RESUMEN
B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP185â206. However, ex-vivoâ T cell proliferation to PLP185â206 peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.
Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular/inmunología , Tejido Linfoide/inmunología , Organogénesis/inmunología , Animales , Linfocitos B/metabolismo , Diferenciación Celular/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Citometría de Flujo , Tejido Linfoide/crecimiento & desarrollo , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Proteína Proteolipídica de la Mielina/química , Proteína Proteolipídica de la Mielina/inmunología , Organogénesis/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunologíaRESUMEN
In conventionally reared mice germinal centers (GCs) are chronically induced in Peyer's patches (PP), mesenteric lymph node (MLN), and isolated lymphoid follicles (ILF) of gut-associated lymphoid tissues (GALT), as a result of continuous B cell stimulation by commensal bacteria. It is generally thought that BCR-mediated antigen recognition controls the recruitment and thus selection of B cells within GALT GCs. However, recent results challenge this view and suggest that engagement of innate immune receptors by microbial antigens promotes B cell recruitment to, and maintenance within, the GC, irrespective of BCR specificity. We propose a scenario in which microbial determinants presented by follicular dendritic cells (FDCs) to innate receptors on B cells within the GC support the survival and concomitant expansion of somatically mutated, IgA-class-switched B cell clones expressing a variety of BCR specificities. From this pool, B cell mutants recognizing gut-derived antigens through their BCR are either, in GCs, drawn into the process of affinity maturation, or, in the lamina propria (LP) of the gut, locally selected to differentiate into plasmablasts, thus contributing to the continuous production of IgA antibodies required for an efficient protection against commensal and pathogenic microorganisms.
Asunto(s)
Linfocitos B/inmunología , Movimiento Celular/inmunología , Centro Germinal/inmunología , Intestinos/inmunología , Animales , Humanos , RatonesRESUMEN
Significant progress has been made in recent years in our understanding of the cellular origin of Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin's lymphoma (HL). It is now clear that in most instances HRS cells represent clonal populations of transformed germinal centre (GC) B cells. While the tumour cells in the lymphocyte predominant type of the disease resemble mutating and antigen-selected GC B cells, there is evidence that HRS cells in classical HL originate from pre-apoptotic GC B cells. HRS cells of the recently defined novel subtype lymphocyte-rich classical HL moleculary resemble HRS cells of the other types of classical HL, but there appear to be phenotypic differences. In rare cases, HRS cells derive from T cells. In contrast to previous speculations, cell fusion apparently does not play a role in the generation of the tumour clone. By gene expression profiling of HL cell lines, it became evident that HRS cells have lost most of the B cell-typical gene expression program, which may explain why these cells can persist without B cell receptor expression and which suggests that at least one of the transforming events involved in HL pathogenesis affects a master regulator of cell lineage identity.
Asunto(s)
Enfermedad de Hodgkin/patología , Animales , Linfocitos B/patología , Células Clonales , Expresión Génica , Reordenamiento Génico , Enfermedad de Hodgkin/genética , Humanos , Células de Reed-Sternberg/patología , Linfocitos T/patologíaRESUMEN
Southern blot analyses of immunoglobulin light chain gene rearrangements in human leukemias and myelomas indicated that lambda loci in kappa-producing cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (KDE). For a systematic analysis of the regulation of light chain rearrangements during early B cell development in normal human B cells also considering functionality of the rearrangements, we used FACS-sorted single naive kappa- and lambda-expressing B cells from peripheral blood of healthy humans. V(kappa)J(kappa) and V(lambda)J(lambda) joints and rearrangements involving the KDE were amplified simultaneously from single cells and sequenced. Whereas only 2 - 3 % of kappa-expressing cells carry V(lambda)J(lambda) joints, nearly all lambda-expressing cells have rearranged kappa loci and indeed carry V(kappa)J(kappa) joints. The V(kappa)J(kappa) joints in lambda-expressing cells exhibit preferential J(kappa)4 and J(kappa)5 over J(kappa)1 and J(kappa)2 usage compared to kappa-expressing cells. Thirty percent of the V(kappa)J(kappa) joints in lambda producers are rearranged in-frame. These data indicate extensive sequential V(kappa)-J(kappa) rearrangements and inactivation of functional V(kappa)J(kappa) joints in lambda-expressing cells, presumably before V(lambda)J(lambda) joining.
