B cells

If a B cell succeeds in producing a functional, non-autoreactive B cell receptor (BCR), it differentiates into a mature, naive B cell. These cells express the BCR as IgM and IgD molecules, recirculate through the body, and account for roughly 60–70% of B cells in adults. Naive B cells are CD27^–, a receptor of the TNF family that plays a role in B cell activation and subsequent immunoglobulin synthesis.

B cell activation requires two signals to obtain the clustering of B cell receptors and their associated signaling molecules. The first signal is initialized by the binding of a specific antigen to the BCR. The second signal is costimulatory and can be T cell–dependent or T cell–independent. For T cell–dependent costimulation, the TCR of T helper cells recognize the same antigen as the respective B cells, which the B cell has processed and presented on its surface in an antigen-MHCII complex beforehand. This in turn activates the T cell, which then provides a second activation signal for the B cell, like the expression of CD40L on the T helper cell surface. CD40L can bind to CD40 on B cells enabling increased clustering and multimerization of the receptors (PMID: 27800605), which triggers a signaling cascade that increases proliferation, possibly induces class switch, and drives differentiation to plasma or memory cells. T cell–independent costimulation normally involves antigens which exhibit a structure that can already multimerize the BCR without the need of T cell support. These antigens display highly repetitive epitope motifs, like bacterial cell wall components (carbohydrates and lipopolysaccharides). Activated B cells display an increase in their expression of CD86, CD80, and MHCII or CD62L, which can be detected by flow cytometry.

GC B cells and post-GC PCs are also CD27⁺ but can be excluded from analysis by gating out CD38⁺⁺ cells.

IgG⁺ memory B cells

Approximately 15–20% of peripheral blood (PB) B cells in adults are IgG⁺ B cells, expressing mainly IgG1, IgG2, or IgG3, and rarely IgG4. Proportions of these cells among memory B cells vary considerably between individuals, likely because of different infection histories. Switching to specific IgG subclasses is influenced by the type of antigen and various immunoregulatory factors

Approximately 20–25% of IgG⁺ memory B cells lack CD27 expression (PMID: 22566870). This subpopulation increases in the elderly suggesting that this cell population may consist of aged or exhausted memory B cells. IgG⁺CD27⁻ B cells are considerably less mutated than their CD27⁺ counterparts, and are more frequently IgG3⁺ and less frequently IgG2⁺. Both types of memory B cells often derive from common GC B cell clones.

In mice and humans, IgG memory B cells have the propensity to differentiate into PCs upon reactivation, and mostly not to reenter GC reactions.
 

IgA⁺ memory B cells

IgA⁺ memory B cells account for approximately 10% of B cells in peripheral blood and mostly express CD27. They are preferentially generated in immune responses in the intestine and other mucosa-associated lymphatic tissues, like Peyer’s patches or mesenteric lymph. 
 

IgE⁺ memory B cells

IgE-expressing memory B cells are hardly detectable in peripheral blood of healthy humans. They are important in immunologic reactions against parasites like helminths and protozoa, and their interaction with mast cells and eosinophils generates allergic reactions. 
 

IgM-only and IgM⁺IgD⁺ memory B cells

Two distinct subpopulations of human IgM-expressing PB B cells with somatically mutated IgV genes exist, IgM-only B cells (IgM⁺IgD^low/−) and IgM⁺IgD⁺ B cells, both expressing CD27 and representing roughly 5% and 15% of B cells, respectively, in PB and secondary lymphoid organs, hence representing a major fraction of the B cell pool. In the course of an adaptive immune response all mature B cells are IgM⁺ or IgD⁺ at first and undergo class switch to other isotypes classes only later depending on the surrounding influences, mediated by cytokines. IgM⁺ cells are mostly involved in immune responses that involve and activate the complement system.
 

Human plasma cells are a heterogeneous cell population comprising several subsets, from short-lived and proliferative plasmablasts in lymphoid tissues, to transitional plasma cells in peripheral blood, and long-lived, non-dividing plasma cells in bone marrow (PMID: 28633925). Plasma cells of all differentiation stages are identified by high-level expression of CD38 (PMID: 28633925). Other surface markers are differentially regulated, depending on stage of differentiation and spatial localization. Plasma cells in blood lack expression of the typical B cell marker CD22 and express lower levels of CD19 and CD20 than mature B cells. They are further characterized as being CD27⁺⁺CD31⁺CD44⁺CD45⁺CD56^–CD62L⁺CD86⁺ and HLA-DR⁺. A subset of CD38⁺ blood plasma cells further expresses the CD138 antigen, whereas all CD38⁺ plasma cells are also CD138⁺ in bone marrow. (PMID:11877292, 16956823,18626062).

