RESUMEN
The AM14 BCR, derived from an autoimmune MRL/lpr mouse, binds autologous IgG2aa/j with low affinity, and as a result, AM14 B cells only proliferate in response to IgG2a immune complexes that incorporate DNA, RNA, or nucleic acid-binding proteins that serve as autoadjuvants. As such, AM14 B cells have served as a useful model for demonstrating the importance of BCR/TLR coengagement in the activation of autoreactive B cells. We now show that the same receptor recognizes an additional murine-encoded Ag, expressed by B6 splenocytes, with sufficient avidity to induce a TLR-independent proliferative response of BALB/c AM14 Vκ8 B cells both in vivo and in vitro. Moreover, detection of this cross-reactive Ag by B6 AM14 Vκ8 B cells promotes an anergic phenotype as reflected by suboptimal responses to BCR cross-linking and the absence of mature B cells in the bone marrow. The B6 Ag further impacts B cell development as shown by a dramatically expanded marginal zone compartment and extensive receptor editing in B6 AM14 Vκ8 mice but not BALB/c AM14 Vκ8 mice. Despite their anergic phenotypes, B6 AM14 Vκ8 B cells can respond robustly to autoantigen/autoadjuvant immune complexes and could therefore participate in both autoimmune responses and host defense.
RESUMEN
UNLABELLED: Virus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimeric proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here, we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in prefusion (pre-F/F) or postfusion (post-F/F) configurations. The structures of the chimeric proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody. Following immunization of mice, without adjuvant, pre-F/F-containing VLPs induced significantly higher neutralizing antibody titers than the post-F/F-containing VLPs or the wild-type F/F-containing VLPs after a single immunization but not after prime and boost immunization. The specificities of anti-F IgG induced by the two mutant VLPs were assessed by enzyme-linked immunosorbent assay (ELISA) using soluble forms of the prefusion and postfusion forms of the F protein as targets. While both types of VLPs stimulated similar levels of IgG specific for the soluble postfusion F protein, titers of IgG specific for prefusion F induced by the pre-F/F-containing VLPs were higher than those induced by post-F/F-containing VLPs. Thus, VLPs containing a stabilized prefusion form of the RSV F protein represent a promising RSV vaccine candidate. IMPORTANCE: The development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle-associated RSV F protein containing mutations that stabilize the structure in a prefusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild-type or postfusion conformation. These findings indicate that the prefusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate.
Asunto(s)
Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Pruebas de Neutralización , Virus de la Enfermedad de Newcastle/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitiales Respiratorios/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) initiates a process generating DNA mutations and breaks in germinal center (GC) B cells that are necessary for somatic hypermutation and class-switch recombination. GC B cells can "tolerate" DNA damage while rapidly proliferating because of partial suppression of the DNA damage response by BCL6. In this study, we develop a model to study the response of mouse GC B cells to endogenous DNA damage. We show that the base excision repair protein apurinic/apyrimidinic endonuclease (APE) 2 protects activated B cells from oxidative damage in vitro. APE2-deficient mice have smaller GCs and reduced Ab responses compared with wild-type mice. DNA double-strand breaks are increased in the rapidly dividing GC centroblasts of APE2-deficient mice, which activate a p53-independent cell cycle checkpoint and a p53-dependent apoptotic response. Proliferative and/or oxidative damage and AID-dependent damage are additive stresses that correlate inversely with GC size in wild-type, AID-, and APE2-deficient mice. Excessive double-strand breaks lead to decreased expression of BCL6, which would enable DNA repair pathways but limit GC cell numbers. These results describe a nonredundant role for APE2 in the protection of GC cells from AID-independent damage, and although GC cells uniquely tolerate DNA damage, we find that the DNA damage response can still regulate GC size through pathways that involve p53 and BCL6.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/inmunología , Daño del ADN , Endonucleasas/inmunología , Centro Germinal/inmunología , Animales , Apoptosis/genética , Apoptosis/inmunología , Linfocitos B/metabolismo , Ciclo Celular/genética , Ciclo Celular/inmunología , Proliferación Celular , Células Cultivadas , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Roturas del ADN de Doble Cadena , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Endonucleasas/deficiencia , Endonucleasas/genética , Citometría de Flujo , Centro Germinal/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Enzimas Multifuncionales , Estrés Oxidativo/inmunología , Proteínas Proto-Oncogénicas c-bcl-6 , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