Asunto(s)
Linfocitos B/fisiología , Reordenamiento Génico , Genes de Inmunoglobulinas , Cadenas Ligeras de Inmunoglobulina/genética , Mapeo Cromosómico , Humanos , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genéticaRESUMEN
In order to determine whether V gene replacement accompanies somatic hypermutation in the germinal center (GC) reaction in the human, we analyzed V(kappa)J(kappa) and V(lambda)J(lambda) joints and the kappa-deleting element in single lambda(+) naive and post GC B cells for rearrangements at the kappa and lambda loci. Among 265 lambda(+) post GC B cells, not a single unequivocal and only two potential examples of a cell that switched to lambda light chain expression after accumulation of (unfavorable) mutations in its productive V(kappa) rearrangement were observed. Taking the PCR efficiency into account, the frequency of such cells is likely below 3 %. In addition, heavy and light chain gene rearrangements were amplified and sequenced from the oligoclonal population of IgD-only peripheral blood post GC B cells which display extensive intraclonal sequence diversity. Among 61 IgD-only B cells belonging to 15 clones with intraclonal diversity, no combination of V gene rearrangements indicating receptor revision during clonal expansion was observed. Moreover, among 124 and 49 V(H) genes amplified from IgD-only and class-switched B cells, respectively, not a single example of V(H) revision through V(H) hybrid generation was detected. These results suggest that in the human GC reaction V gene replacement either does not usually accompany somatic hypermutation or is mostly counterselected.
Asunto(s)
Linfocitos B/fisiología , Reordenamiento Génico , Genes de Inmunoglobulinas , Memoria Inmunológica , Receptores de Antígenos de Linfocitos B/fisiología , Hipermutación Somática de Inmunoglobulina , Mapeo Cromosómico , Centro Germinal/fisiología , Humanos , Cambio de Clase de Inmunoglobulina , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genéticaRESUMEN
Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a peculiar T cell lymphoma, as expanding B cell clones are often present besides the malignant T cell clones. In addition, large numbers of Epstein-Barr virus (EBV)-infected B cells are frequently observed. To analyze the differentiation status and clonal composition of EBV-harboring B cells in AILD, single EBV-infected cells were micromanipulated from lymph nodes of six patients with frequent EBV(+) cells and their rearranged immunoglobulin (Ig) genes analyzed. Most EBV-infected B cells carried mutated Ig genes, indicating that in AILD, EBV preferentially resides in memory and/or germinal center B cells. EBV(+) B cell clones observed in all six cases ranged from small polyclonal to large monoclonal expansions and often showed ongoing somatic hypermutation while EBV(-) B cells showed little tendency for clonal expansion. Surprisingly, many members of expanding B cell clones had acquired destructive mutations in originally functional V gene rearrangements and showed an unfavorable high load of replacement mutations in the framework regions, indicating that they accumulated mutations over repeated rounds of mutation and division while not being selected through their antigen receptor. This sustained selection-free accumulation of somatic mutations is unique to AILD. Moreover, the survival and clonal expansion of "forbidden" (i.e., Ig-deficient) B cells has not been observed before in vivo and thus represents a novel type of viral latency in the B cell compartment. It is likely the interplay between the microenvironment in AILD lymph nodes and the viral transformation that leads to the survival and clonal expansion of Ig-less B cells.
Asunto(s)
Linfocitos B/virología , Reordenamiento Génico de Linfocito B , Herpesvirus Humano 4/aislamiento & purificación , Linfadenopatía Inmunoblástica/inmunología , Linfoma de Células T/inmunología , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD20 , Diferenciación Celular , Células Clonales , Femenino , Genes de Inmunoglobulinas , Genes Codificadores de la Cadena gamma de los Receptores de Linfocito T , Humanos , Linfadenopatía Inmunoblástica/virología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Activación de Linfocitos , Linfoma de Células T/virología , Masculino , Micromanipulación , Persona de Mediana Edad , ARN Viral/aislamiento & purificación , Latencia del VirusRESUMEN
To study homeostasis of peripheral B lymphocytes in the absence of B cell influx from the bone marrow, we generated a mouse mutant in which the recombination-activating gene (RAG)-2 can be inducibly deleted. When RAG-2 was deleted at the age of 8-10 wk, splenic naive follicular B cells were gradually lost over a year of observation, with a half-life of approximately 4.5 mo. By contrast, the pool of marginal zone B cells in the spleen and of B-1 cells in the peritoneal cavity were kept at normal level. In lymph nodes, approximately 90% of the B cells were lost within 4 mo, and B cell numbers remained constant thereafter. Mice in which RAG-2 was deleted at birth maintained a small population of activated B cells with an increased proportion of marginal zone B cells. Additionally, an increase of the pool of IgM secreting cells and B-1a cells was observed.
Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea/citología , Proteínas de Unión al ADN/inmunología , Animales , Linfocitos B/clasificación , Linfocitos B/inmunología , Células de la Médula Ósea/clasificación , Células de la Médula Ósea/inmunología , Diferenciación Celular , Proteínas de Unión al ADN/genética , Femenino , Homeostasis , Inmunoglobulina M/sangre , Inmunofenotipificación , Trabajo de Parto , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Factores de TiempoRESUMEN
To determine the function of immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, we generated mice in which Igalpha ITAM tyrosines were replaced by phenylalanines (Igalpha(FF/FF)). Igalpha(FF/FF) mice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that lambda1 light chain usage was increased. The mutants responded less efficiently to T cell-dependent antigens, whereas T cell-independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igalpha non-ITAM tyrosines. Strikingly, when the Igalpha ITAM mutation was combined with a truncation of Igbeta, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/citología , Citoplasma/metabolismo , Receptores Fc/metabolismo , Tirosina/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD79 , ADN , Ratones , Datos de Secuencia Molecular , Fenilalanina/química , Fosforilación , Receptores Fc/química , Tirosina/químicaRESUMEN
The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Bacteriófago P1/enzimología , Bacteriófago P1/genética , Integrasas/metabolismo , Mutagénesis Sitio-Dirigida/genética , Recombinación Genética/genética , Proteínas Virales/metabolismo , Secuencia de Bases , Western Blotting , Dimerización , Escherichia coli/genética , Genes Reporteros/genética , Genes Virales/genética , Integrasas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Eliminación de Secuencia/genética , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. MATERIALS AND METHODS: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. RESULTS: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. CONCLUSIONS: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.
Asunto(s)
Linaje de la Célula , Centro Germinal/patología , Enfermedad de Hodgkin/patología , Leucemia de Células B/patología , Linfoma Folicular/patología , Neoplasias Primarias Múltiples/patología , Anciano , Anciano de 80 o más Años , Células Clonales , Resultado Fatal , Femenino , Reordenamiento Génico de Cadena Pesada de Linfocito B , Reordenamiento Génico de Cadena Ligera de Linfocito B , Genes de Inmunoglobulinas/genética , Centro Germinal/inmunología , Enfermedad de Hodgkin/genética , Humanos , Región Variable de Inmunoglobulina/genética , Inmunohistoquímica , Inmunofenotipificación , Leucemia de Células B/genética , Linfoma Folicular/genética , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Células de Reed-Sternberg/inmunología , Células de Reed-Sternberg/patologíaRESUMEN
On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)alpha chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7--10 days, but minute amounts of surface-bound TCR beta chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naive T cells decay over time, the decay rates being faster for CD8(+) cells (t(1/2) approximately 16 days) than for CD4(+) cells (t(1/2) approximately 46 days). TCR(+) naïve cells are either maintained (CD8(+)) or decay more slowly (CD4(+); t(1/2) approximately 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8(+) cells; t(1/2) approximately 52 days) or not at all (CD4(+) cells), but at the population level, these cells fail to expand as their TCR(+) counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the alpha beta TCR contributes significantly to T-cell homeostasis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
We have developed a novel inducible Cre mutant with enhanced recombinase activity to mediate genetic switching events. The protein, designated Cre*PR, is composed of a new Cre mutant at the N-terminus followed by the ligand-binding domain (LBD) of the progesterone receptor (PR). The response to low doses of inducer is significantly enhanced by elongating the C-terminus of the PR LBD from amino acid 891 to 914. The mutant Cre lacks the first 18 amino acids and contains a Val-->Ala substitution at position 336, thereby destroying a cryptic splice donor at the 3'-end of CRE: The latter mutation reduces unwanted background recombinase activity in the absence of the synthetic ligand RU486 by a factor of at least 10 to an almost undetectable level. Thus, the recombinase activity turns out to be inducible by a factor of >200. We expect Cre*PR to serve as a valuable tool for conditional expression of genes both in vitro and in vivo.
