RESUMO
The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.
Assuntos
Envelhecimento/imunologia , Apresentação de Antígeno , Linfócitos T CD8-Positivos/enzimologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Antígenos/imunologia , Antígenos/farmacologia , Apresentação Cruzada/efeitos dos fármacos , Feminino , Camundongos , Poli U/imunologia , Poli U/farmacologiaRESUMO
ssRNA can interact with dendritic cells (DCs) through binding to TLR7, inducing secretion of proinflammatory cytokines and type I IFN. Triggering TLR7 enhances cross-priming of CD8(+) T cells, which requires cross-presentation of exogenous Ag to DCs. However, how TLR triggering can affect Ag cross-presentation is still not clear. Using OVA as an Ag model, we observed that stimulation of TLR7 in DCs by polyuridylic acid (polyU), a synthetic ssRNA analog, generates a strong specific cytotoxic response in C57BL/6 mice. PolyU stimulate CD8α(+) DCs to cross-prime naive CD8(+) T cells in a type I IFN-dependent fashion. This enhanced cross-priming is accompanied by a higher density of OVA(256-264)/H-2K(b) complexes on CD8α(+) DCs treated with polyU, as well as by upregulation of costimulatory molecules and increased secretion of proinflammatory cytokines by DCs. Cross-priming of CD8(+) T cells by DCs treated with polyU requires proteasome and Ag translocation to cytosol through the Sec61 channel in DCs. The observed enhancement in OVA cross-presentation with polyU in DCs could be mediated by a limited Ag degradation in endophagosomal compartments and a higher permanence of OVA peptide/MHC class I complexes on DCs. These observations clearly reveal that key steps of Ag processing for cross-presentation can be modulated by TLR ligands, opening new avenues for understanding their mechanisms as adjuvants of the immune response.
Assuntos
Adjuvantes Imunológicos/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Antígenos H-2/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Poli U/farmacologia , Receptor 7 Toll-Like/efeitos dos fármacos , Animais , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Compartimento Celular , Células Cultivadas/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Endossomos/imunologia , Feminino , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Canais de Translocação SEC , Baço/imunologia , Receptor 7 Toll-Like/imunologiaRESUMO
The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263-273). In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.
Assuntos
Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Calmodulina/metabolismo , Caseína Quinase II , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Fosforilação , Poli U/farmacologia , Cloreto de Potássio/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência , Homologia de Sequência de Aminoácidos , Temperatura , Xenopus laevis , Peixe-ZebraRESUMO
Several homologous polynucleotides have been tested as inhibitors on the reactions catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyribonucleotides and 2'-fluorinated polynucleotides as templates. Polynucleotides differentially inhibited the reactions catalyzed by reverse transcriptase in the presence of these synthetic templates. Polyriboadenylic acid (poly(rA), poly(2'-O-methyladenylic acid) (poly(Am)), poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl), polyinosinic acid (poly(rI)) and polyuridylic acid poly(rU)) inhibited the polyribonucleotide-, but not the 2'-fluorinated polynucleotide-directed reverse transcriptase activity.