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T Boon

Publications and source records attributed to T Boon.

At least 109 records · Page 6Linked to original sources
PubMed1995

Structure, chromosomal location, and expression pattern of three mouse genes homologous to the human MAGE genes.

The human MAGE1 gene directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. MAGE1 belongs to a family of genes that are expressed in a number of tumors of various histological types but not in normal tissues except testis. The MAGE genes are arranged in two groups that are located within two different regions of the human X chromosome (Xq26-qter and Xp21.3). By hybridizing mouse genomic libraries with a MAGE1 probe, we identified three homologous genes. Two of these mouse genes, Smage1 and Smage2, are more than 99% identical to each other and encode the same protein of 330 aa. The 5' noncoding region of Smage2 provides the potential for regulating the expression of the gene through several different promoters located in front of alternative first exons. The third gene, Smage3, has the structure of a processed transcript. It codes for a protein with only 11 aa substitutions with respect to the Smage1/2 product. Somatic cell hybrids and interspecific backcross analysis showed that Smage3 is autosomal and that Smage1 and Smage2 are located between the Dmd and the Ar loci on the mouse X chromosome. Since this region is syntenic to the human Xp21.1-p22.1 region, we conclude that Smage1 and Smage2 are homologous to the MAGE-Xp rather than to the MAGE-Xq genes. Smage1/2 transcripts were detected in several tumor and embryonal cell lines but not in normal mouse tissues with the exception of testis. Expression of Smage3 was found in embryos from Day 11 to Day 15.

Amino Acid Sequence↗
PubMed1995

Costimulation with B7-1, IL-6, and IL-12 is sufficient for primary generation of murine antitumor cytolytic T lymphocytes in vitro.

The requirements for generating CD8+ CTLs against the mastocytoma P815 were first defined by using an allogeneic mixed lymphocyte tumor culture (MLTC). Both the expansion of effector lymphocytes and the acquisition of lytic activity were dependent on the presence of accessory cells, but not CD4+ lymphocytes. Several factors were examined for their ability to replace accessory cell function. Expression of B7-1 by P815 was sufficient to induce IL-2 production by CD8+ cells, but substantial proliferation was achieved only if IL-6 was provided as well. Although IL-12 had little effect on the net proliferation of developing effector cells, it increased specific lytic activity 10-fold, acted synergistically with B7-1 in induction of IFN-gamma production during primary stimulation, and resulted in a shift toward a Th1 cytokine profile following secondary stimulation. These costimulatory factors were then studied in a primary syngeneic MLTC by using splenocytes from nonimmunized DBA/2 mice, an approach that had never before succeeded in generating specific CTLs. The combination of B7-1, IL-6, and IL-12 was sufficient to induce P815-specific CTL activity after a 5-day MLTC, which was expanded optimally following secondary stimulation in the presence of B7-1, IL-2, and IL-7. The irrelevant syngeneic tumor L1210, constructed to express B7-1 and the P815-derived tumor Ag gene P1A, also generated CTLs that lysed the parental P815. The combination of B7-1, IL-6, and IL-12 should be useful in the derivation of other tumor-specific CTLs in vitro, and may constitute the optimal stimuli for rapid proliferation and differentiation of helper-independent, tumor-specific CTLs in vivo.

Animals↗
PubMed1995

Differences in the antigens recognized by cytolytic T cells on two successive metastases of a melanoma patient are consistent with immune selection.

We have studied the patterns of antigens recognized by autologous cytolytic T lymphocytes (CTL) on two melanoma cell lines derived from metastases that were removed from patient LB33 at several years distance. Cell line LB33-MEL.A was obtained after surgery in 1988. A large number of CTL clones directed against LB33-MEL.A was obtained with blood lymphocytes collected from the patient in 1990. In vitro selection of melanoma cells that were resistant to these CTL clones indicated that at least five different antigens were recognized on LB33-MEL.A by autologous CTL. Four of these antigens were found to be presented by HLA-A28, B13, B44 and Cw6, respectively. The patient remained disease-free until 1993 when a metastasis was detected and was used to obtain cell line LB33-MEL.B. This cell line proved resistant to lysis by all the CTL clones directed against the LB33-MEL.A cells and showed no expression of HLA class I molecules except for HLA-A24. Using LB33-MEL.B cells to stimulate blood lymphocytes collected from the patient in 1994 we derived CTL clones that lysed these cells. All these CTL clones recognized a new antigen presented by HLA-A24. These results suggest that in patient LB33 the melanoma cells may have lost the expression of several HLA molecules under the selective pressure of an anti-tumor CTL response.