Asunto(s)
Regulación de la Expresión Génica , Integrasas/genética , Integrasas/metabolismo , Mutación/genética , Receptores de Progesterona/química , Receptores de Progesterona/metabolismo , Recombinación Genética/genética , Proteínas Virales , Sustitución de Aminoácidos/genética , Animales , Bacteriófago P1/enzimología , Bacteriófago P1/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Marcación de Gen/métodos , Humanos , Integrasas/biosíntesis , Ligandos , Mifepristona/farmacología , Estructura Terciaria de Proteína , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Progesterona/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sensibilidad y Especificidad , TransfecciónRESUMEN
Hodgkin- and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease of the B lineage are the clonal progeny of antigen-experienced B cells harboring highly mutated immunoglobulin variable (V) region genes. Based on the detection of obviously destructive somatic mutations in a fraction of cases, we speculated that H/RS cells may be derived from a pre-apoptotic germinal center B cell. Seemingly contradicting this speculation, we present here the first case of classical Hodgkin's disease with H/RS cells harboring unmutated, potentially functional V region genes, which may indicate the derivation of the H/RS clone from a naive B cell. However, germinal center founder cells, which have not yet acquired somatic mutations, already have the intrinsic propensity to die by apoptosis. Thus, the rare occurrence of H/RS cells with unmutated V genes is expected if the H/RS cells are derived from the pool of pre-apoptotic germinal center B cells.
Asunto(s)
Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/patología , Región Variable de Inmunoglobulina/genética , Células de Reed-Sternberg/inmunología , Células de Reed-Sternberg/patología , Anciano , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/patología , Femenino , Centro Germinal/inmunología , Centro Germinal/patología , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/virología , Humanos , Inmunofenotipificación , Ganglios Linfáticos/patología , Biología Molecular , Mutación/inmunologíaRESUMEN
The generation of a diverse antigen receptor repertoire is fundamental for the functionality of the adaptive immune system. While the V(D)J recombination process that generates the primary antigen receptor repertoire is understood in great detail, it is still unclear by which mechanism immunoglobulin (Ig) genes are further diversified by somatic hypermutation. Using mouse strains that carry a non-functional, pre-defined V(H)D(H)J(H) gene segment in their IgH locus we demonstrate DNA double-strand breaks (DSBs) in and around V(H)D(H)J(H) in B cells undergoing somatic hypermutation. The generation of these DSBs depends on transcriptional activity, and their distribution along the V(H)D(H)J(H) segment parallels that of point mutations in the hypermutation domain. Furthermore, similar to hot spots of somatic hypermutation, 50-60% of all DSBs occur preferentially at RGYW motifs. DSBs may transiently dissociate the Ig promoter from the intronic enhancer to block further transcription and to initiate an error-prone non-homologous DSB repair pathway. In accord with this model large deletions are frequently produced, along with point mutations, in a V(H)D(H)J(H) segment inserted together with its promoter into the IgH locus in inverted orientation. Our data suggest that DSBs are reaction intermediates of the mechanism underlying somatic hypermutation.
Asunto(s)
Daño del ADN/genética , Inmunoglobulinas/genética , Mutación , Animales , Linfocitos B , Secuencia de Bases , Inversión Cromosómica , Mapeo Cromosómico , ADN , Región Variable de Inmunoglobulina , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Supresoras de Tumor , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/fisiología , Antígenos CD40/inmunología , Ciclo Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Fibroblastos/citología , Fibroblastos/metabolismo , Fase G1 , Marcación de Gen , Interleucina-4/inmunología , Interleucina-4/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Mitógenos/inmunología , Mitógenos/farmacología , Mutación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Fase de Descanso del Ciclo CelularRESUMEN
Progressively transformed germinal centers (PTGCs) are histologic structures mainly composed of small resting B cells and intermingled proliferating centroblast-like cells. The B-cell differentiation processes within PTGCs and their relation to classical germinal centers (GC) and to lymphocyte-predominant Hodgkin disease (LPHD), with which PTGCs are often associated, are largely unknown. To address these issues, single small resting (Ki67-) and proliferating (Ki67+) centroblast-like cells were isolated from 7 PTGCs of 5 lymph nodes, and rearranged immunoglobulin genes were amplified and sequenced. Most small resting B cells were clonally unrelated, and most carried unmutated immunoglobulin gene rearrangements resembling mantle zone B cells. Small resting B cells with mutated immunoglobulin gene rearrangements may represent centrocytes, memory B cells, or both. Among the centroblast-like Ki67+ cells, expanded B-cell clones were observed in 6 of 7 PTGCs analyzed. Clonally related V region genes showed extensive intraclonal diversity, and the mutation pattern indicated stringent selection of the cells for the expression of functional antigen receptors. Thus, somatic hypermutation, clonal expansion, and selection occur also in the disorganized PTGC microenvironment, as in classical GCs. In lymph nodes affected by PTGCs, no clonal expansion across the borders of individual PTGCs was observed, distinguishing PTGCs from LPHD.