B cell immunophenotyping | Cytokine Transcription Tests (2023)

B cells go through a series of developmental stages before and after antigen challenge. As they mature and differentiate, several functionally distinct subpopulations emerge. The differential expression of cell surface and intracellular markers and their unique immunoglobulin and cytokine secretion profiles provide valuable clues about the different properties and functions of different B cell subsets. For example, syndecan-1 (CD138) expression distinguishes circulating plasmablasts and plasma cells ("professional", antibody-secreting B cells) from other developmental and functional subsets. If the development process is not completed successfully, apoptosis occurs in mature B cells. To support the study of B cells using multicolor flow cytometry, BD offers a broad portfolio of B cell phenotyping reagents. They are available in different formats and offer maximum flexibility in panel design. We will continue to add new details as new markers become available. This diagram summarizes the key developmental and differentiation stages and some of the key markers associated with each B cell subset in mice and humans.

conventional B cell maturation

B cells originate from hematopoietic stem cells (HSCs), which reside in the bone marrow and go through the first stages of development there. The immature B cells then migrate to secondary lymphoid tissues, where most develop into mature follicular B cells. When the B cell receptor (BCR) complex of a mature B cell, consisting of a membrane-bound (m) composed of IgM and IgD, binds its cognate foreign antigen, the cell becomes activated and differentiates into an antibody-secreting plasma cell .²

See B cell development poster

B cell subsets with different functions

B cells also follow alternative differentiation pathways to traditional B cells, creating subpopulations with distinct functions and marker expression patterns. For example, rim zone (MC) B cells function as innate cells. Unlike conventional B cells, they can be activated by toll-like receptor (TLR) ligation, bypassing the BCR. They also tend to express CD1d and CD21 but not CD23. A subpopulation of B cells with regulatory function characterized by the ability to secrete IL-10 or TGF-β-1 has been identified.³

B cell maturation test

With a wide range of antibodies against mouse and human markers, BD can support different phenotype panels to study B cells at different developmental stages. To illustrate the utility of differential marker expression in B cell analysis, B cell subsets in mouse bone marrow were analyzed using seven cell surface markers, allowing the differentiation of seven different developmental stages in this tissue. Based on the differential expression of BP1 and CD24, pre-pro-B, pro-B and pre-B cells could be distinguished in the low-positive CD45R/B220 population. Based on the differential expression of IgM and IgD, immature, transitional, early maturing, and late maturing B cells can be distinguished. The expression of the IL-7 receptor CD127 was analyzed in these different subpopulations and found to decrease with increasing B cell maturation.

1. Martínez-Riaño A, Bovolenta ER, Mendoza P, et al. Antigen phagocytosis by B cells is essential for an efficient humoral response.EMBO representative. 2018;19(9):e46016. Number: 10.15252/embr.201846016
2. Yam-Puc JC, Zhang L, Zhang Y, Toellner KM. The role of the B cell receptor in B cell development and antigen-induced differentiation.F1000 resources。 2018；7：429。 doi:10.12688/f1000research.13567.1
3. Matsushita T. Regulatory and effector B cells: friend or foe?Journal of Dermatology。 2019；93(1):2-7。 doi:10.1016/j.jdermsci.2018.11.008

Plasmagenerator

B cells are known for their ability to produce antibodies that are crucial for neutralizing foreign beings or infected cells. Antibody-secreting cells (ASCs), including plasmablasts and plasma cells, lack the CD20 B cell marker and express a combination of CD27, CD38 and CD138 in humans. Plasmablasts play an early role in the adaptive immune response because they produce low-affinity immunoglobulins and have a short lifespan. Plasma cells are more differentiated and include short-lived and long-lived subgroups. Short-lived plasma cells have a lifespan and antibody secretion rate similar to plasma blasts, and although plasma blasts express all immunoglobulin (Ig) isotypes, plasma cells prefer IgM and IgG. Long-lived plasma cells have the longest lifespan, the highest rate of antibody secretion, and immunoglobulin (Ig) isotypes IgG > IgA > IgM.¹

effector B cells

Effector B cells or B-eff play a supportive role in the immune response through antigen presentation and secrete a variety of cytokines (e.g. IL-2, IL-6, GM-CSF, IL-12, IL-17, IFN -γ). , TNF)-a) Induces activation of macrophages (e.g. interferon-γ), differentiation of T cells (IL-6 for Th17) and differentiation of plasma cells to produce antibodies.²In autoimmune diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS), effector B cells also help differentiate plasma cells to produce autoantibodies, resulting in a dysregulated immune response. In MS, CD138+ B cells have been shown to accumulate in the cerebrospinal fluid (CSF) and contribute to the maintenance of chronic inflammation in the CNS through inflammatory cytokine secretion and activation of resident microglia.³

regulatory B cells

While B cells secrete cytokines to regulate immune cell function, they also receive environmental signals from their immune cells to regulate their function. In autoimmunity, BAFF derived from macrophages and dendritic cells can induce a balance between effector B cells and regulatory B cells (B-reg). In SLE, excess BAFF promotes autoantibody production by plasma cells while inhibiting activation of IL-10-producing B-reg. Most IL-10 secreting regulatory B cells can be identified by the expression of CD9 (88%) and tumor necrosis factor receptor 2 (TNFR2).^{2, 4}