UNLABELLED: Immunization with virus-like particles (VLPs) containing the Newcastle disease virus (NDV) core proteins, NP and M, and two chimera proteins (F/F and H/G) containing the respiratory syncytial virus (RSV) F- and G-protein ectodomains fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins, respectively, stimulated durable RSV-neutralizing antibodies, F-protein-specific long-lived, bone marrow-associated plasma cells (LLPCs), and B cell memory, in striking contrast to RSV infection, which did not (M. R. Schmidt, L. W. McGinnes, S. A. Kenward, K. N. Willems, R. T. Woodland, and T. G. Morrison, J. Virol. 86:11654-11662, 2012). Here we report the characterization of a VLP with an RSV F-protein ectodomain fused to the NDV F-protein heptad repeat 2 (HR2), transmembrane, and cytoplasmic domain sequences, creating a chimera with two tandem HR2 domains, one from the RSV F protein and the other from the NDV F-protein ectodomain (F/HR2F). The F/HR2F chimera protein was efficiently assembled into VLPs along with the H/G chimera protein. This VLP (VLP-H/G+F/HR2F) stimulated anti-F-protein and anti-G-protein IgG, durable RSV-neutralizing antibodies, and anti-RSV F-protein-secreting LLPCs. However, the subtypes of anti-F-protein IgG induced were different from those elicited by VLPs containing the F/F chimera (VLP-H/G+F/F). Most importantly, VLP-H/G+F/HR2F did not induce RSV F-protein-specific B cell memory, as shown by the adoptive transfer of B cells from immunized animals to immunodeficient animals. The VLP did, however, induce B cell memory specific to the RSV G protein. Thus, the form of the F protein has a direct role in inducing anti-F-protein B cell memory. IMPORTANCE: The development of vaccines for respiratory syncytial virus (RSV) is hampered by a lack of a clear understanding of the requirements for eliciting protective as well as durable human immune responses to virus antigens. The results of this study indicate that the form of the RSV F protein has a direct and significant impact on the type of anti-F-protein IgG antibodies induced and the generation of F-protein-specific memory. Identification of the conformation of the RSV F protein that most effectively stimulates not only LLPCs and but also memory B cells will be important in the future development of RSV vaccines.
Asunto(s)
Linfocitos B/inmunología , Memoria Inmunológica , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vectores Genéticos , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Pruebas de Neutralización , Virus de la Enfermedad de Newcastle/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Although respiratory syncytial virus (RSV) is a significant human pathogen, no RSV vaccines are available. We have reported that a virus-like particle (VLP) RSV vaccine candidate stimulated, in mice, robust, protective anti-RSV glycoprotein T(H)1 biased immune responses without enhanced respiratory disease upon RSV challenge. We report here an analysis of long-term responses to these VLPs. BALB/c mice immunized, without adjuvant, with VLPs or with infectious RSV generated anti-F and anti-G protein serum antibody responses that were stable over 14 months. Neutralizing antibody titers stimulated by VLPs were robust and durable for 14 months, whereas those of RSV-immunized animals declined significantly by 3 months. F protein-specific antibody-secreting cells were detected in the bone marrows of VLP-immunized mice but not in the marrows of RSV-immunized mice. Adoptive transfer of enriched splenic B cells from VLP-immunized mice into immunodeficient rag(-/-) mice resulted in anti-F and anti-G protein serum IgG antibody responses, in recipient mice, that were protective upon RSV challenge. In contrast, transfer of splenic B cells from RSV-immunized mice produced no detectable serum antibody in the recipients, nor could these mice inhibit RSV replication upon virus challenge. Immunization with VLPs stimulated the formation of germinal center GL7(+) B cells in normal mice. VLP immunization of TCR ßδ(-/-) T-cell-deficient mice did not induce anti-RSV IgG antibodies, results consistent with T-cell-dependent immune responses. These results demonstrate that VLPs are effective in stimulating long-lived RSV-specific, T-cell-dependent neutralizing antibody-secreting cells and RSV-specific memory responses.
