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>
		<li>Use peripheral blood cells from healthy volunteers for memory B cell purification.</li>
		<li>Dilute 7.5 mL of blood with 24.5 mL of RPMI 1640 medium.</li>
		<li>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.</li>
		<li>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&#39;s balanced salt solution.</li>
		<li>Centrifuge the cells for 5 min at 500 x g. Carefully remove the supernatant and keep the pellet.</li>
		<li>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.</li>
		<li>Remove CD2+ cells using anti-CD2 magnetic beads. Add 4 beads for one target cell. Incubate 30 min at 4 &#176;C.
		<ol>
			<li>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 &#176;C (2 &#181;L of antibody for 106 cells).</li>
			<li>Wash twice in PBS/10% goat serum.</li>
			<li>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+.</li>
			<li>Purify MBCs using a cell sorter with a 95% purity.</li>
		</ol>
		</li>
		<li>Perform all the culture steps using IMDM and 10% FCS, supplemented with 50 mg/mL human transferrin and 5 mg/mL human insulin.</li>
		<li>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).
		<ol>
			<li>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 PrePBs11,12 (Figure 1).</li>
			<li>For gene expression profiling, purify CD38-/CD20- PrePBs, at day 4, using a cell sorter (Figure 2).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Plasmablastic cell generation
	<ol>
		<li>At day 4, count the cells.</li>
		<li>Plate cells in 12-well culture plates at 2.5 x 105/mL in 2 mL/well.</li>
		<li>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 &#176;C.</li>
		<li>For gene expression profiling, purify CD38+/CD20- PBs, at day 7, using a cell sorter (Figure 2).</li>
	</ol>
	</li>
	<li>Early plasma cell generation
	<ol>
		<li>At day 7, count the cells.</li>
		<li>Plate cells in 12-well culture plates at 5 x 105/mL in 2 mL/well.</li>
		<li>Differentiate PB in early PCs using fresh culture medium with IL-6 (50 ng/mL), IL-15 (10 ng/mL) and IFN-&#945; (500 U/mL) for 3 days at 37 &#176;C. For gene expression profiling, purify CD20-/CD38+/CD138+ early PCs, at day 10, using a cell sorter (Figure 2).</li>
	</ol>
	</li>
	<li>Long-lived plasma cell generation
	<ol>
		<li>Differentiate Early PCs into long lived PCs by changing the culture conditions.</li>
		<li>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 &#176;C, using IL-6 and stromal cell conditioned medium and APRIL (200 ng/mL).</li>
		<li>Obtain stromal cell-conditioned medium by culturing stromal cells for 5 days with culture medium. Filter the stromal cell culture supernatant (0.2 &#181;m) and freeze.</li>
		<li>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.</li>
		<li>Measure Ig secretion from flow cytometry sorted PCs.</li>
		<li>Culture PCs at 106 cells/mL for 24 h and harvest culture supernatant. Measure IgG and IgA using human enzyme-linked immunosorbent assay (ELISA) kits11,12,13. The median IgG secretion ranged from 10 pg/cell/day at day 10 to 17 pg/cell/day at day 6013.</li>
	</ol>
	</li>
</ol>
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-236014,15. Genomicscape is a freely available webtool.
<ol>
	<li>Selection of B to PC dataset
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Supervised analysis and principal component analysis (PCA)
	<ol>
		<li>Select Analysis Tools | webSAM to load the SAM tool.</li>
		<li>Choose the Human B cells to plasma cells dataset and select the groups of samples to compare.</li>
		<li>Use the different filters available to apply the filtering options of interest.</li>
		<li>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.</li>
		<li>Run principal component analysis by selecting Principal Component Analysis in Analysis Tools menu.</li>
		<li>Choose the Human B cells to plasma cells dataset and select the groups of interest.</li>
		<li>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&#8230;. Genomicscape will compute PCA that could be visualized and downloaded.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:817px;" width="818">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-CD2 magnetic beads</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11159D</td>
			<td style="width:344px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-CD138-APC</td>
			<td style="width:139px;">Beckman-Coulter&#160;</td>
			<td style="width:119px;">B49219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-CD19-APC</td>
			<td style="width:139px;">BD</td>
			<td align="right" style="width:119px;">555415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-CD20-PB</td>
			<td style="width:139px;">Beckman-Coulter&#160;</td>
			<td style="width:119px;">B49208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-CD27-PE</td>
			<td style="width:139px;">BD</td>
			<td align="right" style="width:119px;">555441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-CD38-PE</td>
			<td style="width:139px;">Beckman-Coulter&#160;</td>
			<td style="width:119px;">A07779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-histidine</td>
			<td style="width:139px;">R&#38;D Systems</td>
			<td>MAB050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CpG ODN(PT)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;"></td>
			<td>T*C*G*T*C*G*T*T*T*T*G*T*C* 
			G*T*T*T*T*G*T*C*G*T*T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">human Transferin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3309</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN-&#945;</td>
			<td style="width:139px;">Merck</td>
			<td>Intron A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IMDM</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:119px;">31980-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinan Human CD40L-hi</td>
			<td style="width:139px;">R&#38;D Systems</td>
			<td>2706-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human APRIL</td>
			<td style="width:139px;">R&#38;D Systems</td>
			<td>5860-AP-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human IL-10</td>
			<td style="width:139px;">R&#38;D Systems</td>
			<td>217-IL-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human IL-15</td>
			<td style="width:139px;">Peprotech</td>
			<td>200-15-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Human IL-2 Protein</td>
			<td style="width:139px;">R&#38;D Systems</td>
			<td>202-IL-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Human IL-6</td>
			<td style="width:139px;">Peprotech</td>
			<td>200-06</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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

Research

JoVE Journal

Immunology and Infection

12.0K Views.  Univ Montpellier. Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.