Adult↗
PubMed1995

Individual differences in the orientation of the cytolytic T cell response against mouse tumor P815.

We reported previously that the mouse tumor P815 expresses four distinct antigens (A, B, C, D) recognized by syngeneic cytolytic T lymphocytes (CTL). A fifth P815 antigen (E) was identified by means of a CTL clone derived from tumor-infiltrating lymphocytes. We compared a number of mice for the orientation of their CTL response with respect to the various P815 antigens. Lymphocytes from mice inoculated subcutaneously with living P815 cells were stimulated in vitro with tumor cells and the resulting CTL were tested against targets expressing either antigens A and B or antigens C, D and E. Many mice had an asymmetrical response, some producing CTL directed almost exclusively against antigens A, B and others producing CTL directed almost exclusively against C, D. E. When mice were inoculated into two separate sites, different orientations in the responses of the two local lymph nodes were often observed, suggesting that individual differences in the orientation of the anti-P815 CTL response do not result from preexisting differences between the animals. Asymmetrical CTL responses persisted in mice that were given a second injection of tumor cells. A possible interpretation of our results is that the major component of the CTL response is made of the progeny of a very small number of CTL precursors that happen to be the first to be stimulated by the tumor antigens.

Amino Acid Sequence↗
PubMed1995

CD8+ cell activation to a major mastocytoma rejection antigen, P815AB: requirement for tum- or helper peptides in priming for skin test reactivity to a P815AB-related peptide.

Delayed-type hypersensitivity (DTH) responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum-) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum- antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum- peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8+ cell-mediated DTH to P815AB was unaffected by the ablation of CD4+ cells, the same treatment, or neutralization of IFN-gamma, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4+ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum- cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.

Amino Acid Sequence↗
PubMed1995

Involvement of two Ets binding sites in the transcriptional activation of the MAGE1 gene.

The MAGE1 gene codes for an antigen recognized on melanoma cell line MZ2-MEL by autologous cytolytic T lymphocytes. It is expressed in a number of tumors of different histological origins, but not in normal tissues except in testis. The MAGE1 promoter region was analyzed by performing transient transfections in MZ2-MEL cells with luciferase reporter plasmids. A fragment extending from nucleotide -792 to +118 exhibited high transcriptional activity. By deletional analysis of this fragment, we identified five activating regions designated C, A, B', B, and D. The activity of region A depends on the presence of region B' and vice versa. Two inverted Ets motifs contained in regions B' and B were found to drive 90% of the activity of the MAGE1 promoter in MZ2-MEL cells. Electrophoretic mobility shift assays performed with a nuclear extract from MZ2-MEL cells and with competitor oligonucleotides containing an Ets consensus site showed that nuclear proteins bind to the Ets motif of regions B' and B. Similar experiments suggested that region A binds transcription factors of the Sp1 family. The MAGE1 promoter was found to exert transcriptional activity in tumor cells where the MAGE1 gene is not expressed, suggesting that other mechanisms, such as demethylation, may contribute to the tumor-specific expression of the gene.

Antigens, Neoplasm↗
PubMed1995

BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes.

Several tumor antigens are recognized by autologous cytolytic T lymphocytes (CTL) on human melanoma MZ2-MEL. Some of them are encoded by genes MAGE-1 and MAGE-3, which are not expressed in normal tissues except in testis. Here, we report the identification of a new gene that codes for another of these antigens. This gene, named BAGE, codes for a putative protein of 43 aa and seems to belong to a family of several genes. The antigen recognized by the autologous CTL consists of BAGE-encoded peptide AARAVFLAL bound to an HLA-Cw 1601 molecule. Gene BAGE is expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas, and 6% of non-small cell lung carcinomas. Like the MAGE genes, it is silent in normal tissues with the exception of testis. Because of its tumor-specific expression, the BAGE-encoded antigen may prove useful for cancer immunotherapy.

Amino Acid Sequence↗
PubMed1994

A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.