Asunto(s)
Memoria Inmunológica , Virus de la Enfermedad de Newcastle/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Vacunas Virales/inmunología , Traslado Adoptivo , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Inmunoglobulina G/sangre , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Virus de la Enfermedad de Newcastle/genética , Virus Sincitial Respiratorio Humano/genética , Factores de Tiempo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Virosoma/administración & dosificación , Vacunas de Virosoma/genética , Vacunas de Virosoma/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21(int)-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-kappaB2). Sustained c-Rel activation required de novo c-Rel gene transcription and translation via Btk-dependent mechanisms. Like T1 cells, mature B cells from Btk- and c-Rel-deficient mice also failed to activate these genes. These findings suggest that the gain of survival potential within transitional B cells is dependent on the ability to produce a long-term c-Rel response, which plays a critical role in T2 B cell survival and differentiation in vivo by inducing anti-apoptotic genes, BAFF-R and NF-kappaB2, an essential component for BAFF-R survival signaling. Thus, acquisition of resistance to apoptosis during transitional B cell maturation is achieved by integration of BCR and BAFF-R signals.
Asunto(s)
Receptor del Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Subunidad p52 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Linfocitos B/citología , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-rel/genética , Transducción de Señal/inmunología , Especificidad por Sustrato , Factores de TiempoRESUMEN
The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.
Asunto(s)
Linfocitos B/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Antígenos CD19/biosíntesis , Linfocitos B/citología , Núcleo Celular/metabolismo , Separación Celular , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/metabolismo , Vacunas Neumococicas/uso terapéutico , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/química , Transducción de SeñalRESUMEN
Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid, B-lymphoid, and erythroid lineages, but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization, which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Animales , Antígenos CD34/sangre , Linfocitos B/citología , Linfocitos B/inmunología , Técnicas de Cultivo de Célula/métodos , Femenino , Hematopoyesis , Humanos , Recién Nacido , Subunidad gamma Común de Receptores de Interleucina/genética , Activación de Linfocitos , Linfopoyesis , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas Recombinantes/administración & dosificación , Linfocitos T/citología , Linfocitos T/inmunología , Acondicionamiento Pretrasplante , Trasplante Heterólogo , Factor de Necrosis Tumoral alfa/administración & dosificaciónRESUMEN
We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.
Asunto(s)
Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Proteínas Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas/inmunología , Transducción de Señal/inmunología , Animales , Atrofia/genética , Atrofia/inmunología , Atrofia/patología , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/inmunología , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Centro Germinal/patología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Glucólisis/inmunología , Inmunosupresores/farmacología , Ratones , Ratones Noqueados , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TORRESUMEN
Immunodeficient NOD-scid mice bearing a targeted mutation in the IL2 receptor common gamma chain (Il2rgamma(null)) readily engraft with human stem cells. Here we analyzed human peripheral blood mononuclear cells (PBMC) for their ability to engraft NOD-scid Il2rgamma(null) mice and established engraftment kinetics, optimal cell dose, and the influence of injection route. Even at low PBMC input, NOD-scid Il2rgamma(null) mice reproducibly support high human PBMC engraftment that plateaus within 3-4 weeks. In contrast to previous stocks of immunodeficient mice, we observed low intra- and inter-donor variability of engraftment. NOD-scid Il2rgamma(null) mice rendered hyperglycemic by streptozotocin treatment return to normoglycemia following transplantation with human islets. Interestingly, these human islet grafts are rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide. These data suggest that humanized NOD-scid Il2rgamma(null) mice may represent an important surrogate for investigating in vivo mechanisms of human islet allograft rejection.
Asunto(s)
Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Islotes Pancreáticos/inmunología , Leucocitos Mononucleares/inmunología , Mutación/genética , Animales , Diabetes Mellitus/inmunología , Modelos Animales de Enfermedad , Rechazo de Injerto/inmunología , Humanos , Trasplante de Islotes Pancreáticos/inmunología , Leucocitos Mononucleares/trasplante , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , FenotipoRESUMEN
Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.