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/patología , Enfermedad de Hodgkin/inmunología , Adolescente , Adulto , Linfocitos B/química , Linfocitos B/citología , Diferenciación Celular , Niño , Femenino , Reordenamiento Génico de Linfocito B , Genes de Inmunoglobulinas , Centro Germinal/citología , Centro Germinal/inmunología , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Antígeno Ki-67/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Masculino , Modelos InmunológicosRESUMEN
In most cases, Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (HD) carry rearranged immunoglobulin (Ig) genes and thus derive from B cells. In rare cases, HRS cells originate from T cells. However, based on the unusual immunophenotype of HRS cells, often showing coexpression of markers typical for different hematopoietic lineages, and the regular detection of numerical chromosomal abnormalities, it has been speculated that HRS cells might represent cell fusions. Five cases of HD with 2 rearranged IgH alleles were analyzed for the presence of additional IgH alleles in germline configuration as a potential footprint of a cell fusion between a B and a non-B cell. Similarly, one case of T-cell-derived HD with biallelic T-cell receptor beta (TCRbeta) rearrangements was studied for the presence of unrearranged TCRbeta alleles. In none of the 6 cases was evidence for additional IgH (or TCRbeta) alleles obtained, strongly arguing against a role of cell fusion in HRS cell generation.
Asunto(s)
Enfermedad de Hodgkin/genética , Células de Reed-Sternberg/patología , Fusión Celular , Reordenamiento Génico de Cadena Pesada de Linfocito B , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Mutación de Línea Germinal , Humanos , Fragmentos de InmunoglobulinasRESUMEN
Receptor editing, clonal deletion, and anergy are the mechanisms by which B cells maintain tolerance to self antigens. To determine the extent to which receptor editing shapes the normal antibody repertoire, we generated an immunoglobulin kappa polymorphism that facilitates the detection of editing of immunoglobulin light chains in vivo. We found that B cells are targeted for editing during a 2-hour delay in development at the pre-BII cell stage, and that about 25% of all antibody molecules are produced by gene replacement. These results suggest that receptor editing represents a major force in shaping the antibody repertoire.
Asunto(s)
Linfocitos B/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Autotolerancia , Animales , Afinidad de Anticuerpos , Linfocitos B/metabolismo , Sitios de Unión de Anticuerpos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes de Inmunoglobulinas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Regiones Constantes de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Proteínas Nucleares , Recombinación GenéticaRESUMEN
The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Mutación , Receptores de Antígenos de Linfocitos B/genética , Alelos , Animales , Apoptosis/genética , Apoptosis/inmunología , Linfocitos B/metabolismo , Secuencia de Bases , Señalización del Calcio/genética , Señalización del Calcio/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Cartilla de ADN/genética , Inmunoglobulina D/biosíntesis , Inmunoglobulina M/biosíntesis , Ratones , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Somatic hypermutation specifically modifies rearranged immunoglobulin (Ig) genes in germinal center (GC) B cells. However, the bcl-6 gene can also acquire somatic mutations during the GC reaction, indicating that certain non-Ig genes can be targeted by the somatic hypermutation machinery. The CD95 gene, implicated in negative selection of B lymphocytes in GCs, is specifically expressed by GC B cells and was recently identified as a tumor suppressor gene being frequently mutated in (post) GC B cell lymphomas. In this study, the 5' region (5'R) and/or the last exon coding for the death domain (DD) of the CD95 gene were investigated in naive, GC, and memory B cells from seven healthy donors. About 15% of GC and memory, but not naive, B cells carried mutations within the 5'R (mutation frequency 2.5 x 10(-4) per basepair). Mutations within the DD were very rare but could be efficiently selected by inducing CD95-mediated apoptosis: in 22 apoptosis-resistant cells, 12 DD mutations were found. These results indicate that human B cells can acquire somatic mutations of the CD95 gene during the GC reaction, which potentially confers apoptosis resistance and may counteract negative selection through the CD95 pathway.