Abstract
Upon antigen recognition, naïve B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs), called plasma cells. Increased circulating plasma cells are reported in patients with B cell-associated malignancies, chronic graft-vs.-host disease, and autoimmune disorders. Our aim was to optimize an RNAi-based method that efficiently and reproducibly knocks-down genes of interest in human primary peripheral B cells for the targeted analysis of ASC differentiation. The unique contributions of transcriptional diversity in species-specific regulatory networks and the mechanisms of gene function need to be approached directly in human B cells with tools to hone our basic inferences from animal models to human biology. To date, methods for gene knockdown in human primary B cells, which tend to be more refractory to transfection than immortalized B cell lines, have been limited by losses in cell viability and ineffective penetrance. Our single-step siRNA nucleofector-based approach for human primary naïve B cells demonstrates reproducible knockdown efficiency (~40–60%). We focused on genes already known to play key roles in murine ASC differentiation, such as interferon regulatory factor 4 (IRF4) and AID. This study reports a validated non-viral method of siRNA delivery into human primary B cells that can be applied to study gene regulatory networks that control human ASC differentiation.
Highlights
B lymphocytes are critical members of the adaptive immune system as they are uniquely capable of secreting high titers of antigen-neutralizing antibody
In mice lacking Irf4, B and T cells were unable to proliferate in response to B cell receptor (BCR), T cell receptor (TCR), CD40, or LPS stimulation [25]
We found that interferon regulatory factor 4 (IRF4) expression peaked at 48 h in CD19+IgD+ B cells after stimulation of peripheral blood mononuclear cells (PBMCs) with anti-IgM and the TLR9 agonist CpG-B; AID expression peaked at 72–96 h (Supplementary Figures 2A,B)
Summary
B lymphocytes are critical members of the adaptive immune system as they are uniquely capable of secreting high titers of antigen-neutralizing antibody. B cells and their associated antibody-mediated response to antigen are important in the clearance of viral, bacterial, and fungal pathogens. Recognition of these foreign antigens by B cells triggers rapid proliferation and differentiation to specialized antibody secreting cells (ASCs) known as plasma cells. The process of ASC differentiation is a tightly regulated one that relies on synergistic signaling from multiple pathways [1]. A large gene-regulatory network of transcription factors is required for regulating this multi-step process. One key player in the differentiation of naïve B cells to ASCs is the transcription factor interferon regulatory factor 4 (IRF4).
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