In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Abstract

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals. We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation. Video LinkThe video component of this article can be found at https://www.jove.com/video/58929/ 11,12,13,14,15 . PCs are rare cells and our in vitro differentiation model allows to study human B to PC differentiation. ProtocolThe protocol follows the guidelines in accordance with the Declaration of Helsinki and agreement

Integrated Single-Cell (Phospho-)Protein and RNA Detection Uncovers Phenotypic Characteristics and Active Signal Transduction of Human Antibody-Secreting Cells

“…We adopted an in vitro differentiation protocol of human peripheral B-cells to generate human ASCs ( 6 , 7 , 8 ). Isolated B-cells from healthy donors were stimulated with CpG for 1 day.…”

“…Human PCs only represent ∼0.25% of total bone marrow cells and these cells home to different tissues in the human body, making extensive molecular analysis and functional studies challenging ( 5 ). Protocols for in vitro differentiation of human peripheral B-cells into ASCs ( 6 , 7 , 8 ) form the basis for phenotypic and functional studies ( 9 , 10 ). Studies in mice have shown divergent gene expression profiles for the different Ig class PCs ( 11 ) and different expression profiles were observed comparing human class–switched germinal center B-cells from human tonsils as compared to nonclass–switched cells ( 12 ).…”

High-Throughput Detection of Autoantigen-Specific B Cells Among Distinct Functional Subsets in Autoimmune Donors

“…The limited antibody secretion detected from plasmablasts following this in vitro stimulation points to a need to apply other approaches, such as single-cell BCR sequencing, to examine the antigen-specific plasmablast repertoire. Alternatively, this stimulation protocol could be modified to include factors essential for plasmablast/plasma cell survival and differentiation, such as IL-6, IL-10, IFN-α and APRIL ( 50 , 51 ). Such modifications would need to be separately tested for their impact on other B cell subsets.…”

Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells

“…We adopted an in vitro differentiation protocol of human peripheral B-cells to generate human ASCs (6)(7)(8). Isolated B-cells from healthy donors were stimulated with CpG for 1 day.…”

“…Human PCs only represent ~0.25% of total bone marrow cells and home to different tissues in the human body, making extensive molecular analysis and functional studies challenging (5). Protocols for in vitro differentiation of human peripheral B-cells into ASCs (6)(7)(8) form the basis for phenotypic and functional studies (9,10). Studies in mice have shown divergent gene expression profiles for the different Ig class PCs (11) and different expression profiles were observed comparing human class-switched germinal center (GC) B-cells from human tonsils as compared to non-class switched cells (12).…”