RESUMO
Gene gun-mediated biolistic DNA vaccination with beta-galactosidase (betaGal)-encoding plasmid vectors efficiently modulated antigen-induced immune responses in an animal model of type I allergy, including the inhibition of immunoglobulin E (IgE) production. Here we show that CD4(+) as well as CD8(+) T cells from mice biolistically transfected with a plasmid encoding betaGal under the control of the fascin promoter (pFascin-betaGal) are capable of inhibiting betaGal-specific IgE production after adoptive transfer into naïve recipients. Moreover, suppression of IgE production was dependent on interferon (IFN)-gamma. To analyse the modalities of activation of CD4(+) and CD8(+) T cells regarding the localization of antigen synthesis following gene gun-mediated DNA immunization, we used the fascin promoter and the keratin 5 promoter (pK5-betaGal) to direct betaGal production mainly to dendritic cells (DCs) and to keratinocytes, respectively. Gene gun-mediated DNA immunization with each vector induced considerable activation of betaGal-specific CD8(+) cytotoxic T cells. Cytokine production by re-stimulated CD4(+) T cells in draining lymph nodes and immunoglobulin isotype profiles in sera of immunized mice indicated that immunization with pFascin-betaGal induced a T helper type 1 (Th1)-biased immune response, whereas immunization with pK5-betaGal generated a mixed Th1/Th2 immune response. Nevertheless, DNA vaccination with pFascin-betaGal and pK5-betaGal, respectively, efficiently inhibited specific IgE production in the mouse model of type I allergy. In conclusion, our data show that uptake of exogenous antigen produced by keratinocytes and its presentation by untransfected DCs as well as the presentation of antigen synthesized endogenously in DCs represent equivalent pathways for efficient priming of cellular immune responses.
Assuntos
Apresentação de Antígeno , Biolística , Hipersensibilidade/terapia , Células de Langerhans/imunologia , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/genética , Apresentação Cruzada/imunologia , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Feminino , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Queratina-15 , Queratina-5/imunologia , Queratina-5/metabolismo , Queratinócitos/imunologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/imunologia , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas de DNA/imunologia , beta-Galactosidase/genéticaRESUMO
Chronic inflammatory diseases may develop when regulatory T cells (Tregs) fail to control the balance between tolerance and immunity. Alternatively, activated immune cells might prevent the induction or activation of Tregs in such diseases. In this study, we demonstrate that trans-signaling into T cells via the soluble IL-6 receptor completely abrogates the de novo induction of adaptive Tregs. Mechanistically, IL-6 trans-signaling augmented the expression of the TGF-beta signaling inhibitor SMAD7. Consequently, SMAD7 overexpression in T cells using newly created transgenic mice rendered CD4(+)CD25(-) T cells resistant to the induction of FoxP3. Finally, IL-6 trans-signaling inhibited Treg-mediated suppression in a murine model of colitis. In summary, IL-6 trans-signaling into T cells emerges as a key pathway for blockade of the development of adaptive Tregs and thus may play a pivotal role in shifting the balance between effector and regulatory T cell numbers in chronic inflammatory and autoimmune diseases.
Assuntos
Colite/imunologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Receptores de Interleucina-6/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doença Crônica , Colite/genética , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/imunologia , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos Transgênicos , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/genética , Proteína Smad7/genética , Proteína Smad7/imunologia , Proteína Smad7/metabolismo , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
CD4+ CD25+ regulatory T cells (Tregs) are crucial for the maintenance of immunological tolerance. Recent data indicate that Tregs not only develop in the thymus during ontogeny but can also differentiate from naive T cells in the periphery. The following protocol describes a method by which Tregs are generated in vitro by stimulation of naive T cells in the presence of transforming growth factor beta (Ti-Tregs). In vitro-induced regulatory T cells express markers of conventional Treg such as CD25 and the genetic program committing transcription factor FoxP3. Functionally the in vitro-generated Ti-Tregs suppress T-cell activation and proliferation while in vivo these cells have been proven to control inflammation in different animal models, suggesting a potential use of these cells for immunotherapy. The protocol can be completed within 5 days.