RESUMEN
Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell-deficiency syndrome in mice caused by mutations in Atp11c, which encodes a member of the P4 ATPase family thought to serve as 'flippases' that concentrate aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11C resulted in a lower rate of phosphatidylserine translocation in pro-B cells and much lower pre-B cell and B cell numbers despite expression of pre-rearranged immunoglobulin transgenes or enforced expression of the prosurvival protein Bcl-2 to prevent apoptosis and abolished pre-B cell population expansion in response to a transgene encoding interleukin 7. The only other abnormalities we noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. Our results identify an intimate connection between phospholipid transport and B lymphocyte function.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Endocitosis/inmunología , Fosfoserina/inmunología , Adenosina Trifosfatasas/genética , Animales , Linfocitos B/enzimología , Secuencia de Bases , Femenino , Citometría de Flujo , Genes bcl-2/inmunología , Interleucina-7/genética , Interleucina-7/inmunología , Hígado/citología , Hígado/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis/inmunología , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Effective vaccine adjuvants must induce expression of major histocompatibility (MHC) class II proteins and the costimulatory molecule CD86 on dendritic cells (DCs). However, some adjuvants elicit production of cytokines resulting in adverse inflammatory consequences. Development of agents that selectively increase MHC class II and CD86 expression without triggering unwanted cytokine production requires a better understanding of the molecular mechanisms influencing the production and degradation of MHC class II and CD86 in DCs. Here, we investigate how CD83, an immunoglobulin protein expressed on the surface of mature DCs, promotes MHC class II and CD86 expression. Using mice with an N-ethyl-N-nitrosourea-induced mutation eliminating the transmembrane (TM) region of CD83, we found that the TM domain of CD83 enhances MHC class II and CD86 expression by blocking MHC class II association with the ubiquitin ligase MARCH1. The TM region of CD83 blocks interleukin 10-driven, MARCH1-dependent ubiquitination and degradation of MHC class II and CD86 in DCs. Exploiting this posttranslational pathway for boosting MHC class II and CD86 expression on DCs may provide an opportunity to enhance the immunogenicity of vaccines.
Asunto(s)
Antígenos CD/inmunología , Antígeno B7-2/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunoglobulinas/inmunología , Interleucina-10/inmunología , Glicoproteínas de Membrana/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Secuencia de Bases , Membrana Celular/inmunología , Células Dendríticas/inmunología , Células HEK293 , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/genética , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Ubiquitina-Proteína Ligasas/inmunología , Antígeno CD83RESUMEN
To identify genes and mechanisms involved in humoral immunity, we did a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified had loss-of-function mutations in the gene encoding a previously obscure member of a family of Rho-Rac GTP-exchange factors, DOCK8. DOCK8-mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutations disrupted accumulation of the integrin ligand ICAM-1 in the B cell immunological synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to Dock8 mutation provides evidence that organization of the immunological synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.
Asunto(s)
Formación de Anticuerpos , Linfocitos B/inmunología , Centro Germinal/inmunología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Mutación , Sinapsis/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Secuencia de Bases , Centro Germinal/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Alineación de SecuenciaRESUMEN
BCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologic blockade of BCR proximal signaling pathways results in a global "back-differentiation" response in which immature B cells down-regulate genes important for the mature B cell program and up-regulate genes characteristic of earlier stages of B cell development. These observations led us to test the hypothesis that self-Ag-induced down-regulation of the BCR, and not self-Ag-induced positive signals, lead to Rag induction and hence receptor editing. Supporting this hypothesis, we found that immature B cells from xid (x-linked immunodeficiency) mice induce re-expression of a Rag2-GFP bacterial artificial chromosome reporter as well as wild-type immature B cells following Ag incubation. Incubation of immature B cells with self-Ag leads to a striking reversal in differentiation to the pro-/pre-B stage of development, consistent with the idea that back-differentiation results in the reinduction of genes required for L chain rearrangement and receptor editing. Importantly, Rag induction, the back-differentiation response to Ag, and editing in immature and pre-B cells are inhibited by a combination of phorbol ester and calcium ionophore, agents that bypass proximal signaling pathways and mimic BCR signaling. Thus, mimicking positive BCR signals actually inhibits receptor editing. These findings support a model whereby Ag-induced receptor editing is inhibited by BCR basal signaling on developing B cells; BCR down-regulation removes this basal signal, thereby initiating receptor editing.
