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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 encoding BLIMP1 and XBP1 PC TF will extinguish B cell genes and induce a coordinated antibody-secreting cell transcriptional program^3,4,5. 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 chain^2,3,4. 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 immunoglobulins^6,7. The TF XBP1 plays a major role in this cellular adaptation^8,9,10.

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 vivo^11,12,13. 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 combination^11,12. 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 vivo^{11,12,13,14,15}. PCs are rare cells and our in vitro differentiation model allows to study human B to PC differentiation.

Protokół

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 medium.
   3. Add 12.5 mL of room temperature density gradient (e.g., Ficoll) to a sterile 50 mL conical tube. Gently overlay the density gradient with the 32 mL of diluted blood. Minimize the mixing of the two phases.
   4. Centrifuge 20 min at 500 x g with the brake off. Collect the PBMCs from the diluted plasma/density gradient interface and place the cells in a sterile 50 mL tube and complete to 45 mL with Hank's balanced salt solution.
   5. Centrifuge the cells for 5 min at 500 x g. Carefully remove the supernatant and keep the pellet.
   6. Add 45 mL of RPMI1640/10% fetal calf serum (FCS) and centrifuge 5 min at 500 x g. Carefully remove the supernatant and keep the pellet. Add 30 mL of PBS/2% FCS.
   7. Remove CD2+ cells using anti-CD2 magnetic beads. Add 4 beads for one target cell. Incubate 30 min at 4 °C.
      1. Separate bead-bound cells from unbound cells using a magnet for cell separation application. Collect unbound cells and incubate the cell pellet with anti CD19 APC and anti CD27 PE antibodies for 15 minutes at 4 °C (2 µL of antibody for 10⁶ cells).
      2. Wash twice in PBS/10% goat serum.
      3. Remove contaminating events on both FSC and SSC plots. Plot singlets on FSC-A vs FSC-H and SSC-A vs SSC-H plots to remove debris and to select the total leukocyte population. Select memory B cells on CD19/CD27 and CD19⁺CD27⁺.
      4. Purify MBCs using a cell sorter with a 95% purity.
   8. Perform all the culture steps using IMDM and 10% FCS, supplemented with 50 mg/mL human transferrin and 5 mg/mL human insulin.
   9. Plate purified MBCs in six-well culture plates (1.5 x 10⁵ MBCs/mL in 5 mL/well), for 4 days, with IL-2 (20 U/mL), IL-10 (50 ng/mL), and IL-15 (10 ng/mL).
      1. Add Phosphorothioate CpG ODN 2006, histidine-tagged recombinant human soluble CD40L (sCD40L; 50 ng/mL) and anti-polyhistidine mAb (5 mg/mL) for MBCs activation (Figure 1). Activation by sCD40L or ODN only with the same cytokine cocktail yields to lower amplification¹². The combination of activation signals described here leaded to the maximum number of activated B cells and PrePBs^11,12 (Figure 1).
      2. For gene expression profiling, purify CD38^-/CD20^- PrePBs, at day 4, using a cell sorter (Figure 2).
2. Plasmablastic cell generation
   1. At day 4, count the cells.
   2. Plate cells in 12-well culture plates at 2.5 x 10⁵/mL in 2 mL/well.
   3. Induce differentiation by removal of CpG oligonucleotides and sCD40L and addition of a new culture medium with a new cytokine cocktail including IL-2 (20 U/mL), IL-6 (50 ng/mL), IL-10 (50 ng/mL) and IL-15 (10 ng/mL) (Figure 1). Culture cells for 3 days (from day 4 to day 7) at 37 °C.
   4. For gene expression profiling, purify CD38⁺/CD20^- PBs, at day 7, using a cell sorter (Figure 2).
3. Early plasma cell generation
   1. At day 7, count the cells.
   2. Plate cells in 12-well culture plates at 5 x 10⁵/mL in 2 mL/well.
   3. Differentiate PB in early PCs using fresh culture medium with IL-6 (50 ng/mL), IL-15 (10 ng/mL) and IFN-α (500 U/mL) for 3 days at 37 °C. For gene expression profiling, purify CD20^-/CD38⁺/CD138⁺ early PCs, at day 10, using a cell sorter (Figure 2).
4. Long-lived plasma cell generation
   1. Differentiate Early PCs into long lived PCs by changing the culture conditions.
   2. Culture the cells in 12-well culture plates at 5 x 10⁵/mL in 2 mL/well or in 24-well culture plates in 1 mL/well at 37 °C, using IL-6 and stromal cell conditioned medium and APRIL (200 ng/mL).
   3. Obtain stromal cell-conditioned medium by culturing stromal cells for 5 days with culture medium. Filter the stromal cell culture supernatant (0.2 µm) and freeze.
   4. Add 50% of stromal cell-conditioned media to PC cultures with renewal every week. Generated long-lived PCs could be maintained for months¹³. Long term survival of PCs could be obtained with IL-6 and APRIL only as reported¹³.
   5. Measure Ig secretion from flow cytometry sorted PCs.
   6. Culture PCs at 10⁶ cells/mL for 24 h and harvest culture supernatant. Measure IgG and IgA using human enzyme-linked immunosorbent assay (ELISA) kits^11,12,13. The median IgG secretion ranged from 10 pg/cell/day at day 10 to 17 pg/cell/day at day 60¹³.

2. Molecular Atlas of B to PC Differentiation

NOTE: We built a convenient and open access bioinformatics tools to extract and visualize the most prominent information from Affymetrix GEP data related to PC differentiation (GenomicScape)¹⁵. GEP are publicly available from ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) including purified MBCs, PrePBs, PBs and EPCs: E-MTAB-1771, E-MEXP-2360 and E-MEXP-3034 and BMPC E-MEXP-2360^14,15. Genomicscape is a freely available webtool.

1. Selection of B to PC dataset
   1. Select B to PC dataset in the Browse Data menu in GenomicScape open access bioinformatic tool (http://www.genomicscape.com) called Human B cells to plasma cells. Visualization of gene expression profile of unique gene or a list of genes is available using the Expression-Coexpression Report tool in the Analysis Tools available in the home page.
2. Supervised analysis and principal component analysis (PCA)
   1. Select Analysis Tools | webSAM to load the SAM tool.
   2. Choose the Human B cells to plasma cells dataset and select the groups of samples to compare.
   3. Use the different filters available to apply the filtering options of interest.
   4. To compare gene expression profiles with SAM tool, modify the filtering options including Fold change, number of permutations, False discovery rate and the type of comparison. GenomicScape will compute the analysis and provide genes differentially expressed between the selected groups.
   5. Run principal component analysis by selecting Principal Component Analysis in Analysis Tools menu.
   6. Choose the Human B cells to plasma cells dataset and select the groups of interest.
   7. Paste a list of genes of interest or select genes according to the variance. To paste a list of genes, select To analyse a list of genes/ncRNAs, click here…. Genomicscape will compute PCA that could be visualized and downloaded.

Wyniki

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...

Dyskusje

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...

Materiały

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