StraightFrom® MicroBeads allow magnetic cell separation directly from whole blood, bone marrow, and buffy coat. Pre-enrichment of white blood cells by density gradient centrifugation or other methods is not required. The purified cells are well-suited for subsequent flow cytometry analysis, molecular biology studies, and functional studies.

StraightFrom Whole Blood CD19 MicroBeads allow the isolation of CD19⁺ cells directly from 0.25–15 mL whole blood or bone marrow.  Cells in the sample are labeled with Whole Blood MicroBeads and subsequently purified using the autoMACS® Pro Separator, the MultiMACS™ Cell24 Separator Plus with the Whole Blood Column Kit, or manual separators. 

Similarly effective is the StraightFrom Buffy Coat CD19 MicroBead Kit that enables gentle and fast magnetic isolation of B cells directly from buffy coat. An entire buffy coat from a blood donation of up to 500 mL can be processed in one run. No washing or cell counting is needed; after adding Buffy Coat MicroBeads directly to the sample, labeled cells are separated over Whole Blood Columns using the MultiMACS Cell24 Separator Plus for fast and convenient semi-automated processing, or manually using MACS Separators. 

If the starting material is LRSC, the StraightFrom LRSC CD19 MicroBead Kit is perfectly suited for fast and automated isolation of CD19⁺ cells.
 

Alternatively, the MACSxpress® Cell Isolation Kits allow the isolation of untouched target cells directly from freshly drawn anticoagulated whole blood, buffy coat, or LRSC by depletion of non-target cells. MACSxpress Whole Blood Kits are ideal for the processing of larger sample volumes (total capacity 3×30 mL). Specialized MACSxpress Kits enable the isolation of  B cells from an entire buffy coat or LRSC.

The MACSxpress Whole Blood B Cell Isolation Kit,  human was developed for the isolation of B cells directly from  anticoagulated whole blood without density gradient centrifugation. While non-target cells are removed by immunomagnetic depletion, erythrocytes are sedimented to yield untouched B cells of high purity.

The MACSxpress Whole Blood Pan B Cell Isolation Kit,  human enables the isolation of untouched B cells including activated CD43⁺ B cells. 
The CD43 marker is often used for the isolation of untouched B cells by depletion of non-target cells since it is expressed on nearly all lymphocytes, except B cells. However, as CD43 can be up-regulated on B cells upon activation, these activated B cells would be removed as well. The MACSxpress Whole Blood Pan B Cell Isolation Kit avoids depletion of the activated CD43⁺ B cells.

Blood samples from patients with B cell chronic lymphocytic leukemia (B-CLL) often have variable and high B cell frequencies, depending on the treatment/disease status of the patient. Thus, Miltenyi Biotec developed the MACSxpress Whole Blood B-CLL Cell Isolation Kit, human, a specialized kit to isolate untouched B cells from such samples.

Instead of working directly with whole blood or buffy coat, the blood samples can be processed using density gradient centrifugation to pre-enrich peripheral blood mononuclear cells (PBMCs) as starting material for subsequent B cell isolation. 
CD19 MicroBeads, CD20 MicroBeads, and CD22 MicroBeads,  human allow positive selection and depletion of B cells from PBMCs. The lineage markers CD19, 20, and 22 are expressed on all peripheral blood B cells and are down-regulated when the B cells differentiate into plasma cells.
The B Cell Isolation Kit II, human enables the isolation of untouched B cells. This kit contains a cocktail of biotinylated antibodies and Anti-Biotin MicroBeads for magnetic labeling and subsequent depletion of non–B cells.
The B-CLL Cell Isolation Kit,  human is designed for the isolation of untouched B cells from tumor samples. During the course of some diseases such as B-CLL, malignant B cells express CD43. The depletion cocktail of the kit has been specifically developed for the isolation of malignant B cells and therefore does not contain a CD43 antibody.

