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A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma.

Mandruzzato S, Brasseur F, Andry G, Boon T, van der Bruggen P - J. Exp. Med. (1997)

Bottom Line: The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8.The antigenic peptide is a nonamer presented by HLA-B*3503.The five last amino acids are encoded by the extension of the reading frame caused by the mutation.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Brussels Branch, and Unité de Génétique Cellulaire, Université Catholique de Louvain, B-1200 Brussels, Belgium.

ABSTRACT
Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and beta-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.

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Related in: MedlinePlus

Lysis by CTL 121 of autologous EBV-transformed B cell line  (BB49-EBV) incubated with synthetic peptide FPSDSWCYF. 1,000  chromium-labeled BB49-EBV cells were incubated at room temperature  for 30 min with the indicated peptides at various concentrations, before  adding an equal volume containing 10,000 CTL 121 and 50,000 unlabeled K562. Chromium release was measured after 4 h.
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Figure 4: Lysis by CTL 121 of autologous EBV-transformed B cell line (BB49-EBV) incubated with synthetic peptide FPSDSWCYF. 1,000 chromium-labeled BB49-EBV cells were incubated at room temperature for 30 min with the indicated peptides at various concentrations, before adding an equal volume containing 10,000 CTL 121 and 50,000 unlabeled K562. Chromium release was measured after 4 h.

Mentions: cDNA 668 is 2647 bp long. To identify the region encoding the peptide presented to CTL 121, exonuclease III was used to generate progressive deletions from the 3′ end of the cDNA. A large number of truncated cDNA clones were cotransfected into COS-7 cells together with the HLA-B*3503 cDNA to test their ability to code for the antigen. Sequence comparison of the longest negative cDNA clone (nucleotides 1–1,446) with the shortest positive clone (nucleotides 1–1,600) indicated that the COOH-terminus of the antigenic peptide had to be encoded between nucleotides 1,446 and 1,600. In this region of the putative protein, we searched for peptides bearing the HLA-B35 binding motif, i.e., Pro in position 2, and Tyr or hydrophobic residues at the COOH terminus of the peptide (33, 34). Two peptides corresponding to this motif were identified, namely nonapeptide FPSDSWCYF (amino acids 476–484) and octapeptide FPSDSWCY (amino acids 476–483). They were synthesized and tested for recognition. Only nonapeptide FPSDSWCYF sensitized BB49-EBV to lysis by CTL 121. Half-maximal lysis was obtained with only 3 nM of peptide (Fig. 4).


A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma.

Mandruzzato S, Brasseur F, Andry G, Boon T, van der Bruggen P - J. Exp. Med. (1997)

Lysis by CTL 121 of autologous EBV-transformed B cell line  (BB49-EBV) incubated with synthetic peptide FPSDSWCYF. 1,000  chromium-labeled BB49-EBV cells were incubated at room temperature  for 30 min with the indicated peptides at various concentrations, before  adding an equal volume containing 10,000 CTL 121 and 50,000 unlabeled K562. Chromium release was measured after 4 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199018&req=5

Figure 4: Lysis by CTL 121 of autologous EBV-transformed B cell line (BB49-EBV) incubated with synthetic peptide FPSDSWCYF. 1,000 chromium-labeled BB49-EBV cells were incubated at room temperature for 30 min with the indicated peptides at various concentrations, before adding an equal volume containing 10,000 CTL 121 and 50,000 unlabeled K562. Chromium release was measured after 4 h.
Mentions: cDNA 668 is 2647 bp long. To identify the region encoding the peptide presented to CTL 121, exonuclease III was used to generate progressive deletions from the 3′ end of the cDNA. A large number of truncated cDNA clones were cotransfected into COS-7 cells together with the HLA-B*3503 cDNA to test their ability to code for the antigen. Sequence comparison of the longest negative cDNA clone (nucleotides 1–1,446) with the shortest positive clone (nucleotides 1–1,600) indicated that the COOH-terminus of the antigenic peptide had to be encoded between nucleotides 1,446 and 1,600. In this region of the putative protein, we searched for peptides bearing the HLA-B35 binding motif, i.e., Pro in position 2, and Tyr or hydrophobic residues at the COOH terminus of the peptide (33, 34). Two peptides corresponding to this motif were identified, namely nonapeptide FPSDSWCYF (amino acids 476–484) and octapeptide FPSDSWCY (amino acids 476–483). They were synthesized and tested for recognition. Only nonapeptide FPSDSWCYF sensitized BB49-EBV to lysis by CTL 121. Half-maximal lysis was obtained with only 3 nM of peptide (Fig. 4).

Bottom Line: The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8.The antigenic peptide is a nonamer presented by HLA-B*3503.The five last amino acids are encoded by the extension of the reading frame caused by the mutation.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Brussels Branch, and Unité de Génétique Cellulaire, Université Catholique de Louvain, B-1200 Brussels, Belgium.

ABSTRACT
Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and beta-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.

Show MeSH
Related in: MedlinePlus