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

Summary

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

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.

Introduction

The differentiation of B cells to plasma cells (PCs) is essential for humoral immunity and protect the host against infections¹. B to PC differentiation is associated with major changes in transcription capacity and metabolism to accommodate to antibody secretion.The transcription factors that control B to PC differentiation have been extensively studied and revealed exclusive networks including B- and PC-specific transcription factors (TFs)². In B cells, PAX5, BCL6 and BACH2 TFs are the guardians of B cell identity^2,3. Induction of IRF4, PRDM1 ....

Protocol

The protocol follows the guidelines in accordance with the Declaration of Helsinki and agreement of the Montpellier University Hospital Centre for Biological Resources.

1. In Vitro Normal Plasma Cell Differentiation Model

NOTE: PCs are generated through a four-step culture^11,12,13.

1. B cell amplification and differentiation
   1. Use peripheral blood cells from healthy volunteers for memory B cell purification.
   2. Dilute 7.5 mL of blood with 24.5 mL of RPMI 1640 m....

Results

The overall procedure of in vitro normal PC differentiation is represented in Figure 1. Using the protocol presented here, we could generate adequate quantity of cells that could not be obtained with ex vivo human samples. Although the role of the complex network of transcription factors involved in PC differentiation has been investigated, the mechanisms regulating key PC differentiation transcription networks remain poorly known. Cellular differentiation is.......

Discussion

In human, PC are rare cells with differentiation stages taking place in anatomic places that hamper full biological characterization. We have developed an in vitro B to PC differentiation model using multi-step culture systems where various combinations of activation molecules and cytokines are subsequently applied in order to reproduce the sequential cell differentiation occurring in the different organs/tissues in vivo^11,12,13.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-CD2 magnetic beads	Invitrogen	11159D
Anti-CD138-APC	Beckman-Coulter	B49219
Anti-CD19-APC	BD	555415
Anti-CD20-PB	Beckman-Coulter	B49208
Anti-CD27-PE	BD	555441
Anti-CD38-PE	Beckman-Coulter	A07779
Anti-histidine	R&D Systems	MAB050
CpG ODN(PT)	Sigma		T*C*G*T*C*G*T*T*T*T*G*T*C* G*T*T*T*T*G*T*C*G*T*T
human Transferin	Sigma-Aldrich	T3309
IFN-α	Merck	Intron A
IMDM	Gibco	31980-022
Recombinan Human CD40L-hi	R&D Systems	2706-CL
Recombinant Human APRIL	R&D Systems	5860-AP-010
Recombinant Human IL-10	R&D Systems	217-IL-
Recombinant Human IL-15	Peprotech	200-15-10ug
Recombinant Human IL-2 Protein	R&D Systems	202-IL-
Recombinant Human IL-6	Peprotech	200-06