Asunto(s)
Linfocitos B/enzimología , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/fisiología , Agammaglobulinemia Tirosina Quinasa , Animales , Receptor del Factor Activador de Células B/deficiencia , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/fisiología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Naïve peripheral B cells survive in vivo because of active stimulation by the TNF superfamily ligand B lymphocyte stimulator (BLyS/BAFF). Although the survival promoting properties of BLyS are well known, the signal pathways and molecular effectors that characterize this stimulation are still being elucidated. In this communication, we discuss the signal cascades that effect BLyS dependent survival and the regulation of BLyS induced signaling. We also examine the role of BLyS as a growth factor and propose that BLyS induced metabolic enhancement optimizes the B cell response to BCR and TLR-dependent signaling.
Asunto(s)
Factor Activador de Células B/fisiología , Receptor del Factor Activador de Células B/fisiología , Subgrupos de Linfocitos B/citología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Empalme Alternativo , Animales , Apoptosis , Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/inmunología , Antígeno de Maduración de Linfocitos B/inmunología , Antígeno de Maduración de Linfocitos B/fisiología , Subgrupos de Linfocitos B/inmunología , Supervivencia Celular , Homeostasis , Humanos , Cambio de Clase de Inmunoglobulina , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos A , Modelos Inmunológicos , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/fisiología , Proteínas Quinasas/fisiología , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal , Factores de Transcripción/fisiología , Transfección , Proteína Activadora Transmembrana y Interactiva del CAML/inmunología , Proteína Activadora Transmembrana y Interactiva del CAML/fisiología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/fisiologíaRESUMEN
Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cmu) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/genética , ADN Glicosilasas/genética , Reordenamiento Génico de Linfocito B/genética , Cadenas mu de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/genética , Animales , Linfocitos B/citología , Citidina Desaminasa/inmunología , ADN/genética , ADN/inmunología , ADN Glicosilasas/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Reordenamiento Génico de Linfocito B/inmunología , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Cadenas mu de Inmunoglobulina/inmunología , Ratones , Ratones Mutantes , Recombinación Genética/genética , Recombinación Genética/inmunología , Hipermutación Somática de Inmunoglobulina/inmunología , Bazo/citología , Bazo/inmunología , Uracil-ADN GlicosidasaRESUMEN
As an update to previously published recommendations for the management of Helicobacter pylori infection, an evidence-based appraisal of 14 topics was undertaken in a consensus conference sponsored by the Canadian Helicobacter Study Group. The goal was to update guidelines based on the best available evidence using an established and uniform methodology to address and formulate recommendations for each topic. The degree of consensus for each recommendation is also presented. The clinical issues addressed and recommendations made were: population-based screening for H. pylori in asymptomatic children to prevent gastric cancer is not warranted; testing for H. pylori in children should be considered if there is a family history of gastric cancer; the goal of diagnostic interventions should be to determine the cause of presenting gastrointestinal symptoms and not the presence of H. pylori infection; recurrent abdominal pain of childhood is not an indication to test for H. pylori infection; H. pylori testing is not required in patients with newly diagnosed gastroesophageal reflux disease; H. pylori testing may be considered before the use of long-term proton pump inhibitor therapy; testing for H. pylori infection should be considered in children with refractory iron deficiency anemia when no other cause has been found; when investigation of pediatric patients with persistent or severe upper abdominal symptoms is indicated, upper endoscopy with biopsy is the investigation of choice; the 13C-urea breath test is currently the best noninvasive diagnostic test for H. pylori infection in children; there is currently insufficient evidence to recommend stool antigen tests as acceptable diagnostic tools for H. pylori infection; serological antibody tests are not recommended as diagnostic tools for H. pylori infection in children; first-line therapy for H. pylori infection in children is a twice-daily, triple-drug regimen comprised of a proton pump inhibitor plus two antibiotics (clarithromycin plus amoxicillin or metronidazole); the optimal treatment period for H. pylori infection in children is 14 days; and H. pylori culture and antibiotic sensitivity testing should be made available to monitor population antibiotic resistance and manage treatment failures.
