RESUMO
Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.
Assuntos
Animais , Feminino , Camundongos , Antígenos de Protozoários/genética , Genes Codificadores dos Receptores de Linfócitos T/genética , Glicoproteínas/genética , Epitopos Imunodominantes/genética , Interferon gama/genética , Neuraminidase/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos , Antígenos de Protozoários/imunologia , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Hibridomas/metabolismo , Epitopos Imunodominantes/imunologia , Interferon gama/imunologia , Interferon gama , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neuraminidase/imunologia , Neuraminidase/metabolismo , Transcrição GênicaRESUMO
Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.
Assuntos
Antígenos de Protozoários/genética , Genes Codificadores dos Receptores de Linfócitos T/genética , Glicoproteínas/genética , Epitopos Imunodominantes/genética , Interferon gama/genética , Neuraminidase/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/imunologia , Feminino , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Hibridomas/metabolismo , Epitopos Imunodominantes/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neuraminidase/imunologia , Neuraminidase/metabolismo , Transcrição GênicaRESUMO
We have recently generated CD4 clones from BALB/c mice immunized with a plasmid DNA containing the gene encoding for the catalytic domain of trans-sialidase, an important enzyme expressed on the surface of Trypanosoma cruzi trypomastigotes. These clones allowed us to study in vitro the interaction between T cells and T. cruzi-infected macrophages. A cytotoxic CD4 clone of the Th1 type effectively activated macrophages to kill intracellular amastigote forms of T. cruzi. In contrast, CD4 Th2-like clones were much less efficient, being unable to activate macrophages to significantly reduce parasite development. We found that the anti-parasitic activity of Th1 cells was completely suppressed by the presence of nitric oxide synthase inhibitors. Also, we observed that anti-IFN-gamma antibodies significantly inhibited the anti-parasitic activity of these cells. We conclude that trypomastigote-specific Th1 cells activate macrophages to kill intracellular amastigotes of T. cruzi by a mechanism exclusively dependent on the induction of nitric oxide synthesis.
Assuntos
Macrófagos/imunologia , Óxido Nítrico/fisiologia , Células Th1/imunologia , Células Th2/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Células Clonais , Citocinas/biossíntese , Citocinas/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Ativação de Macrófagos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neuraminidase/genética , Neuraminidase/imunologia , Óxido Nítrico/biossíntese , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.