Asunto(s)
Antígenos/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunología , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/inmunología , Femenino , Genes Reporteros/genética , Humanos , Ionóforos/farmacología , Cinética , Masculino , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ésteres del Forbol/farmacología , Receptores Fc/inmunología , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Divergent hypotheses exist to explain how signaling by the B cell receptor (BCR) is initiated after antigen binding and how it is qualitatively altered in anergic B cells to selectively uncouple from nuclear factor kappaB and c-Jun N-terminal kinase pathways while continuing to activate extracellular signal-regulated kinase and calcium-nuclear factor of activated T cell pathways. Here we find that BCRs on anergic cells are endocytosed at a very enhanced rate upon binding antigen, resulting in a large steady-state pool of intracellularly sequestered receptors that appear to be continuously cycling between surface and intracellular compartments. This endocytic mechanism is exquisitely sensitive to the lowering of plasma membrane cholesterol by methyl-beta-cyclodextrin, and, when blocked in this way, the sequestered BCRs return to the cell surface and RelA nuclear accumulation is stimulated. In contrast, when plasma membrane cholesterol is lowered and GM1 sphingolipid markers of membrane rafts are depleted in naive B cells, this does not diminish BCR signaling to calcium or RelA. These results provide a possible explanation for the signaling changes in clonal anergy and indicate that a chief function of membrane cholesterol in B cells is not to initiate BCR signaling, but instead to terminate a subset of signals by rapid receptor internalization.
Asunto(s)
Linfocitos B/inmunología , Colesterol/fisiología , Anergia Clonal/inmunología , Lípidos de la Membrana/fisiología , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/metabolismo , Anergia Clonal/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/genética , Transducción de Señal/genéticaRESUMEN
In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was "knocked-in" at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was approximately 6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.
Asunto(s)
Linfocitos B/inmunología , Anergia Clonal/inmunología , Supresión Clonal/inmunología , Modelos Inmunológicos , Edición de ARN/inmunología , Autotolerancia/inmunología , Animales , Formación de Anticuerpos/genética , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Anergia Clonal/genética , Supresión Clonal/genética , Humanos , Cinética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muramidasa/genética , Muramidasa/inmunología , Muramidasa/metabolismo , Edición de ARN/genética , Quimera por Radiación/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Autotolerancia/genética , Solubilidad , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking "back-differentiation" of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulinas/fisiología , Transducción de Señal/inmunología , Androstadienos/farmacología , Animales , Linfocitos B/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células Cultivadas , Reordenamiento Génico , Reordenamiento Génico de Linfocito B , Proteínas Fluorescentes Verdes/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Inmunoglobulina M/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , WortmaninaRESUMEN
Expression of the c-myc gene is frequently dysregulated in malignant tumors and translocations of c-myc into the Ig H chain locus are associated with Burkitt's-type lymphoma. There is indirect evidence that bcl-x, an anti-apoptotic member of the bcl-2 gene family, may also contribute to a variety of B lymphoid tumors. In this study, we show that mice transgenic for both B cell-restricted c-myc and bcl-x(L) developed aggressive, acute leukemias expressing early B lineage and stem cell surface markers. Of interest, the tumor cells proliferated and differentiated down the B cell developmental pathway following in vitro treatment with IL-7. Analysis of sorted leukemic cells from spleen indicated constitutive expression of sterile micro and kappa transcripts in combination with evidence for D-J(H) DNA rearrangements. Several B cell-specific genes were either not expressed or were expressed at low levels in primary tumor cells and were induced following culture with IL-7. IL-7 also increased V-Jkappa and V-DJ(H) rearrangements. These data demonstrate oncogenic synergy between c-myc and bcl-x(L) in a new mouse model for acute lymphoblastic leukemia. Tumors in these animals target an early stage in B cell development characterized by the expression of both B lineage and stem cell genes.
Asunto(s)
Genes myc/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Células Cultivadas , Reordenamiento Génico , Genes de Inmunoglobulinas , Inmunofenotipificación , Interleucina-7/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Proteína bcl-XRESUMEN
Receptor editing is an important mechanism to modify the Ag specificity of newly developing B cells that are reactive with self-Ags. Previous studies have suggested that late immature B cells, bearing high levels of IgM on their cell surface, are unable to initiate receptor editing and instead are deleted by apoptosis. Using the hen egg lysozyme transgenic system, we show that IgM(high) late-immature B cells are fully capable of receptor editing to soluble self-Ag. This was demonstrated by the induction of new endogenous light chain locus rearrangements and by a single-cell flow cytometric assay using a recombination-activating gene 2-green fluorescence protein reporter transgene. These studies suggest that the developmental window available for immature B cells to edit their Ig receptors, at least in response to certain soluble Ags, extends through the IgM(high) late immature B cell stage.
Asunto(s)
Autoantígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Inmunoglobulina D , Cadenas Ligeras de Inmunoglobulina/metabolismo , Inmunoglobulina M/biosíntesis , Edición de ARN/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Subgrupos de Linfocitos B/citología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Inmunoglobulina D/biosíntesis , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Muramidasa/genética , Muramidasa/inmunología , Muramidasa/metabolismo , Edición de ARN/genética , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Antígenos de Linfocitos B/genética , Receptores Fc/biosíntesis , Receptores Fc/genética , Receptores Fc/metabolismo , SolubilidadRESUMEN
The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment.