Context of MUC1 epitope: immunogenicity.

MUC1 is a glycoprotein found at the secretory poles of normal cells but is hypoglycosylated on the entire surface of cell membranes of adenocarcinomas. In order to determine the influence on the immune response of peptide context for epitope presentation, peripheral blood mononuclear cells (PBMC) from patients with adenocarcinomas, were stimulated with MUC1 peptides derived from the 20 amino acids (aa) long sequence that is characteristic of the MUC1 Variable Number of Tandem Repeats (VNTR). In the seven peptides tested, the T-cell tumor-specific epitope (cTSE) was surrounded by variable numbers of aa and repeated up to 5 times in the same peptide. The results of this study indicate that cultures stimulated with peptide 610 (GSTAPPAHGVTS APDTRPAP) showed the highest specific killing of the MUC1-expressing breast cancer MCF-7 cells. Peptide 610 is also superior to the other peptides in inducing better production of the type 1 cytokines, tissue necrosis factor alpha and interferon gamma. In conclusion, context of the epitope and not sequence alone determines immunogenicity.

Cytotoxic T lymphocytes from humans with adenocarcinomas stimulated by native MUC1 mucin and a mucin peptide mutated at a glycosylation site.

MUC1 mucin peptides stimulated cytotoxic T lymphocytes (CTL) from humans with adenocarcinomas. Peripheral blood mononuclear cells, tumor-draining lymph node cells, or tumor-infiltrating lymphocytes were stimulated using mono-nuclear cells from humans with adenocarcinomas of breast or ovary, respectively, using (a) a native MUC1 mucin tandem repeat peptide of 20 amino acids (MUC1-mtr1) plus recombinant human interleukin-2 (IL-2), (b) the mutated (T3N) MUC1-mtr1 plus IL-2, or (c) immobilized anti-CD3 plus IL-2, or (d) IL-2 alone. The CTL stimulated by each of these four conditions were predominately CD4+. However, the CTL stimulated by either the native MUC1-mtr1 or (T3N) MUC1-mtr1 showed 5-10 times greater cytotoxicity of a breast cancer cell line that expresses MUC1 compared to CTL stimulated by either anti-CD3 + IL-2 or IL-2 alone. Each incubation condition generated CTL with different variable beta gene families of T-cell receptors, implying an oligoclonal expansion of a limited CTL repertoire for each. Thus, peptide-stimulated T cells showed expression of cytotoxic cells, which was not induced by nonspecific (anti-CD3 or IL-2) stimulation.

Design and expression of a synthetic mucin gene fragment in Escherichia coli.

Adenocarcinomas of glandular tissues produce a hypoglycosylated form of a normal glycoprotein (mucin) that elicits an immune response. A tumor-specific epitope of mucin occurs in a 20-amino-acid, tandemly repeated domain of human MUC1 mucin. A synthetic gene encoding five tandem repeats of the tumor-specific epitope of human mucin (m5tr) was designed for efficient cloning and expression in Escherichia coli for subsequent use in preparing reagent quantities of the mucin 5 tandem repeat (mtr5) polypeptide. The synthetic gene was cloned in the correct reading frame into the maltose-binding protein (MBP) fusion expression vector pMAL-p2. Bacterial clones containing the mucin synthetic gene (m5tr) were shown to produce the intended recombinant fusion protein, MBP-mtr5. The fusion protein represents a significant fraction of the cell protein, 50% or more of which is secreted into the periplasm. The MBP-mtr5 protein is largely intact and easily prepared in sufficient quantity and purity for preliminary structure-function studies.

Regulation of expression of TL genes of the mouse Mhc.

The expression of thymus leukemia (TL) antigens and genes in thymocytes and activated T cells was examined by immunoprecipitation, flow cytometric, northern, and nuclear run-off transcription analyses. Cell surface forms of TL were detectable by immunoprecipitation on activated peripheral T cells from Tla haplotypes except Tla(b), in agreement with expression observed on thymocytes. Approximately 40%-50% of concanavalin A (Con A) or anti-CD3-activated T cells were TL+, with expression detected on both the CD4 and CD8 subsets by dual-color analysis. Activated T cells expressed detectable levels of TL mRNA 48 h after stimulation, but no TL transcripts were detectable in unstimulated splenocytes. However, TL mRNA expression in mature activated T cells did not precisely mimic thymocyte expression: the level of expression was considerably lower in activated T cells, and in most haplotypes the transcripts produced in activated T cells appeared to represent a subset of the transcripts produced in thymocytes. By run-off transcription assays in isolated nuclei, TL gene expression was detected in activated but not resting T cells indicating that lack of expression of TL in resting T cells is not due to message instability. These data demonstrate that TL genes are inducible and transcriptionally regulated.