RESUMO
Two spontaneous mouse leukemias were adapted to culture. In agreement with most reported observations on spontaneous tumors, injection of irradiated cells of the malignant culture cell lines failed to protect mice against these leukemias. These cell lines were treated in vitro with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and stable immunogenic variants (tum-) were obtained, that failed to form progressive tumors in syngeneic CBA/Ht mice. Mice that had rejected tum- variants showed a significant degree of resistance to challenge not only with the original malignant cell line but also with the original transplantable tumor. No protection was observed against syngeneic tumor cells other than those of the parental tumor. These results indicate that these two spontaneous leukemias carry a specific transplantation antigen that can be the target of a rejection response by syngeneic mice. In confirmation of this, we found that lymphocytes of CBA/Ht mice that had rejected tum- variants could be restimulated in vitro so as to develop a cytolytic activity directed against an antigen that was specific for the original tumor cell line.
Assuntos
Leucemia Experimental/imunologia , Mutação , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular , Células Clonais/imunologia , Feminino , Leucemia Experimental/genética , Masculino , Metilnitronitrosoguanidina/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Transplante de Neoplasias , Linfócitos T Citotóxicos/imunologiaRESUMO
Tumor cell variants that were rejected by syngeneic mice (tum-) were obtained from mastocytoma P815 by mutagenesis (as described in the accompanying report (13). A considerable T lymphocyte-mediated lysis was observed upon incubation of these tum- variants with peritoneal exudate cells collected a few days after an intraperitoneal challenge of immune animals. Spleen cells from these animals were cytolytic after stimulation in vitro with the immunizing variant. New antigens, absent from the original P815 tum+ cells, were detected on 15 of the 21 tum- variants that were tested. All these antigens appeared to be different. No new antigen was detected on any of 10 mutagenized P815 clones that had retained their ability to form tumors. We compared the evidence obtained in vivo and in vitro for the presence of specific antigens on five tum- variants. Three variants were shown both in vivo and in vitro to carry an individual antigen. One showed no specificity either in vivo or in vitro. However, for one variant, no specificity was observed in vivo, although cytolysis tests demonstrated the existence of a singular antigenic specificity.
Assuntos
Citotoxicidade Imunológica , Variação Genética , Sarcoma de Mastócitos/imunologia , Mutação , Sarcoma Experimental/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Superfície/genética , Rejeição de Enxerto , Camundongos , Camundongos Endogâmicos DBA , Transplante de Neoplasias , Transplante IsogênicoRESUMO
We showed previously that mouse mastocytoma P815 expresses several distinct antigens that are recognized by cytolytic T lymphocytes (CTL) of syngeneic DBA/2 mice. Antigens P815A and P815B are usually lost jointly and are targets for immune rejection responses in vivo. We used a cosmid library and a CTL stimulation assay to obtain transfectants expressing tumor rejection antigen P815A. From these transfectants we retrieved gene P1A which transferred the expression of both P815A and B. This gene is unrelated to three previously isolated genes coding for tum-antigens. It encodes a putative protein of 224 amino acids which contains two highly acidic domains showing homology with similar regions of nuclear proteins. The P1A gene expressed by tumor P815 is completely identical to the gene present in normal DBA/2 cells. Expression of the gene was tested by Northern blots. Cells from liver, spleen, and a number of mast cell lines were negative, but mast cell line L138.8A produced a high level of P1A message and was lysed by CTL directed against antigens P815A and B. We conclude that major tumor rejection antigens of P815 are encoded by a gene showing little or no expression in most normal cells of adult mice.
Assuntos
Antígenos de Neoplasias/genética , Sarcoma de Mastócitos/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Sequência de Bases , Northern Blotting , Clonagem Molecular , Citotoxicidade Imunológica , Expressão Gênica , Genes , Antígenos H-2/imunologia , Imunidade Celular , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Mapeamento por Restrição , Células Tumorais Cultivadas/imunologiaRESUMO
Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.
Assuntos
Antígenos de Neoplasias/genética , Antígeno HLA-A2/imunologia , Melanoma/imunologia , Monofenol Mono-Oxigenase/genética , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular , Células Clonais , Clonagem Molecular , DNA , Feminino , Humanos , Melanoma/patologia , Monofenol Mono-Oxigenase/imunologia , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Mutagen treatment of mouse tumor cell line P815 produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These tum- antigens are recognized by cytotoxic T lymphocytes (CTL) but induce no detectable antibody response. By transfecting P815 cell line P1.HTR with DNA of tum- variant P198, we obtained transfectants expressing tum- antigen P198 that could be identified on the basis of their ability to stimulate anti-P198 CTL. This was repeated with DNA of a cosmid library derived from variant P198, and a cosmid carrying the sequence encoding antigen P198 was recovered from a transfectant. Gene P198 is 3 kb long and contains eight exons. It shows no homology with previously identified tum- gene P91A, nor with any gene presently recorded in the data banks. The long open reading frame codes for a 23.5-kD protein. The antigenic allele of gene P198 differs from the normal allele by a point mutation located in exon 7. This mutation causes an Ala to Thr change, and was shown by site-directed mutagenesis to be responsible for the expression of the antigen. An 11-amino acid synthetic peptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P198 CTL. The homologous peptide corresponding to the normal sequence of the gene did not, but it was able to compete for binding to major histocompatibility complex molecule Kd. We conclude that tum- mutation P198 generates a new epitope recognized by syngeneic T cells. As observed with gene P91A, we found that a fragment of gene P198 that contained only exons 3-7, cloned in nonexpression vectors, transferred efficiently the expression of the antigen.
Assuntos
Antígenos de Histocompatibilidade/genética , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cosmídeos , Epitopos/imunologia , Expressão Gênica , Antígenos de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/imunologia , Mapeamento por Restrição , Linfócitos T Citotóxicos/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
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.
Assuntos
Antígenos de Diferenciação/genética , Antígenos de Neoplasias/genética , Antígeno HLA-A2/imunologia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/análise , Expressão Gênica , Humanos , Melanoma/genética , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Células Tumorais CultivadasRESUMO
We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.
Assuntos
Antígenos de Neoplasias/genética , Antígeno HLA-A1/imunologia , Melanoma/imunologia , Oligopeptídeos/análise , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência MolecularRESUMO
T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.
Assuntos
Antígeno HLA-A2 , Melanoma/imunologia , Proteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/imunologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Antígenos de Neoplasias/imunologia , Asparagina/metabolismo , Ácido Aspártico/biossíntese , Células Clonais , Epitopos , Humanos , Melanoma/enzimologia , Modelos Biológicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
A cytolytic T lymphocyte (CTL) clone that lyses many HLA-A2 melanomas was derived from a population of tumor-infiltrating lymphocytes of an HLA-A2 melanoma patient. The gene coding for the antigen recognized by this CTL was identified by transfection of a cDNA library. It is the gene which has been reported to code for N-acetylglucosaminyltransferase V (GnT-V). Remarkably, the antigenic peptide recognized by the CTL is encoded by a sequence located in an intron. In contrast to the fully spliced GnT-V mRNA, which was found in a wide range of normal and tumoral tissues, the mRNA containing the intron region coding for the antigen was not found at a significant level in normal tissues. This mRNA was observed to be present in about 50% of melanomas. Our results suggest that a promoter located near the end of the relevant intron is activated in melanoma cells, resulting in the production of an mRNA coding for the antigen.
Assuntos
Antígenos de Neoplasias/genética , Antígeno HLA-A2/imunologia , Íntrons , Linfócitos do Interstício Tumoral/imunologia , Melanoma/genética , Melanoma/imunologia , N-Acetilglucosaminiltransferases/genética , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Antígenos de Neoplasias/biossíntese , Sequência de Bases , Técnicas de Cocultura , Citotoxicidade Imunológica , Primers do DNA , DNA Complementar , Feminino , Expressão Gênica , Biblioteca Gênica , Humanos , Masculino , Melanoma/enzimologia , Dados de Sequência Molecular , Neoplasias/enzimologia , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Técnica de Amplificação ao Acaso de DNA Polimórfico , Mapeamento por Restrição , Transfecção , Células Tumorais CultivadasRESUMO
Primed syngeneic or unprimed allogeneic mouse spleen cells were stimulated with azaguanine-resistant P815 tumor cells that were killed by the addition of aminopterin to the stimulation medium. The recovery of lymphocytes and their cytolytic activity and specificity were similar to those obtained after stimulation with irradiated cells. This method conveniently replaces the inactivation of stimulatory cells by irradiation or mitomycin treatment. Moreover, it has the advantage of inactivating not only the stimulatory cells but also the tumor cells that often contaminate the spleens of tumor-bearing animals, provided these animals have been inoculated with azaguanine-resistant tumor cell mutants.
