RESUMO
Compared with naïve B cells, the B cell receptor (BCR) signal in germinal center (GC) B cells is attenuated; however, the significance of this signaling attenuation has not been well defined. Here, to investigate the role of attenuation of BCR signaling, we employed a Csk mutant mouse model in which Csk deficiency in GC B cells resulted in augmentation of net BCR signaling with no apparent effect on antigen presentation. We found that Csk is required for GC maintenance and efficient antibody affinity maturation. Mechanistically, ROS-induced apoptosis was exacerbated concomitantly with mitochondrial dysfunction in Csk-deficient GC B cells. Hence, our data suggest that attenuation of the BCR signal restrains hyper-ROS production, thereby protecting GC B cells from apoptosis and contributing to efficient affinity maturation.
Assuntos
Linfócitos B , Centro Germinativo , Proteínas Tirosina Quinases , Espécies Reativas de Oxigênio , Receptores de Antígenos de Linfócitos B , Animais , Camundongos , Afinidade de Anticorpos , Apoptose , Linfócitos B/imunologia , Linfócitos B/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased â¼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Animais , Camundongos , Humanos , Retroalimentação , Células B de Memória , SARS-CoV-2 , COVID-19/prevenção & controle , RNA Mensageiro , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells; conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate.
Assuntos
Linfócitos B/imunologia , Reprogramação Celular/imunologia , Centro Germinativo/imunologia , Memória Imunológica , Transdução de Sinais/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Reprogramação Celular/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/genética , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Germinal center (GC) B cells cycle between two states, the light zone (LZ) and the dark zone (DZ), and in the latter they proliferate and hypermutate their immunoglobulin genes. How this functional transition takes place is still controversial. In this study, we demonstrate that ablation of Foxo1 after GC development led to the loss of the DZ GC B cells and disruption of the GC architecture, which is consistent with recent studies. Mechanistically, even upon provision of adequate T cell help, Foxo1-deficient GC B cells showed less proliferative expansion than controls. Moreover, we found that the transcription factor BATF was transiently induced in LZ GC B cells in a Foxo1-dependent manner and that deletion of BATF similarly led to GC disruption. Thus, our results are consistent with a model where the switch from the LZ to the DZ is triggered after receipt of T cell help, and suggest that Foxo1-mediated BATF up-regulation is at least partly involved in this switch.
Assuntos
Linfócitos B/imunologia , Proteína Forkhead Box O1/fisiologia , Centro Germinativo/imunologia , Ativação Linfocitária , Linfócitos T/fisiologia , Animais , Linfócitos B/citologia , Proliferação de Células , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth-Holm-Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.