RESUMEN
One of the predominant VH genes utilized to encode the anti-BrMRBC specificity is a member of the small VH11 family rearranged to JH1. Using the polymerase chain reaction (PCR) we have determined that the frequency of B cells with a VH11 rearrangement is 10-20 times higher in Ly-1 B than in Ly-1- "conventional" B cells regardless of location (spleen or peritoneal cavity). Conventional B cells rearrange this gene at comparable levels in pre-B cells and in mature B cells utilizing all JH gene segments. In contrast, the increased levels of VH11 rearrangement in Ly-1 B are restricted to JH1 (and some JH2) and therefore appear to be the result of selection. Furthermore, most peritoneal Ly-1 B cells with VH11 rearrangements fall in a fraction stained by anti-BrMRBC antibody, likely bearing multivalent natural (likely self) antigen constitutively bound to their surface Ig receptors. Thus, we suggest that autoantigens are largely responsible for the accumulation of autoantibody specificities in the Ly-1 B cell lineage with time, whereas they do not exert this effect in the conventional B cells.
Asunto(s)
Antígenos Ly/inmunología , Linfocitos B/inmunología , Reordenamiento Génico de Linfocito B/genética , Región Variable de Inmunoglobulina/genética , Selección Genética , Animales , Autoanticuerpos/biosíntesis , Separación Celular , Citometría de Flujo , Reordenamiento Génico de Linfocito B/inmunología , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Sondas de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFL16.1, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement, but no V-D-J. Finally, functional analysis demonstrates that the proliferative response to IL-7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A.
Asunto(s)
Antígenos CD , Linfocitos B/citología , Células de la Médula Ósea , Células Madre Hematopoyéticas/citología , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Secuencia de Bases , Biotina/metabolismo , Médula Ósea/inmunología , Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , ADN/análisis , ADN/genética , Femenino , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B/genética , Reordenamiento Génico de Cadena Ligera de Linfocito B/inmunología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Leucosialina , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fenotipo , Ficoeritrina/metabolismo , Reacción en Cadena de la Polimerasa , Sialoglicoproteínas/metabolismoRESUMEN
The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin mu heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre-B cell progression as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 mu heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Feto , Regulación del Desarrollo de la Expresión Génica/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/biosíntesis , Cadenas mu de Inmunoglobulina/genética , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Transgénicos , Pruebas de Precipitina , Receptores de Antígenos de Linfocitos B/genética , Células Madre/inmunología , TransfecciónRESUMEN
Lymphocyte development is critically influenced by self-antigens. T cells are subject to both positive and negative selection, depending on their degree of self-reactivity. Although B cells are subject to negative selection, it has been difficult to test whether self-antigen plays any positive role in B cell development. A murine model system of naturally generated autoreactive B cells with a germ line gene-encoded specificity for the Thy-1 (CD90) glycoprotein was developed, in which the presence of self-antigen promotes B cell accumulation and serum autoantibody secretion. Thus, B cells can be subject to positive selection, generated, and maintained on the basis of their autoreactivity.
Asunto(s)
Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Antígenos Thy-1/inmunología , Envejecimiento/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Antígenos CD5/análisis , Genes de Inmunoglobulinas , Hibridomas , Inmunidad Innata , Vigilancia Inmunológica , Ratones , Ratones SCID , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
A common feature of B-cell chronic lymphocytic leukemia (CLL) is chromosomal loss of 13q14, containing the miR15a/16-1 locus controlling B-cell proliferation. However, CLL etiology remains unclear. CLL is an adult leukemia with an incidence that increases with advancing age. A unique feature of CLL is biased B-cell antigen receptor (BCR) usage, autoreactivity with polyreactivity and CD5 expression, all suggest a role for the BCR in driving CLL pathogenesis. Among human CLLs, BCRs autoreactive with non-muscle myosin IIA (AMyIIA) are recurrent. Here we identify an unmutated AMyIIA BCR in mouse, with distinctive CDR3 segments capable of promoting leukemogenesis. B cells with this AMyIIA BCR are generated by BCR-dependent signaling during B-1 fetal/neonatal development with CD5 induction, but not in adults. These early-generated AMyIIA B-1 B cells self-renew, increase during aging and can progress to become monoclonal B-cell lymphocytosis, followed by aggressive CLL in aged mice, often with the loss of a chromosomal region containing the miR15a/16-1 locus of varying length, as in human CLL. Thus, the ability to generate this defined autoreactive BCR by B-1 B cells is a key predisposing step in mice, promoting progression to chronic leukemia.