Assuntos
Azaguanina/farmacologia , Citotoxicidade Imunológica , Sarcoma de Mastócitos/imunologia , Linfócitos T/imunologia , Aminopterina/farmacologia , Animais , Transformação Celular Neoplásica , Meios de Cultura , Hipoxantinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mutação , Timidina/farmacologiaAssuntos
Antígenos de Neoplasias/fisiologia , Antígenos de Histocompatibilidade/fisiologia , Monitorização Imunológica , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Clonagem Molecular , Antígenos de Histocompatibilidade/genética , Tolerância Imunológica , Camundongos , Dados de Sequência Molecular , Neoplasias Experimentais/imunologiaRESUMO
Several independent groups have developed methods aimed at identifying antigens that are expressed on human tumors and can be recognized by T lymphocytes. The positively identified antigens can be presently classified as follows: 1) antigens derived from genes whose functions are still unknown that are expressed in significant proportions of tumors of various histologic types but not expressed in most normal tissues, 2) differentiation antigens expressed in melanomas, 3) mucins, 4) viral antigens, and 5) products of abnormal genes expressed in tumors. These results are presented with explanations about the methodologies used to identify these antigens in the perspective of their use in immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Antígenos Virais/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Neoplasias/microbiologia , Linfócitos T/microbiologiaRESUMO
It is now widely accepted that cytolytic T cells recognize their antigens in the form of small peptides bound to major histocompatibility complex molecules at the surface of the target cells. We present here the hypothesis that, when these antigenic peptides are derived from the cellular genome, they are not degradation products of cellular proteins but can be generated directly by the autonomous transcription and translation of short subgenic regions that we propose to name "peptons". We discuss some consequences of the notion that antigenic peptides can be produced in the absence of synthesis of messenger RNA and protein from the corresponding genes.
Assuntos
Antígenos/genética , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos/imunologia , Modelos Biológicos , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Transcrição GênicaRESUMO
We reported previously that, by mutagenesis of a malignant teratocarcinoma cell line, it is possible to obtain a number of variant clones that are incapable of forming progressive tumors. Each of these "tum-" variants is rejected in syngeneic mice and stimulates the production of immune memory cells (self-protection). We show here that four different tum- clones confer an immune protection against each other although this cross-protection is invariably weaker than the self-protection. Moreover, mice immunized with living tum- cells are partially protected against the original malignant teratocarcinoma cells, even though the latter cells are incapable of conferring any immune protection when injected after being killed by irradiation. These results indicate that each tum- variant carries at least one specific transplantation antigen that is absent from the original tumor cell line and from most other tum- variants. Other tumor-specific transplantation antigens are probably present on all the tum- variants and also on the malignant teratocarcinoma cell line.
Assuntos
Imunização , Teratoma/imunologia , Animais , Linhagem Celular , Reações Cruzadas , Rejeição de Enxerto , Camundongos , Camundongos Endogâmicos , Mutação , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Teratoma/genética , Transplante IsogênicoRESUMO
The nonimmunogenic thymic leukemia TH, obtained in mouse strain CBA/Ht, was adapted to culture. By in vitro treatment of a clonal TH cell line with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, stable variant cell clones (tum-) were obtained that elicited a rejection response in syngeneic mice. Mice that had rejected a tum- variant were partially protected against a challenge with the original tumor. When spleen cells of these animals were restimulated in vitro, cytolytic T cells were obtained that were directed against an antigen present on the original tumor. The existence of these cytolytic effectors was confirmed by clonal analysis of the cytolytic response. No immune protection against TH was observed in mice that had been immunized with irradiated cells of the original TH tumor. These results confirm that tum- variants can elicit a syngeneic rejection response against tumors that are apparently devoid of transplantation immunogenicity. The experimental conditions and the results make it likely but not certain that the tumor-associated antigen detected on leukemia TH was present on the primary tumor.
Assuntos
Leucemia Experimental/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular , Citotoxicidade Imunológica , Feminino , Tolerância Imunológica , Imunização , Leucemia Experimental/genética , Masculino , Camundongos , Camundongos Endogâmicos CBA , Mutação , Baço/imunologia , Linfócitos T/imunologiaRESUMO
A tk- cell line derived from mouse mastocytoma P815 was transfected with a plasmid carrying a thymidine kinase gene. The tk+ cells were obtained at a frequency of 10(-6). From some of these tk+ transfectants it was possible to select tk- cells with BrdU so that these cells could be submitted again to transfection with the thymidine kinase gene. By repeating cycles of transfection and tk+ selection followed by reverse selection with BrdU, it was possible to obtain a stable variant having a 100-fold increase in transfection efficiency. This HTR (high transfection) variant shows a high efficiency of transfection (10(-4)) for the neomycin-resistance gene as well as for the thymidine kinase gene.
Assuntos
Clonagem Molecular , Genes , Transfecção , Animais , Linhagem Celular , DNA/genética , Resistência a Medicamentos , Eletroforese em Gel de Ágar , Marcadores Genéticos , Sarcoma de Mastócitos/genética , Camundongos , Camundongos Endogâmicos DBA , Neomicina/farmacologia , Hibridização de Ácido Nucleico , Plasmídeos , Seleção Genética , Timidina Quinase/genéticaRESUMO
HLA-Cw*05 is one of the least polymorphic subgroups of HLA-C; so far only two alleles, namely Cw*0501 and Cw*0502, have been reported. We report here the identification of a third allele, Cw*0503, in a Caucasian individual. Cw*0503 is closely related to Cw*0501 with only six nucleotide substitutions clustering over a fragment of 48 nucleotides at the beginning of exon 4. All these six substitutions at the same positions have been found only in HLA-B*44 alleles, suggesting that Cw*0503 is a result of recombination between Cw*0501 and one of B*44 alleles.
Assuntos
Antígenos HLA-C/genética , Polimorfismo Genético , Alelos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Éxons , Humanos , Melanoma/genética , Melanoma/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Linfócitos T Citotóxicos/imunologiaRESUMO
As the result of P1-mediated cotransduction with serB from Salmonella potsdam to the Escherichia coli/Salmonella typhimurium hybird 4617, one recombinant, L4004, was isolated which had a restriction-modification (R--M) system different from the SB and SP systems of its parents, and was designated SQ. The genes of SQ were allelic to those of the SB system of S. typhimurium and were shown by complementation experiments to be functionally related to those of the K system of E. coli. Evidence that the SQ system in L4004 arose as the result of a recombination event within the hsdS genes of SB and SP is discussed.
Assuntos
DNA Bacteriano/metabolismo , Genes , Recombinação Genética , Salmonella typhimurium/metabolismo , Salmonella/metabolismo , Alelos , Enzimas de Restrição do DNA/biossíntese , Escherichia coli/metabolismo , Mutação , Transdução GenéticaRESUMO
It has been reported that, by mutagenesis of a malignant mouse teratocarcinoma cell line, it is possible to obtain cell variants that are incapable of forming progressive tumors in syngeneic mice. These variants, which were called "tum-," are eliminated from the host by an immune rejection process. We report here that similar variant cell clones can be obtained at high frequency from a Lewis lung carcinoma cell line treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Syngeneic C57BL/6 mice reject these tum- clones and acquire a strong radioresistant immune protection against the immunizing clone. When the challenging tum- clone differs from the immunizing clone, a weaker radioresistant immune protection can be demonstrated with some, but not all, combinations. All the tum- clones induce a significant protection against the original Lewis lung malignant cells. These results imply that each Lewis lung tum- variant carries on its surface a singular antigen in addition to one or more weak antigens already present on the original tumor cell line. This antigenic pattern is similar to that found on teratocarcinoma tum- variants. Our results suggest that the procedure of using a mutagen in order to generate tum- variants carrying new transplantation antigens may be generally applicable to cancer cells.
Assuntos
Variação Genética , Neoplasias Pulmonares/imunologia , Animais , Linhagem Celular , Metilnitronitrosoguanidina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação/efeitos dos fármacos , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Baço/imunologia , Baço/transplante , Transplante IsogênicoRESUMO
Immunogenic tumor cell variant P35 was obtained by mutagen treatment of mouse mastocytoma P815. It expresses a potent new antigen recognized by syngeneic cytolytic T lymphocytes (CTL). This antigen is the result of a point mutation in a gene that is expressed by most healthy cells. A decapeptide encoded by the region spanning the mutation sensitized P815 cells to the relevant CTL, whereas the homologous decapeptide corresponding to the normal sequence did not. Only the mutant decapeptide was capable of enhancing the expression of the Dd-presenting molecule at the cell surface, indicating that the mutation generates a motif which enables the antigenic peptide to bind to Dd.