Name: in vitro differentiation model of human normal memory b cells to long-lived plasma cells
Uploaded: 2019-01-20T14:30:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells at JoVE.com

This work was supported by grants from French INCA (Institut National du Cancer) Institute (PLBIO15-256), ANR (Tie-Skip) and ITMO Cancer (MM&#38;TT).

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 culture11,12,13.
<ol>
	<li>B cell amplification and differentiation
	<ol>
...

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 vivo11,12,13...

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 identity2,3. Induction of IRF4, PRDM1 encoding BLIMP1 and XBP1 PC TF will extinguish B cell genes and induce a coordinated antibody-secreting cell transcriptional program3,4,5. These coordinated transcriptional changes are associated with Ig genes transcription activation together with a switch from the membrane-bound form to the secreted form of the immunoglobulin heavy chain2,3,4. B to PC differentiation is linked with induction of genes involved in endoplasmic reticulum and Golgi apparatus functions concomitant with unfolded protein response (UPR) activation known to play a key role in PC by accommodating the synthesis of secreted immunoglobulins6,7. The TF XBP1 plays a major role in this cellular adaptation8,9,10.
B cells and PCs are key players of humoral immunity. Understanding the biological processes that control the production and the survival of normal plasma cells is critical in therapeutic interventions that need to ensure efficient immune responses and prevent autoimmunity or immune deficiency. PC are rare cells with early differentiation stages taking place in anatomic locations that hamper full biological characterization, particularly in human. Using multi-step culture systems, we have reported an in vitro B to PC differentiation model. This model reproduces the sequential cell differentiation and maturation occurring in the different organs in vivo11,12,13.&#160;In a first step, memory B cells are first activated for four days by CD40 ligand, oligodeoxynucleotides and cytokine combination and differentiate into preplasmablasts (PrePBs). In a second step, preplasmablasts are induced to differentiate into plasmablasts (PBs) by removing CD40L and oligodeoxynucleotides stimulation and changing the cytokine combination. In a third step, plasmablasts are induced to differentiate into early PCs by changing the cytokine combination11,12. A fourth step was introduced to get fully mature PCs by culturing these early PCs with bone marrow stromal cells conditioned medium or selected growth factors13. These mature PCs could survive several months in vitro and secrete high amounts of immunoglobulin (Figure 1). Interestingly, our in vitro model recapitulates the coordinated transcriptional changes and the phenotype of the different B to PC stages that can be detected in vivo11,12,13,14,15. PCs are rare cells and our in vitro differentiation model allows to study human B to PC differentiation.

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in vitro differentiation model of human normal memory b cells to long-lived plasma cells

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.&#160;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.

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...

Watch this Scientific Journal Video about In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells at JoVE.com

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

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

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 ...

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