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2.
Tissue Antigens ; 62(5): 426-32, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14617050

RESUMO

'Cancer-germline' genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in other normal tissues. They encode shared tumor-specific antigens, which have been used in small therapeutic vaccination trials of cancer patients. Gene MAGE-4, which is expressed in more than 50% of carcinomas of esophagus, head and neck, lung, and bladder, has two known alleles. Using PCR amplifications and digestions of the amplified product, we found that one third of the MAGE-4-positive samples expressed MAGE-4a. We folded HLA-A1 tetramers with peptide MAGE-4a169-177 EVDPASNTY, which is homologous to MAGE-1- and MAGE-3-encoded peptides recognized on HLA-A1 by cytolytic T lymphocytes. Blood lymphocytes from an individual without cancer were directly labelled with these A1/MAGE-4 tetramers. The very rare cells that were stained were sorted by flow cytometry and cloned. We isolated a cytolytic T-lymphocyte clone that lyzed specifically cells pulsed with this MAGE-4 peptide and HLA-A1 tumor cells expressing MAGE-4a, demonstrating that this antigenic peptide is processed efficiently in tumor cells. This peptide might therefore be useful for therapeutic antitumoral vaccination.


Assuntos
Proteínas de Neoplasias/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias , Citometria de Fluxo , Antígeno HLA-A1/imunologia , Humanos , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células Tumorais Cultivadas
3.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 3): 529-30, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11856845

RESUMO

Human interleukin-22, a novel member of the cytokine family, has been crystallized in hanging drops using the vapour-diffusion technique. Preliminary X-ray diffraction experiments using synchrotron radiation reveal that the protein crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.44, b = 61.62, c = 73.43 A, and diffracts beyond 2.00 A resolution.


Assuntos
Interleucinas/química , Cristalização , Cristalografia por Raios X , Humanos , Conformação Proteica , Proteínas Recombinantes/química , Interleucina 22
4.
Proc Natl Acad Sci U S A ; 98(18): 10290-5, 2001 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-11517302

RESUMO

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen. A monoclonal CTL response against a MAGE-3 antigen was observed in a melanoma patient, who showed partial rejection of a large metastasis after treatment with a vaccine containing only the tumor-specific antigenic peptide. Tetramer analysis after in vitro restimulation indicated that about 1/40,000 postimmunization CD8(+) blood lymphocytes were directed against the antigen. The same TCR was present in all of the positive microcultures. TCR evaluation carried out directly on blood lymphocytes by PCR amplification led to a similar frequency estimate after immunization, whereas the TCR was not found among 2.5 x 10(6) CD8(+) lymphocytes collected before immunization. Our results prove unambiguously that vaccines containing only a tumor-specific antigenic peptide can elicit a CTL response. Even though they provide no information about the effector mechanisms responsible for the observed reduction in tumor mass in this patient, they would suggest that low-level CTL responses can initiate tumor rejection.


Assuntos
Antígenos de Neoplasias , Vacinas Anticâncer/uso terapêutico , Melanoma/imunologia , Melanoma/terapia , Proteínas de Neoplasias/uso terapêutico , Linfócitos T Citotóxicos/imunologia , Sequência de Bases , Vacinas Anticâncer/genética , Células Clonais/imunologia , Citotoxicidade Imunológica , Primers do DNA/genética , Humanos , Imunofenotipagem , Técnicas In Vitro , Melanoma/genética , Melanoma/secundário , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Vacinação
5.
J Immunol ; 166(12): 7090-5, 2001 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-11390453

RESUMO

The class II cytokine receptor family includes the receptors for IFN-alphabeta, IFN-gamma, IL-10, and IL-10-related T cell-derived inducible factor/IL-22. By screening genomic DNA databases, we identified a gene encoding a protein of 231 aa, showing 33 and 34% amino acid identity with the extracellular domains of the IL-22 receptor and of the IL-20R/cytokine receptor family 2-8, respectively, but lacking the transmembrane and cytoplasmic domains. A lower but significant sequence identity was found with other members of this family such as the IL-10R (29%), cytokine receptor family 2-4/IL-10Rbeta (30%), tissue factor (26%), and the four IFN receptor chains (23-25%). This gene is located on chromosome 6q24, at 35 kb from the IFNGR1 gene, and is expressed in various tissues with maximal expression in breast, lungs, and colon. The recombinant protein was found to bind IL-10-related T cell-derived inducible factor/IL-22, and to inhibit the activity of this cytokine on hepatocytes and intestinal epithelial cells. We propose to name this natural cytokine antagonist IL-22BP for IL-22 binding protein.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Interleucina-10/química , Interleucinas/antagonistas & inibidores , Interleucinas/metabolismo , Receptores de Superfície Celular , Receptores de Interleucina/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular , Clonagem Molecular , Células HT29 , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Receptores de Citocinas/química , Receptores de Citocinas/classificação , Receptores de Citocinas/genética , Células Tumorais Cultivadas , Interleucina 22
6.
Cancer Res ; 61(9): 3718-24, 2001 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11325844

