RESUMEN
Somatic rearrangement of immunoglobulin (Ig) genes is a key step during B cell development. Using pro-B cells lacking the phosphatase Pten (phosphatase and tensin homolog), which negatively regulates phosphoinositide-3-kinase (PI3K) signaling, we show that PI3K signaling inhibits Ig gene rearrangement by suppressing the expression of the transcription factor Ikaros. Further analysis revealed that the transcription factor FoxO1 is crucial for Ikaros expression and that PI3K-mediated down-regulation of FoxO1 suppresses Ikaros expression. Interestingly, FoxO1 did not influence Ikaros transcription; instead, FoxO1 is essential for proper Ikaros mRNA splicing, as FoxO1-deficient cells contain aberrantly processed Ikaros transcripts. Moreover, FoxO1-induced Ikaros expression was sufficient only for proximal V(H) to DJ(H) gene rearrangement. Simultaneous expression of the transcription factor Pax5 was needed for the activation of distal V(H) genes; however, Pax5 did not induce any Ig gene rearrangement in the absence of Ikaros. Together, our results suggest that ordered Ig gene rearrangement is regulated by distinct activities of Ikaros, which mediates proximal V(H) to DJ(H) gene rearrangement downstream of FoxO1 and cooperates with Pax5 to activate the rearrangement of distal V(H) genes.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/fisiología , Genes de Inmunoglobulinas/genética , Factor de Transcripción Ikaros/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Empalme del ARN/fisiología , Recombinación V(D)J/fisiología , Animales , Cartilla de ADN/genética , Citometría de Flujo , Proteína Forkhead Box O1 , Regulación de la Expresión Génica/genética , Factor de Transcripción Ikaros/genética , Immunoblotting , Ratones , Factor de Transcripción PAX5/metabolismo , Fosfohidrolasa PTEN/metabolismo , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Empalme del ARN/genética , Transducción GenéticaRESUMEN
Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock-in model system of the 3-83 autoreactive B-cell antigen receptor (BCR) in combination with precursor-BCR (pre-BCR) deficiency, we show here that the 3-83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto-antigen. Experiments with mixed bone marrow chimeras showed that combining the 3-83 BCR with the corresponding auto-antigen resulted in efficient reconstitution of B-cell development in immune-deficient mice. These results suggest that B cells are positively selected by recognition of self-antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre-BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Linfocitos B/metabolismo , Células Cultivadas , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Células Precursoras de Linfocitos B/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Developing B cells express distinct classes of B cell antigen receptors (BCRs) that differ in their heavy chain (HC). Although only muHC is expressed in early stages, deltaHC-containing BCRs dominate on the surface of mature B cells. The reason for the tightly regulated expression of these receptors is poorly understood. Here we show that muHC was specifically required for precursor BCR (pre-BCR) function and that deltaHC was unable to form a functional pre-BCR. A conserved asparagine (N)-linked glycosylation site at position 46 (N46) in the first conserved domain of muHC was absolutely required for pre-BCR function, and swapping that domain with deltaHC resulted in a functional deltaHC-containing pre-BCR. When tested in the context of the BCR, muHC with a mutant N46 showed normal function, which indicated that N46-glycosylation is specifically required for pre-BCR function. Our results suggest an unexpected mode of pre-BCR function, in which binding of the surrogate light chain to N46 mediates autonomous crosslinking and, concomitantly, receptor formation.
Asunto(s)
Linfocitos B/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Receptores de Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Asparagina/inmunología , Linfocitos B/citología , Glicosilación , Ratones , Ratones NoqueadosRESUMEN
The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component lambda5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors.