RESUMO
IgE antibodies may elicit potent allergic reactions, and their production is tightly controlled. The tendency to generate IgE has been thought to reflect the balance between type 1 and type 2 cytokines, with the latter promoting IgE. Here, we reevaluated this paradigm by a direct cellular analysis, demonstrating that IgE production was not limited to type 2 immune responses yet was generally constrained in vivo. IL-21 was a critical negative regulator of IgE responses, whereas IFN-γ, IL-6, and IL-10 were dispensable. Follicular helper T cells were the primary source of IL-21 that inhibited IgE responses by directly engaging the IL-21 receptor on B cells and triggering STAT3-dependent signaling. We reconciled previous discordant results between mouse and human B cells and revealed that the inhibition of IgE class switch recombination by IL-21 was attenuated by CD40 signaling, whereas IgG1 class switch recombination was potentiated by IL-21 in the context of limited IL-4. These findings establish key features of the extrinsic regulation of IgE production by cytokines.
Assuntos
Linfócitos B/imunologia , Switching de Imunoglobulina/genética , Imunoglobulina E/genética , Interleucinas/metabolismo , Animais , Apoptose , Antígenos CD40/metabolismo , Contagem de Células , Centro Germinativo/citologia , Humanos , Imunidade , Imunoglobulina G/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Linfonodos/citologia , Camundongos , Modelos Biológicos , Plasmócitos/metabolismo , Receptores de Interleucina-21/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células T Auxiliares Foliculares/metabolismoRESUMO
Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.
