RESUMEN
The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4(+) T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4(+) T cells in prediabetic 3A9-TCR × insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4(+) cells or the numbers of islet-specific CD4(+) or CD4(+)Foxp3(+) T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-ß-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4(+) cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4(+) cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Predisposición Genética a la Enfermedad , Islotes Pancreáticos/inmunología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autoanticuerpos/biosíntesis , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Anergia Clonal/genética , Anergia Clonal/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Progresión de la Enfermedad , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/fisiología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Páncreas/inmunología , Páncreas/metabolismo , Páncreas/patología , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaRESUMEN
The use of proofreading DNA polymerases in genotyping assays offers the prospect of improved performance. To this end, we have recently used compatible DNA polymerases, protected primers, and substrates to implement proofreading single base extension (P-SBE) and proofreading allele-specific extension (PRASE) assays. Key aspects of the P-SBE and related proofreading assay formats are described here. For transduction of genotyping reactions into physical signals, electrochemical SBE implementations may offer simple, inexpensive assays in electrode array or electrophoretic formats. We have developed electrochemically-labeled nucleotides and electrode detection methods with a view to these applications. Detection of electrochemically-labeled SBE products on an oligonucleotide-modified gold electrode surface is demonstrated.