It has been reported previously that antitumor cytolytic T lymphocyte (CTL) clones can be isolated from blood lymphocytes of HLA-A2 melanoma patients, after stimulation in vitro with autologous tumor cells, and that some of these CTL clones lyse most HLA-A2 melanomas. A first antigen recognized by such CTL clones was previously shown to be encoded by the tyrosinase gene. We report here the identification of another gene that also directs the expression of an antigen recognized on most melanomas by CTL clones that are restricted by HLA-A2. The gene, designated Melan-A, is unrelated to any known gene. It is 18 kb long and comprises five exons. Like the tyrosinase gene, it is expressed in most melanoma tumor samples and, among normal cells, only in melanocytes.

Amino Acid Sequence↗
PubMed1994

Expression of mage genes by non-small-cell lung carcinomas.

Human gene MAGE-I codes for an antigen that is recognized on melanoma cells by autologous cytolytic T lymphocytes (CTL). This antigen is potentially useful as a target for cancer immunotherapy because gene MAGE-I is not expressed in any normal tissues except the testis. We tested 46 surgical samples of non-small-cell lung carcinomas and observed MAGE-I expression in 16 of them (35%). Genes MAGE-2 and 3, which are closely related to MAGE-I, were expressed by a similar proportion of these tumors. Some small-cell lung tumors also express MAGE genes. The proportion of tumors expressing MAGE-I suggests that lung tumor patients may constitute the largest group of patients potentially eligible for pilot studies involving MAGE-I immunization.

Base Sequence↗
PubMed1994

Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.

Adult↗
PubMed1994

Identification of the MAGE-1 gene product by monoclonal and polyclonal antibodies.

The human MAGE-1 gene encodes a melanoma peptide antigen recognized by autologous cytotoxic T lymphocytes. To produce antibodies against the MAGE-1 gene product, several approaches were taken. Three oligopeptides were synthesized based on predicted MAGE-1 amino acid sequences and were used to generate rabbit anti-peptide anti-sera. In addition, a truncated MAGE-1 cDNA was cloned into an Escherichia coli expression vector, and recombinant protein was produced and purified. All three rabbit anti-peptide antisera showed reactivity against the immunizing peptide, and one reacted with the recombinant MAGE-1 protein by immunoblotting, but none reacted with cell lysates from MAGE-1 mRNA-positive cells. The recombinant MAGE-1 protein was then used for the generation of mouse monoclonal and rabbit polyclonal antibodies. One IgG1 monoclonal antibody, MA454, as well as rabbit polyclonal antisera recognized a 46-kDa protein in extracts of MAGE-1 mRNA-positive melanoma cell lines. The antibodies showed no apparent cross-reactivity with products of the closely related MAGE-2 and MAGE-3 genes. Serological typing of normal and tumor cell lysates was in full agreement with mRNA analysis, showing expression of MAGE-1 protein in MAGE-1 mRNA-positive testis and a subset of melanomas but not in MAGE-1 mRNA-negative normal or tumor tissues. Transfection of the MAGE-1 gene into a MAGE-1 mRNA-negative melanoma cell line resulted in the expression of the 46-kDa protein, confirming the identity of this protein as the MAGE-1 gene product.

Amino Acid Sequence↗
PubMed1994

Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes.

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368-376. Both peptides contain consensus motifs of HLA-A2 binding peptides.

Amino Acid Sequence↗
PubMed1994

Use of a skin test assay to determine tumor-specific CD8+ T cell reactivity.

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum-) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum- peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8+ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2-6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8+ anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.

Animals↗
PubMed1994

Autologous cytolytic T lymphocytes recognize a MAGE-1 nonapeptide on melanomas expressing HLA-Cw*1601.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, which codes for antigen MZ2-E which is presented by HLA-A1. Gene MAGE-1 is expressed in many tumors of several types but not in normal tissues except for testis. We show here that gene MAGE-1 directs the expression of another antigen recognized by CTL on the MZ2-MEL cells. This antigen, which was named MZ2-Bb, consists of MAGE-1-encoded peptide SAYGEPRKL bound to major histocompatibility molecule HLA-Cw*1601. The HLA-Cw*1601 allele was found to be expressed by 7 out of 99 individuals from a Caucasian population. Our results extend the range of tumor patients who could be eligible for immunization against MAGE antigens.

Amino Acid Sequence↗