A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma - PubMed (original) (raw)

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

S Mandruzzato et al. J Exp Med. 1997.

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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Figures

Figure 1

Figure 1

Specific lysis of the autologous tumor cells by CTL 121. Squamous cell carcinoma line of patient BB49 (BB49-SCCHN), autologous EBV-transformed B cell line (BB49-EBV), and autologous PHA-treated blood lymphocytes (BB49-PBL) were used as 51Cr-labeled target cells in the presence of a 50-fold excess of unlabeled K562. Chromium release was measured after 4 h.

Figure 2

Figure 2

Recognition of BB49-SCCHN by CTL 121 is inhibited by anti-HLA-B,C mAb. 1,500 CTL were incubated with 20,000 BB49-SCCHN in microwells for 4 h without mAb (medium) or in the presence of either mAb W6/32 (_anti_-HLA–class I), B1.23.2 (anti-HLA-B,C), BB7-2 (anti-HLA-A2), or 2B6 (_anti_-_HLA_-DR). TNF production was estimated by testing the toxicity of the supernatant for TNF-sensitive WEHI-164.13 cells.

Figure 3

Figure 3

Stimulation of CTL 121 by COS-7 cells transiently cotransfected with cDNA clone 668 and with a sequence encoding an HLA-B35 molecule. 1,500 CTL were added into microwells containing 10,000 BB49-SCCHN or 20,000 transfected COS-7 cells. The production of TNF was measured after 24 h of coculture by testing the toxicity of the supernatants for the TNF-sensitive WEHI-164.13 cells. (A) COS-7 cells were cotransfected with cDNA 668 and a cDNA coding for HLA-B35 obtained from the RNA of patient LB1047. The autologous BB49-SCCHN cells were used as a positive control for stimulation. COS-7 cells, cotransfected with HLA-B35 and nonrelevant cDNA clone F10, were used as a negative control. (B) COS-7 cells were cotransfected with cDNA 668 and either a cDNA coding for the autologous HLA-B*3503 molecule or with a cDNA coding for the allogeneic HLA-B*3501 molecule. Stimulator cells included the autologous BB49-SCCHN cells as a positive control and COS-7 cells transfected with either cDNA 668, the HLA-B*3503 cDNA, or the HLA-B*3501 cDNA alone as negative controls.

Figure 4

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.

Figure 5

Figure 5

Sequence of the open reading frame and of the putative protein encoded by cDNA 668. The nucleotides are numbered starting from nucleotide 70 of the cDNA 668. The translated Alu sequences are underlined. The sequence corresponding to the peptide recognized by CTL 121 is boxed. For _MACHα_1 and FLICE, only the stop codon TGA and the differences in nucleotides are indicated. There are two single nucleotide differences with FLICE (This sequence data is available from EMBL/GenBank/DDBJ under accession number U58143), giving rise to two different amino acids, and a single conservative difference with _MACHα_1 (This sequence data is available from EMBL/GenBank/DDBJ under accession number X98172). The 5′-untranslated region of cDNA 668 is identical to that of _MACHα_2. Therefore, compared with the cDNA sequences of CASP-8 already described, cDNA 668 is representative of a new type of transcript.

Figure 6

Figure 6

Autoradiography showing the sequence ladder in the region coding for the antigenic peptide in the squamous cell carcinoma line of patient BB49 (BB49-SCCHN), autologous PHA-treated blood lymphocytes (BB49-PBL), autologous EBV-transformed B cell line (BB49-EBV), and the tumor sample resected from patient BB49. (A) PCR products were obtained by amplification with specific primers on genomic DNA. (B) The RT-PCR product was obtained by amplification with specific primers on RNA extracted from the tumor mass resected from patient BB49. The PCR fragments were purified and directly sequenced. The arrow indicates the mutation G to C in one of the alleles.

Figure 7

Figure 7

Triggering of apoptosis in cells transiently transfected with cDNA 668 or the equivalent wild-type sequence, cDNA 668-wt. 293-EBNA cells were transiently transfected by lipofectAMINE. Transfection marker β-galactosidase, inserted into expression vector pcDNA3, was cotransfected with expression vector pcDNAI/Amp either empty, containing cDNA 668, or containing cDNA 668-wt (wt for wild type). cDNA 668-wt was obtained by site-directed mutagenesis performed on cDNA 668, in which the TCA codon was replaced by stop codon TGA. The experiment was performed in triplicate in two different microplates. (A) After 18 h, cells were fixed in one plate and stained with X-gal. A total number of 300 blue cells were counted. The x-axis gives the percentage of blue apoptotic cells. (B) In the other plate, the extent of cell death was quantitate using an ELISA-based assay in which nucleosomes in the supernatant of the transfected cells are purified by a histone-specific antibody and detected photometrically with an anti-DNA antibody coupled to peroxidase.

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