The table below gives an overview of different options for isolating B cell subsets from various starting materials using different isolation strategies.

Naive B cells

The MACSxpress Naive B Cell Isolation Kit, human was developed for the isolation of untouched naive B cells from up to 30 mL anticoagulated whole blood. Non-target cells are removed by immunomagnetic depletion and erythrocytes are sedimented to yield untouched naive B cells of purity.

If PBMCs are used as starting material, the Naive B Cell Isolation Kit II, human is the right choice. The kit delivers highly pure naive B cells by depletion of non-target cells.
 

Memory B cells
 

The Memory B Cell Isolation Kit, human isolates all memory B cells from human PBMCs. First, unwanted cells are depleted. Subsequently, memory B cells are isolated by positive selection with CD27 MicroBeads.
Miltenyi Biotec also offers solution for the isolation of specific memory B cell subsets. The Switched Memory B Cell Isolation Kit,  human isolates all switched memory B cells from human PBMCs by depletion of IgD⁺ and IgM⁺ memory B cells and other non-target cells.

The IgG⁺ Memory B Cell Isolation Kit, human and the IgM⁺ Memory B Cell Isolation Kit, human deliver highly pure fractions of IgG⁺ and IgM⁺ memory B cells from human PBMCs, respectively. Isolation is done by depletion of unwanted cells followed by positive selection with Anti-IgG or Anti-IgM MicroBeads.
 

Plasma cells

When B cells develop into plasma cells, they down-regulate expression of CD19 and CD20 and become negative for CD22. Plasma cells of all differentiation stages are identified by the expression of high levels of CD38. The Plasma Cell Isolation Kit II, human isolates plasma cells from human PBMCs or bone marrow by depletion of non-plasma cells in a first step. In the second step, pre-enriched plasma cells are directly labeled with CD38 MicroBeads and isolated by positive selection.
 

Other B cell subset

Other B cell subsets, like activated B cells, can be isolated using a combination of the B Cell Isolation Kit II, human (negative selection) with any other MicroBeads for positive selection of a distinctive marker. For example, CD69⁺ B cells can be isolated by the B Cell Isolation Kit II, human followed by CD69 MicroBeads.
 

Besides their function as antigen-presenting cells (APC) or antibody factories, B cells regulate the immune response by secreting various cytokines. However, in contrast to cytokine-secreting T cells, naive B cells do not secrete many cytokines upon activation. To become cytokine-producing cells, they require additional stimulation from the surrounding microenvironment or from B differential stages.  Cytokine production in B cells is heterogeneous and dependent on stimuli.  B cells primed by T_H1 cells and antigen (Be-1 cells) secrete cytokines associated with type 1 immune responses, such as IFN-γ and IL-12. B cells primed by T_H2 cells and antigen (Be-2 cells) make IL-2, IL-13, and IL-4. Regulatory B cells, or B10 cells, are identified by their ability to secrete IL-10 or TGF-β1. In this way, B cells play a potentially important role in regulating the adaptive immune response before developing into plasma cells. Cytokine secretion can be measured in different ways. 

MACS Cytokine Secretion Assays are designed for highly sensitive analysis of viable cytokine-secreting cells, allowing the detection of secreted cytokines at a single-cell level and simultaneous cell phenotyping. A range of Cytokine Secretion Assay – Cell Enrichment and Detection Kits also enable enrichment of viable cytokine-secreting cells.
Cytokines also play a role in class switch determination of the B cell isotype. The following table summarizes the effects of the most important cytokines for class switching.
 

B cell frequency in human peripheral blood is often too low to obtain sufficient cell numbers for research purposes. Therefore, expansion is a crucial step in the analysis of B cells, and is usually achieved by stimulation of CD40. Binding of CD40L to CD40 is important in the interaction between T cells and antigen-presenting cells and is involved in B cell differentiation and proliferation, isotype class-switching, and protection of B cells from apoptosis, among other functions.  

With the B Cell Expansion Kit, human, B cells are expanded by up to 200-fold in 14 days. The kit contains the CD40-Ligand and a cross-linking antibody for multimerization (Human CD40-Ligand Multimer Kit) to increase the biological activity of the CD40-Ligand, as well as premium-grade Human IL-4 and StemMACS™ HSC Expansion Medium XF, human. B cell expansion is done by restimulation on days 7 and 10 of culture.