1. Tellier J, Nat SL. Plasma cells: programming of the antibody secretion machinery.European Journal of Immunology。 2019；49(1):30-37。 doi:10.1002/use.201847517
2. Matsushita T. Regulatory and effector B cells: friend or foe?Journal of Dermatology。 2019；93(1):2-7。 doi:10.1016/j.jdermsci.2018.11.008
3. Knier B, Hiltensperger M, Sie C, et al. Myeloid-derived suppressor cells control the accumulation of B cells in the central nervous system during autoimmunity.natural immunology. 2018;19(12):1341-1351. Number: 10.1038/s41590-018-0237-5
4. Fillatreau S. Regulatory functions of B cells and regulatory plasma cells.Biomedical Journal。 2019；42(4):233-242。 doi:10.1016/j.bj.2019.05.008

antibody production

Measuring the type and amount of immunoglobulins and cytokines secreted by B cells can provide information about the quality and quantity of their immune response - properties that often change in disease states. Methods that enable multiplex measurements are increasingly being used to measure immunoglobulins (Igs) and cytokines. In addition to the benefits of multiplexing, they also offer some unique advantages over traditionally used techniques.

BD^®Cytometry bead arrays: multiplex quantification

BD® Cytometry Bead Arrays (CBAs)is a bead-based immunoassay that can simultaneously quantify multiple analytes in the same sample. BD^®The CBA Human Immunoglobulin Flex Set System provides ready-to-use reagents that serve as building blocks for multiplex quantification of multiple Ig subclasses. Because the number and type of antibodies in a given immune response can vary widely, measuring these parameters can provide information about reactions after immunization or vaccination, or serve as indicators for assessing immunoglobulin deficiencies. e.g. BD^®The CBA assay was used to analyze Igs and cytokines secreted by B cells during inflammation.¹

cytokine production

Depending on their activation state, B cells secrete different cytokines.

BD^®CBA Assay for Cytokines

BD^®The CBA Flex Kit for Cytokines offers an open and configurable menu of bead-based reagents designed for easy and efficient multiplexing. Its distinctive features include a variety of human and mouse cytokines. Data comparison results with BD^®The NIBSC/WHO international standard CBA standard can be used as a guide to facilitate the comparison of cytokine concentration values ​​determined by different laboratories or methods. Measurement of secreted cytokines can also provide information about the activity of different functional B cell subsets in the sample, such as e.g. B. B effector 1 or -2, or regulatory B cells (B-reg) secreting anti-inflammatory cytokines such as IL-10 .BD^®The CBA Flex kit was used to quantify the secretion of several B cell-associated cytokines and chemokines following manipulation of depleted tissue-like memory B cells in chronically virus-infected individuals such as patients.Plasmodium falciparum。^{2, 3}

intracellular signaling

The development, activation, and differentiation of B cells involves many changes in intracellular molecules, including transcription factors, phospho-signaling proteins, and cytokines. BD offers unique solutions that enable the analysis of these intracellular molecules, enabling researchers to decipher the interconnected pathways that regulate B cell biology. Multicolor flow cytometry is a powerful technique for analyzing intracellular molecules. Simultaneous analysis of markers from specific cell subsets can provide information about the differentiation and activation status of different B cell populations. While cell surface staining techniques are relatively standard, optimal staining of intracellular markers often depends on the biological properties of the protein of interest.

To facilitate the detection of these specific intracellular molecules using flow cytometry, BD has developed monoclonal antibodies, proprietary buffer systems and kits. Flow cytometry has several advantages over commonly used methods such aswestern spot- Multiple parameters of heterogeneous samples such as blood can be measured simultaneously. This saves valuable samples and provides detailed results at the cellular level.

Expression of Bcl6 in mouse lymph node cells

Mouse lymph node cells were stained with anti-CD45R/B220, CD4, IgD, and CD95, fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set, and stained with anti-Bcl-6 Alexa Fluor™ 488. Flow cytometry with BD^®LSR II system. Bcl-6 expression was observed in germinal center B cells.