Asunto(s)
Deleción Cromosómica , Trastornos de los Cromosomas , Leucemia Linfocítica Crónica de Células B/etiología , Animales , Linfocitos B/patología , Autorrenovación de las Células , Cromosomas Humanos Par 13 , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Miosina Tipo IIA no Muscular/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , SinteníaRESUMEN
The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a longstanding puzzle in immunology. In the past few years it has become clear that production of mu heavy chain and subsequent assembly with surrogate light chain to form the pre-B cell receptor complex is critical to promote development of adult B cell precursors in mouse bone marrow. Recently we found that instead of promoting pre-B cell expansion as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver, providing a previously unrecognized mechanism for alteration of the B cell repertoire with age. The consequence is very distinct primary repertoires for development of fetal B1 cells and adult bone marrow B2 cells.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Subgrupos de Linfocitos B/citología , Médula Ósea/inmunología , Reordenamiento Génico de Linfocito B , Hematopoyesis Extramedular/inmunología , Células Madre Hematopoyéticas/citología , Sistema Inmunológico/crecimiento & desarrollo , Hígado/embriología , Receptores de Antígenos de Linfocitos B/inmunología , Adulto , Animales , Subgrupos de Linfocitos B/inmunología , Linaje de la Célula , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Células Madre Hematopoyéticas/inmunología , Humanos , Sistema Inmunológico/embriología , Región Variable de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Hígado/citología , Hígado/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/genética , Proteínas Recombinantes de Fusión/inmunología , TransfecciónAsunto(s)
Linfocitos B/citología , Animales , Antígenos CD , Linfocitos B/inmunología , Linfocitos B/trasplante , Antígenos CD5 , Diferenciación Celular , Trasplante de Tejido Fetal , Feto/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Ratones , Ratones SCIDRESUMEN
By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).
Asunto(s)
Antígenos Ly , Linfocitos B/análisis , Antígenos de Grupos Sanguíneos/genética , Genes de Inmunoglobulinas , Hibridomas/análisis , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Isoanticuerpos/genética , Alelos , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Linfocitos B/metabolismo , Secuencia de Bases , Antígenos de Grupos Sanguíneos/inmunología , Bromelaínas , Clonación Molecular , Femenino , Hibridomas/metabolismo , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Cadenas kappa de Inmunoglobulina/genética , Isoanticuerpos/biosíntesis , Isoanticuerpos/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Fenotipo , Polimorfismo GenéticoRESUMEN
Although immature/transitional peripheral B cells may remain susceptible to selection pressures before full maturation, the nature and timing of these selection events remain unclear. We show that correlated expression of surface (s) IgM (sIgM), CD23, and AA4 defines three nonproliferative subpopulations of immature/transitional peripheral B cells. We designate these populations transitional (T) 1 (AA4(+)CD23(-)sIgM(high)), T2 (AA4(+)CD23(+)sIgM(high)), and T3 (AA4(+)CD23(+)sIgM(low)). Cells within all three subsets are functionally immature as judged by their failure to proliferate following sIgM cross-linking in vitro, and their rapid rate of turnover in vivo as assessed by 5-bromo-2'-deoxyuridine labeling. These labeling studies also reveal measurable cell loss at both the T1-T2 and T2-T3 transitions, suggesting the existence of multiple selection points within the peripheral immature B cell pool. Furthermore, we find that Btk-deficient (xid) mice exhibit an incomplete developmental block at the T2-T3 transition within the immature B cell pool. This contrasts markedly with lyn(-/-) mice, which exhibit depressed numbers but normal ratios of each immature peripheral B cell subset and severely reduced numbers of mature B cells. Together, these data provide evidence for multiple selection points among immature peripheral B cells, suggesting that the B cell repertoire is shaped by multiple unique selection events that occur within the immature/transitional peripheral B cell pool.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Receptores de Hialuranos , Glicoproteínas de Membrana , Bazo/inmunología , Agammaglobulinemia Tirosina Quinasa , Animales , Subgrupos de Linfocitos B/clasificación , Bromodesoxiuridina/química , Linaje de la Célula , Células Cultivadas , Femenino , Inmunoglobulina M/metabolismo , Inmunofenotipificación , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Mitocondriales , Mutación , Proteínas Tirosina Quinasas/genética , Receptores de Complemento/metabolismo , Receptores de IgE/metabolismo , Células Madre/inmunología , Familia-src Quinasas/genéticaRESUMEN
The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.
Asunto(s)
Linfocitos B/inmunología , Proteínas de Unión al ADN/genética , Animales , Linfocitos B/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/farmacología , Sinergismo Farmacológico , Reordenamiento Génico de Linfocito B/genética , Reordenamiento Génico de Linfocito B/inmunología , Ratones , Ratones Mutantes , Ratones Transgénicos , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/farmacología , Transcripción Genética/genética , Transcripción Genética/inmunologíaRESUMEN
We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1-) mice. Complementation with a membrane mu heavy chain (mu HC) gene allows progression of developmentally arrested Rag-1- pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated mu HC and kappa light chain (kappa LC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally nature Rag-1- peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2k antibody. This experimental system demonstrates the competence of the mu HC and kappa LC to direct and regulate the sequential stages of B-cell differentiation, defines the time at which negative selection of self-reactive B cells occurs, and shows that elimination of these cells occurs equally well in the absence of Rag-1 as in its presence. These data also support the hypothesis that Rag-1 directly participates in the V(D)J recombination process.