RESUMO

We have identified an antigen recognized by autologous CTL on the lung carcinoma cells of a patient who enjoyed a favorable clinical evolution, being alive 10 years after partial resection of the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and encoded by a mutated sequence in the gene coding for malic enzyme, an essential enzyme that converts malate to pyruvate. In the tumor cell line derived from the patient, only the mutated malic enzyme allele is expressed, because of a loss of heterozygosity in the region of chromosome 6 that contains this locus. Tetramers of soluble HLA-A2 molecules loaded with the antigenic peptide stained approximately 0.4% of the patient's blood CD8 T cells. When these cells were stimulated in clonal conditions, 25% of them proliferated, and the resulting clones were lytic and specific for the mutated malic enzyme peptide. T-cell receptor analysis indicated that almost all of these antimalic CTLs shared the same receptor. Antimalic T cells were consistently found in blood samples collected from the patient between 1990 and 1999, at frequencies ranging from 0.1 to 0.4% of the CD8 cells. Their frequency appeared to double within 2 weeks after intradermal inoculation of lethally irradiated autologous tumor cells. These results indicate that nonmelanoma cancer patients may also have a high frequency of blood CTLs directed against a tumor-specific antigen.


Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Carcinoma de Células Escamosas/imunologia , Antígeno HLA-A2/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/sangue , Sequência de Bases , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 6 , DNA Complementar/genética , DNA de Neoplasias/genética , Epitopos/imunologia , Antígeno HLA-A2/sangue , Antígeno HLA-A2/genética , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/genética , Malato Desidrogenase/genética , Malato Desidrogenase/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação Puntual , Transfecção
7.
Proc Natl Acad Sci U S A ; 97(18): 10144-9, 2000 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-10954742

RESUMO

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, alpha1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the beta chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.


Assuntos
Citocinas/fisiologia , Linfócitos T/imunologia , Proteínas de Fase Aguda/genética , Sequência de Aminoácidos , Animais , Carcinoma Hepatocelular , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Citocinas/química , Citocinas/genética , Citocinas/farmacologia , DNA Complementar , Escherichia coli , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/imunologia , Neoplasias Hepáticas , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transativadores/metabolismo , Células Tumorais Cultivadas
8.
J Immunol ; 164(11): 6057-66, 2000 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10820291

RESUMO

We have identified an Ag recognized by autologous CTL on the melanoma cells of a patient who enjoyed an unusually favorable clinical evolution. The antigenic peptide, which is presented by HLA-A28 molecules, is encoded by a mutated sequence in a new gene. This gene, which was named MUM-3, is expressed ubiquitously and shows homology with the RNA helicase gene family. Limiting dilution analysis indicated that at least 0.15% of the blood CD8 T cells were tumor-specific CTL precursors. The MUM-3 Ag was recognized by 90% of these CTL, indicating that it is the dominant target Ag of the tumor-specific CTL response. The high frequency of anti-MUM-3 CTL was confirmed with tetramers of soluble HLA-A28 molecules loaded with the antigenic peptide. MUM-3 tetramers stained 1.2% of blood CD8 cells, a frequency that has never been reported for T cells directed against a strictly tumor-specific Ag. To confirm these results, the CD8 T cells that were clearly labeled with tetramers were restimulated in clonal conditions. About 90% of these cells proliferated, and all the resulting clones proved lytic and MUM-3 specific. By improving the conditions used for the in vitro restimulation of CTL precursors by the tumor cells, the same frequency could be obtained in limiting dilution analysis. These results show that some cancer patients have a high frequency of circulating CTL that are directed against a strictly tumor-specific Ag. These CTL are responsive to restimulation in vitro and are easily detected with tetramers. Such responses may therefore be an achievable goal for therapeutic vaccination with tumor-specific Ags.


Assuntos
Antígenos de Neoplasias/imunologia , Melanoma/genética , Melanoma/imunologia , Mutação Puntual/imunologia , RNA Helicases/genética , RNA Helicases/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/isolamento & purificação , Sequência de Bases , Testes Imunológicos de Citotoxicidade , Antígenos HLA-A/imunologia , Humanos , Ativação Linfocitária , Contagem de Linfócitos , Melanoma/enzimologia , Melanoma/secundário , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Solubilidade , Células-Tronco/imunologia , Células-Tronco/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/metabolismo , Células Tumorais Cultivadas
9.
Mol Cell Biol ; 7(7): 2552-7, 1987 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3302681

RESUMO

The Saccharomyces cerevisiae ILV1 gene, encoding threonine dehydratase (EC 4.2.1.16) was fused to the transferred DNA nopaline synthase promoter and the 3' noncoding region of the octopine synthase gene. It was introduced, by Agrobacterium tumefaciens-mediated gene transfer, into an isoleucine-requiring Nicotiana plumbaginifolia auxotroph deficient in threonine dehydratase. Functional complementation by the ILV1 gene product was demonstrated by the selection of several transformed lines on a medium without isoleucine and by the identification in these lines of the yeast threonine dehydratase activity. This is the first example illustrating the complementation of a plant auxotroph by transfection with a cloned gene.


Assuntos
Plantas/genética , Saccharomyces cerevisiae/genética , Treonina Desidratase/genética , Teste de Complementação Genética , Vetores Genéticos , Mutação , Plantas/enzimologia , Plantas Tóxicas , Rhizobium/genética , Treonina Desidratase/deficiência , Nicotiana/enzimologia , Nicotiana/genética , Transformação Genética
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