Asunto(s)
Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/aislamiento & purificación , Gastropatías/microbiología , Adolescente , Anemia Ferropénica/diagnóstico , Pruebas Respiratorias , Niño , Endoscopía Gastrointestinal , Medicina Basada en la Evidencia , Infecciones por Helicobacter/tratamiento farmacológico , Humanos , Tamizaje Masivo , Gastropatías/tratamiento farmacológico , Neoplasias Gástricas/genéticaRESUMEN
Naïve B cells introduced into a lymphopenic host undergo antigen-independent proliferation which is inhibited in a cell dose dependent manner by feedback from mature B cells. Homeostatic proliferation is a generalized lymphocyte property with B cells sharing many of the inductive and regulatory characteristics established for naïve and memory CD4+ and CD8+ T cells and NK cells. In this communication we discuss the cytokine requirements for B cell HP, extend the murine studies to human cells, and propose the hypothesis that B cell HP may provide an antigen-independent mechanism for maintaining B cell memory.
Asunto(s)
Linfocitos B/inmunología , Homeostasis , Activación de Linfocitos , Animales , Linfocitos B/fisiología , Humanos , Memoria Inmunológica , RatonesRESUMEN
Many microbial pathogens employ antigenic variation as a strategy to evade the immune system, posing a challenge in vaccine development. To understand the requirements for immunity against such pathogens, we studied Borrelia hermsii, a relapsing fever bacterium. We found that mice deficient in T, follicular B, marginal zone B, or B1a lymphocytes resolved B. hersmii bacteremia and became resistant to reinfection. The resolution of bacteremia coincided with an expansion and persistence of B1b lymphocytes, and purified B1b lymphocytes from convalescent wild-type or TCR-betaxdelta-/- mice conferred immunity to Rag1-/- mice. The B1b lymphocytes in the reconstituted Rag1-/- mice provided long-lasting immunity by rapidly generating B. hermsii-specific IgM but not IgG upon bacterial challenge. Unmutated IgM is sufficient to eliminate B. hermsii, because AID-/- mice deficient in somatic hypermutation and class switch recombination efficiently resolved all bacteremic episodes. These data demonstrate that B1b lymphocytes can provide long-lasting T cell-independent IgM memory.
Asunto(s)
Antígenos T-Independientes/inmunología , Linfocitos B/inmunología , Infecciones por Borrelia/inmunología , Memoria Inmunológica , Traslado Adoptivo , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoglobulina M/sangre , RatonesRESUMEN
The rate of pathogen clearance is a critical determinant of morbidity and mortality. We sought to characterize the immune response responsible for the remarkably rapid clearance of individual episodes of bacteremia caused by the relapsing fever bacterium, Borrelia hermsii. SCID or Rag(-/-) mice were incapable of resolving B. hermsii infection, indicating a critical role for T and/or B cells. TCR(-/-) mice, which lack T cells, and IL-7(-/-) mice, which are deficient in both T cells and follicular B cells, but not in B1 cells and splenic marginal zone (MZ) B cells, efficiently cleared B. hermsii. These findings suggested that B1 cells and/or MZ B cells, two B cell subsets that are known to participate in rapid, T-independent responses, might be involved. The efficient resolution of the episodes of moderate level bacteremia by splenectomized mice suggested that MZ B cells do not play the primary role in clearance of this bacterium. In contrast, xid mice, which are deficient in B1 cells, suffered more severe episodes of bacteremia than wild-type mice. The hypothesis that B1 cells are critical for clearance of B. hermsii was further supported by a selective expansion of the B1b (i.e., IgM(high), IgD(-/low), Mac1(+) CD23(-), and CD5(-)) cell subset in infected xid mice, which coincided with the eventual resolution of infection. Finally, mice selectively incapable of secreting IgM, the dominant isotype produced by B1 cells, were completely unable to clear B. hermsii. Together these results support the model that B1b cells generate the T-independent IgM required for the control and resolution of relapsing fever borreliosis.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Inmunoglobulina M/fisiología , Fiebre Recurrente/inmunología , Animales , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/microbiología , Bacteriemia/genética , Bacteriemia/inmunología , Bacteriemia/microbiología , Actividad Bactericida de la Sangre/genética , Actividad Bactericida de la Sangre/inmunología , Borrelia/crecimiento & desarrollo , Borrelia/inmunología , Borrelia/aislamiento & purificación , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Inmunidad Innata/genética , Inmunoglobulina M/biosíntesis , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Mutantes , Ratones SCID , Fiebre Recurrente/genética , Fiebre Recurrente/microbiología , Bazo/citología , Bazo/inmunología , Bazo/microbiología , Linfocitos T/inmunología , Linfocitos T/microbiologíaRESUMEN
