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

Plasma cells are rare cells with 30 differentiation stages taking place in anatomic locations that impair full biological characterization, particularly in human. Our in vitro B2 plasma cell differentiation model reproduces the sequential cell differentiation and maturation occurring in the different organs in vivo. This in vitro model recapitulates the coordinated transcriptional changes and the phenotype of the different B2 plasma cell stages that can be detected in vivo.

We also built an open-access bioinformatic tools to analyze the most prominent informations from exploration data related to plasma cell differentiation. Demonstrating the procedure will be Hugues de Boussac, a postdoc from my laboratory. To begin, obtain peripheral blood cells from healthy volunteers for memory B cell purification.

Dilute 7.5 milliliters of blood with 24.5 milliliters of RPMI 1640 medium. Next, add 12.5 milliliters of room-temperature density gradient to a sterile 50-milliliter conical tube. Gently overlay the density gradient with the 32 milliliters of diluted blood, making sure to minimize the mixing of the two phases.

Centrifuge for 20 minutes at 500 times gravity with the brake off. Collect the peripheral blood mononuclear cells from the diluted plasma and density gradient interface. Transfer the cells to a sterile 50-milliliter tube and fill the tube up to 45 milliliters with Hank's Balanced Salt Solution.

Centrifuge at 500 times gravity for five minutes. Carefully remove the supernatant and keep the pellet. Then, add 45 milliliters of RPMI 1640 medium, supplemented with 10%FCS.

Centrifuge at 500 times gravity for five minutes. Carefully remove the supernatant and keep the pellet. Add 30 milliliters of PBS, supplemented with 2%FCS.

Use anti-CD magnetic beads to remove CD2-positive cells by adding four beads for one target cell. Incubate at four degrees Celsius for 30 minutes. Using a magnet for cell separation application, separate the bead-bound cells from the unbound cells.

Collect the unbound cells and incubate them with anti-CD19 APC and anti-CD27 PE antibodies for fifteen minutes at four degrees Celsius. Wash the cells twice in PBS containing 10%goat serum. Then, use the cell sorter to purify the memory B cells to a 95%purity.

After this, add soluble CD40 ligand and anti-polyhistidine monoclonal antibody for memory B cell activation. Plate the purified memory B cells in six-well culture plates for four days with interleukin-2, interleukin-10 and interleukin-15. For gene expression profiling, use a cell sorter to purify the CD38-negative CD20-negative preplasmablasts at day four.

On day four, count the cells and plate them in 12-well culture plates at a cell density of 250, 000 cells per milliliter by adding two milliliters of cell suspension into each well. To induce differentiation, remove the CPG oligonucleotides and soluble CD40 ligand and add new culture medium containing a new cytokine cocktail including interleukin-2, interleukin-6, interleukin-10 and interleukin-15. Culture the cells for three days at 37 degrees Celsius.

For gene expression profiling, use a cell sorter to purify the CD38-positive, CD20-negative plasmablasts at day seven. On day seven, count the cells. Plate the cells in 12-well culture plates at a cell density of 500, 000 cells per well, by adding two milliliters of cell suspension to each well.

Differentiate the plasmablasts into early plasma cells using fresh culture medium containing interleukin-6, interleukin-15, and interferon-alpha for three days at 37 degrees Celsius. For gene expression profiling, use a cell sorter to purify the CD20-negative, CD38-positive, CD138-positive early plasma cells at day 10. First, culture stromal cells for five days with culture medium.

Use a 0.2-microliter filter to filter the stromal cell culture supernatant to obtain stromal cell conditioned medium and freeze until ready to use. Next, culture the early plasma cells in 12-well culture plates, using the previously-obtained stromal cell medium with interleukin-6 and APRIL at a cell density of 500, 000 cells per well, by adding two milliliters of cell suspension to each well. Incubate at 37 degrees Celsius, renewing the stromal cell conditioned medium every week.

Then, measure the immunoglobulin secretion from flow cytometry sorted plasma cells. Culture the plasma cells at a density of one million cells per milliliter for 24 hours and harvest the culture supernatant. Using an ELISA kit, measure the immunoglobulin G, and immunoglobulin A.First, launch the GenomicScape open access bioinformatics web tool.

In the browse data menu, select the dataset named human B cells to plasma cells. Visualization of the gene expression profile of a unique gene or list of genes is available using the Expression/Coexpression Report tool in the Analysis Tools. Next, select Analysis Tools webSAM to load the SAM tool.

Choose the human B cells to plasma cells dataset and select the group of samples to compare. Use the different filters available to apply the filtering options of interest. To compare gene expression profiles with the SAM tool, modify the filtering options including fold change, number of permutations, false discovery rate, and the type of comparison.

The software will compute the analysis and provide genes differentially expressed between the selected groups. In this study, the expression changes of the epigenetic factors in normal plasma cell differentiation are investigated. Using multi-class SAM analysis, 71 genes are seen to be significantly differentially expressed between the memory B cells, the preplasmablasts, the plasmablasts, the early plasma cells and the bone marrow plasma cells with a false discovery rate of under 5%The memory B cell stage is characterized by the overexpression of 23 epigenetic player genes, including 39.13%of histone acetyltransferases, 30.43%of histone methyltransferases, 21.74%of histone demethylases, 4.35%of methyl binding proteins, and 4.35%of histone deacetylases.

14 epigenetic player genes are overexpressed significantly at the preplasmablast stage, including 50%of histone methyltransferases, 14.28%of DNA methyltransferases, 21.43%of histone deacetylases, 7.14%of histone demethylases, and 17.14%of histone acetyltransferases. Following this procedure, several analyses to identify and understand modifications in chromatin structure and architecture during these B2 plasma cell differentiation could be performed. This in vitro differentiation model will allow to generate enough cells to complete this molecular analysis to reveal both the pathways involved in this whole modeling and their downstream transcriptional impact during B2 plasma cell differentiation.

This knowledge derived from this research could inform and instruct on diagnostic and therapeutic strategies for plasma cell disorders, such as in the case of multiple myeloma, a cancer without a definitive cure, for which better prognosis and improved therapeutic strategies are critically needed. Characterizing and understanding the epigenetic remodeling structural and architectural changes that affect chromatin during B2 plasma cell differentiation will be of particular interest. This could be done using gene sequencing, ERRBS, ATAC sequencing, IC, and RNA sequencing.

A moderate heterogeneity could be observed in the percentage of activated B cells, preplasmablasts, plasmablasts and plasma cells, depending on healthy donor's blood, used for memory B cell purification.