RESUMEN
The B-cell receptor (BCR) transmits a tonic survival signal in the absence of antigen stimulation and an antigen-triggered survival signal. Mature B cells express two types of BCR, IgM and IgD, but it remains unclear how B-cell survival is differentially regulated by these two receptors. We found that, whereas cross-linking IgM on spleen B cells greatly enhanced their survival, cross-linking IgD did not enhance, but rather decreased, their survival. Consistently, cross-linking both IgM and IgD only moderately enhanced B-cell survival, suggesting that IgM and IgD play opposing roles in B-cell survival induced by BCR stimulation. Based on these and additional experimental results, we present a mathematical model integrating IgM- and IgD-mediated survival signals. Our model shows that IgD can transmit a tonic survival signal in the absence of antigen stimulation but cross-linking IgD not only does not generate a survival signal but also disrupts its tonic signal, resulting in inhibition of B-cell survival. These results suggest that IgD attenuates BCR-induced survival in mature B cells, presumably to restrain B-cell response to weak and/or self-antigens and prevent nonspecific B-cell activation and autoimmunity.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Supervivencia Celular , Inmunoglobulina D/metabolismo , Inmunoglobulina M/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Teóricos , Transducción de Señal , Bazo/citologíaRESUMEN
B-1 cells are innate-like B-cell population and produce natural antibodies that contribute to the first line of host defense. There are two subsets of B-1 cells: B-1a and B-1b. B-1a cells are the main producer of poly-reactive and autoreactive natural IgM antibodies, whereas B-1b cells can respond specifically to T-cell-independent antigens. Despite the functional significance of B-1a and B-1b cells, little information is available about what regulates the development of these two subsets. We found that Kelch-like protein 14 (KLHL14) was expressed at high levels in B cells but only at low levels in a few non-lymphoid tissues. Although mice lacking KLHL14 died right after birth, the heterozygotes developed normally with no gross abnormalities by appearance. B-cell development in the bone marrow and maturation and activation in the spleen were not affected in the heterozygous mice. However, the number of peritoneal B-1a cells was significantly reduced while B-1b cells were increased in Klhl14 heterozygous mice compared with wild-type (WT) mice. Consistently, Rag1-/- mice reconstituted with Klhl14-/- fetal liver cells had a more severe reduction of B-1a and an increase of B-1b cells in the peritoneal cavity. KLHL14 did not affect the turnover or apoptosis of B-1a and B-1b cells in vivo. Moreover, Klhl14-/- fetal liver contained a similar proportion and absolute numbers of the B-1 progenitor cells as did WT fetal liver. These results suggest that KLHL14 promotes B-1a development in mice.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Subgrupos de Linfocitos B/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficienciaRESUMEN
Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. How AID is targeted to the Ig V gene and switch region to trigger SHM and CSR remains elusive. Primary B cells stimulated with CD40L plus IL-4 or LPS plus IL-4 undergo efficient CSR, but it has been difficult to induce SHM in these cells. In the current study, we used B cells from B1-8hi mice carrying a prerecombined VH186.2DFL16.1JH2 Ab gene to investigate the induction of SHM under in vitro culture conditions. B1-8hi splenic B cells stimulated with CD40L plus IL-4 or LPS plus IL-4 underwent robust CSR to IgG1, but failed to generate SHM in the VH186.2 gene. Remarkably, ectopic expression of AID in AID-deficient, but not wild-type, B1-8hi B cells induced efficient SHM at a rate close to that observed in germinal center B cells. We further established an AID-deficient CH12 B lymphoma line and found that ectopic expression of AID in the mutant line, but not in AID-sufficient CH12 cells, induced efficient point mutations and deletions in the V gene. These results demonstrate that the endogenous AID in ex vivo-activated primary B and B lymphoma cells not only cannot induce SHM but also inhibit the induction of SHM by the exogenous AID. Our results further suggest that the spatiotemporal distribution and/or posttranslational modification of AID strongly affects the induction of SHM in ex vivo-activated primary B cells.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/inmunología , Inmunoglobulina G/inmunología , Activación de Linfocitos , Hipermutación Somática de Inmunoglobulina , Animales , Línea Celular Tumoral , Citidina Desaminasa/genética , Inmunoglobulina G/genética , Ratones , Ratones Noqueados , Mutación PuntualRESUMEN
This paper reports a twisting microfluidic method utilising a centrifuge-based fluid extruding system in a planetary centrifuge which simultaneously generates an orbital rotation and an axial spin. In this method, fluid extrusion from a micro-scale capillary to an 'open-space' solution or air enables release of the fluid from the capillary-based microchannel, which physically means that there is a release of fluids from a confined low-Reynolds-number environment to an open non-low-Reynolds-number environment. As a result, the extruded fluids are separated from the axial spin of the capillary, and the difference in the angular rates of the axial spin between the capillary and the extruded fluids produces the 'twisting' of the fluid. In this study, we achieve control of the twist of highly viscous fluids, and we construct a simple physical model for the fluid twist. In addition, we demonstrate the formation of twisted hydrogel microstructures (stripe-patterned microbeads and multi-helical microfibres) with control over the stripe pattern and the helical pitch length. We believe that this method will enable the generation of more sophisticated microstructures which cannot easily be formed by usual channel-based microfluidic devices. This method can also provide advanced control of microfluids, as in the case of rapid mixing of highly viscous fluids. This method can contribute to a wide range of applications in materials science, biophysics, biomedical science, and microengineering in the future.
