlyA(s) secretion efficiencies were increased by at least 10-fold. As pCH1A in the parental strain, pYL3E was stable in vitro in the absence of antibiotic selection and in vivo neither did plasmids interfere in their stabilities. Increased hIL-6-HlyA(s) expression did not adversely interfere with bacterial growth. Comparative immunization experiments in mice with oral application of the different hIL-6-secreting strains revealed that increased in situ hIL-6-production influenced systemic antibody responses against Salmonella antigens but had no marked effect on mucosal responses. In mice immunized with X4064(pCH1A+pYL3E) significantly higher sera IgG and IgA titers for lipopolysaccharide (LPS) were found compared to mice immunized with X4064(pCH1A) and a hIL-6-negative control strain. Higher sera antibody titers were accompanied by increased numbers of IgG- and IgA-specific antibody-secreting cells in spleens and Peyer's patches, respectively. These data suggest that systemic antibody responses against Salmonella LPS are largely effected by IL-6 and, moreover, the amount and the cellular location of recombinantly expressed IL-6 appears to be crucial for enhancement of immune responses.