Naive peripheral B cells are maintained in sufficient numbers and diversity to mount effective immune responses against infectious agents. However, the size and repertoire of this B cell pool is constantly diminished by normal cell turnover and Ag activation. Homeostatic (Ag-independent) proliferation in response to B cell depletion is one mechanism to compensate for this cell loss. We have used purified CFSE-labeled B cells and an adoptive transfer model system to show that immature and mature B cells divide in a variety of B cell-deficient (scid, xid, IL-7(-/-), and sublethally irradiated) hosts. Homeostatic B cell proliferation is T cell independent, and B cells that have replicated by this mechanism retain the antigenic phenotype of naive B cells. Replication is significantly reduced in B cell-sufficient normal or B cell-reconstituted immunodeficient recipients by the action of competing mature follicular B cells. Using xid mice and transcription factor knockouts, we show that the activation signal(s) that lead to homeostatic B cell proliferation require Bruton's tyrosine kinase; however, c-Rel, a Bruton's tyrosine kinase-induced NF-kappaB/Rel transcription factor critical for Ag and mitogen stimulation, is dispensable, indicating the uniqueness of this activation pathway. Survival and replication signals can also be separated, because the transcription factor p50 (NF-kappaB1), which is required for the survival of peripheral B cells, is not necessary for homeostatic replication. Homeostatic B cell proliferation provides an Ag-independent mechanism for the maintenance and expansion of naive B cells selected into the mature B cell pool.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Homeostasis/inmunología , Interfase/inmunología , Linfopenia/inmunología , Traslado Adoptivo , Agammaglobulinemia Tirosina Quinasa , Animales , Subgrupos de Linfocitos B/patología , Subgrupos de Linfocitos B/trasplante , Diferenciación Celular/genética , Diferenciación Celular/inmunología , División Celular/genética , División Celular/inmunología , Retroalimentación , Femenino , Homeostasis/genética , Inmunofenotipificación , Interleucina-17/deficiencia , Interleucina-17/genética , Interfase/genética , Linfopenia/genética , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , FN-kappa B/deficiencia , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-rel/deficiencia , Proteínas Proto-Oncogénicas c-rel/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Bazo/citología , Bazo/inmunología , Bazo/patologíaRESUMEN
B cell susceptibility to Fas-mediated apoptosis is regulated in a receptor-specific fashion. CD40 engagement produces marked sensitivity to Fas killing, whereas surface Ig (sIg) engagement blocks Fas signaling for cell death in otherwise sensitive, CD40-stimulated B cell targets, and thus, induces a state of Fas resistance. The signaling mediator, Bruton's tyrosine kinase (Btk), is required for certain sIg-triggered responses, and Btk is reported to directly bind Fas and block Fas-mediated apoptosis. For these reasons, the role of Btk as a mediator of sIg-induced Fas resistance was examined. Dysfunction of Btk through mutation, and absence of Btk through deletion did not interfere with induction of Fas resistance by anti-Ig. This may be due, at least in part, to induction of Btk-dependent Bcl-2 family members by anti-Ig after CD40 ligand treatment. However, the susceptibility to Fas-mediated apoptosis of B cell targets stimulated by CD40 ligand alone was increased in the absence of Btk. These results indicate that Fas resistance produced by sIg triggering does not require Btk, but suggests that in certain situations Btk modulates B cell susceptibility to Fas killing.