RESUMEN
Hypomorphic RAG1 or RAG2 mutations cause primary immunodeficiencies and can lead to autoimmunity, but the underlying mechanisms are elusive. We report here a patient carrying a c.116+2T>G homozygous splice site mutation in the first intron of RAG1, which led to aberrant splicing and greatly reduced RAG1 protein expression. B cell development was blocked at both the pro-B to pre-B transition and the pre-B to immature B cell differentiation step. The patient B cells had reduced B cell receptor repertoire diversity and decreased complementarity determining region 3 lengths. Despite B cell lymphopenia, the patient had abundant plasma cells in the BM and produced large quantities of IgM and IgG Abs, including autoantibodies. The proportion of naive B cells was reduced while the frequency of IgD-CD27- double-negative (DN) B cells, which quickly differentiated into Ab-secreting plasma cells upon stimulation, was greatly increased. Immune phenotype analysis of 52 patients with primary immunodeficiency revealed a strong association of the increased proportion of DN B and memory B cells with decreased number and proportion of naive B cells. These results suggest that the lymphopenic environment triggered naive B cell differentiation into DN B and memory B cells, leading to increased Ab production.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/genética , Linfocitos B/inmunología , Granuloma/genética , Proteínas de Homeodominio/genética , Síndromes de Inmunodeficiencia/genética , Linfopoyesis/genética , Receptores de Antígenos de Linfocitos B/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Niño , Trasplante de Células Madre de Sangre del Cordón Umbilical , Resultado Fatal , Granuloma/inmunología , Granuloma/terapia , Proteínas de Homeodominio/metabolismo , Homocigoto , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/terapia , Memoria Inmunológica/inmunología , Linfopenia/genética , Linfopenia/inmunología , Linfopoyesis/inmunología , Masculino , Células Plasmáticas/inmunología , Sitios de Empalme de ARN/genética , Recombinación V(D)J/genéticaRESUMEN
CD40 ligand (CD40 L) expressed by activated T cells interacts with CD40 on B cells and triggers B cell survival, proliferation and differentiation. Deficiency in CD40 L or CD40 in humans causes hyper IgM syndrome due to a defect in T-B interaction that is essential for Ig gene class switch recombination (CSR). CD40 L belongs to the tumor necrosis factor family and normally forms a homotrimer on the cell surface, which is important for its biological activity. To generate a multimeric CD40 L that can be used to stimulate both mouse and human B cells, we fused the extracellular domain of mouse CD40 L, which is known to also bind human CD40, with streptavidin (SA) that forms a stable tetramer under physiological conditions. As expected, 293 T cells transiently transfected with an SA-CD40 L expression vector secreted tetrameric SA-CD40 L in the culture supernatant. The secreted SA-CD40 L exhibited > 25-fold stronger activities in inducing the survival, activation and proliferation of both mouse and human primary B cells than did an agonistic anti-mouse or anti-human CD40 antibody. In the presence of IL-4, SA-CD40 L also induced efficient CSR and plasma cell differentiation in both mouse and human B cells. Moreover, administration of SA-CD40 L in mice induced activation and proliferation of spleen B cells in vivo. These results demonstrate that the SA-CD40 L fusion protein generated in the present study recapitulates the function of membrane-bound trimeric CD40 L and has potent biological activities in both mouse and human primary B cells.
Asunto(s)
Ligando de CD40/farmacología , Diferenciación Celular/efectos de los fármacos , Células Plasmáticas/inmunología , Proteínas Recombinantes de Fusión/farmacología , Animales , Antígenos CD40/inmunología , Ligando de CD40/genética , Ligando de CD40/inmunología , Diferenciación Celular/inmunología , Femenino , Humanos , Síndrome de Inmunodeficiencia con Hiper-IgM/tratamiento farmacológico , Síndrome de Inmunodeficiencia con Hiper-IgM/genética , Síndrome de Inmunodeficiencia con Hiper-IgM/inmunología , Síndrome de Inmunodeficiencia con Hiper-IgM/patología , Masculino , Ratones , Células Plasmáticas/patología , Dominios Proteicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.
Asunto(s)
Metilación de ADN/genética , Pulmón/metabolismo , PPAR gamma/genética , Proteína Proto-Oncogénica c-ets-1/genética , Receptor gamma X Retinoide/genética , Células Epiteliales Alveolares/metabolismo , Linaje de la Célula/genética , Epigénesis Genética , GTP Fosfohidrolasas/genética , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Humanos , Captura por Microdisección con Láser , Pulmón/citología , Regiones Promotoras Genéticas , Transducción de Señal , Linfocitos T/metabolismo , Transcriptoma/genética , Secuenciación Completa del GenomaRESUMEN
The BCR constitutively transmits a "tonic" survival signal in the absence of exogenous antigen-binding. However, the strength of tonic BCR signal and its relationship with antigen-triggered survival signal are poorly understood. We found that primary B cells expressing high levels of BCR had elevated BCR tonic signal and increased survival compared with those expressing low levels of BCR. In addition, we found that crosslinking BCR with low doses of F(ab')2 α-IgM antibodies did not enhance, but rather decreased, B cell survival and that only when most of the BCR were occupied by F(ab')2 α-IgM antibodies was B cell survival enhanced. Based on these experimental results, we present a mathematical model integrating tonic and antigen-triggered BCR signals. Our model indicates that the signal generated from crosslinked BCR is 4.3 times as strong as the tonic signal generated from free BCR and that the threshold of B cell activation corresponds to the signal generated by crosslinking 61% of the surface BCR. This model also allows the prediction of the survival probability of a B cell based on its initial BCR level and the strength and duration of antigen stimulation, and fits with the mechanism of B cell tolerance.