Detection of transcription factors

BD Pharmingen™ Transcription Factor Buffer permeabilizes cells sufficiently to allow exposure of core epitopes, yet is mild enough to allow detection of most cell surface proteins. It can be used alone or in combination with cell surface markers and cytokines for enhanced recognition of multiple transcription factors. BD Biosciences offers a broad portfolio of B-cell associated flow-validated transcription factor antibodies including unique specificities. Some of these, such as Oct-2 and Pax-5, are expressed throughout the follicle or in the germinal center (GC) stage. Bcl-6 is characteristic of proliferating GC-B cells, while others such as Blimp-1 and XPB-1 are typical plasma cells.

Detection of phosphoproteins

BD Phosflow™ antibodies are monoclonal phosphoepitope specific antibodies validated for flow cytometry detection. The recommended permeabilization buffer for most BD Phosflow™ antibodies is BD Phosflow™ Perm Buffer III, although alternative permeabilization buffers are available to meet specific experimental needs.

A set of BD Phosflow™ antibodies is specific for the analysis of signaling pathways involved in B cell development and activation including phosphorylated Akt, phosphorylated Btk, phosphorylated Syk, phosphorylated BLNK, phosphorylated CD20/BL-CAM, phosphorylated IKKγ, phospho-NFκB and phospho-mTOR.

cytokine detection

By using protein transport inhibitors to prevent secretion, researchers can detect cytokines in cells that produce them. This allows for an easy determination of whether cytokine production by an activated cell population is the result of a small number of cells producing large amounts of cytokines or a large cell population producing small amounts of cytokines per cell. Cell. BD kits, buffer systems and protocols for intracellular cytokine staining include BD Cytofix/Cytoperm™ fixation/permeabilization solution, which is suitable for staining of most cytokines and cell surface markers. A variety of direct conjugates are available covering cytokines commonly used to distinguish between B-reg and the secretion of B-effector-1 (Be-1) and B-effector-2 (Be-2) cells become.

1. Lu Y, Liu F, Li C, Chen Y, Weng D, Chen J. IL-10-producing B cells suppress activation of effector T cells and promote regulatory T cells in crystalline silica-induced inflammatory responses in vitro.inflammatory mediator. 2017;2017:8415094. Number: 10.1155/2017/8415094
2. Kardava L, Moir S, Wang W, et al. Attenuation of HIV-associated human B cell depletion by siRNA downregulation of inhibitory receptors.Clinical Investment Journal. 2011;121(7):2614-2624. Number: 10.1172/JCI45685
3. Ambegaonkar AA, Kwak K, Sohn H, Manzella-Lapeira J, Brzostowski J, Pierce SK. In chronic human infectious diseases, B cells express inhibitory receptors that limit responses to membrane-associated antigens.scientific progress。 2020;6(30):eaba6493。 doi:10.1126/sciadv.aba6493

Multicolor immunophenotyping methods are appropriate for studying B cells because they inherently require the use of many more markers than T cells to define the basic subpopulations present in most samples.

spine marking

Extensive B cell research uses cell surface markers that define the major B cell subsets and form the "backbone" or nucleus from which the panels perform more detailed analysis. A key marker of the B cell lineage is the lineage marker CD19, which is expressed by almost all cells of the B cell lineage. The mouse traditionally uses CD45R/B220. Most panels also include surface-expressed IgD and IgM, since BCR isotypes provide information about the stage of B-cell differentiation. To provide flexibility in panel design, BD offers all of these markers in a variety of formats.

polychromatic flow

When measuring a set of five markers on two lasers, commonly used fluorochromes leak into other fluorescence channels and need to be compensated. Researchers with access to four or five laser instruments can use this feature to simplify setup and minimize the need for compensation.

Device structure for multicolor flow

Instrument setup for multicolor experiments using antibody panels with more than four colors can be time consuming. Five-laser instruments are typically equipped with a 355 nm UV laser and are commonly used for side group analysis, calcium measurements with Indo-1, or viability staining.

To enable users to get the most out of this device for phenotyping experiments, BD has developed a new UV-excited polymer dye, BD Horizon Brilliant™ Ultraviolet 395 (BUV395).

Antibodies conjugated to BUV395 were combined in a panel, with each fluorochrome excited by a different laser, resulting in minimal or no cross-contamination between them. This is exemplified by a five-color panel that distinguishes between naïve and memory B cells. Using this panel requires minimal setup and requires little compensation.

In the example shown, B cell subsets in mouse spleen were analyzed using nine cell surface markers. They allow the different developmental stages of this tissue to be distinguished. Follicles B I and II and rim cell subsets (rim zone and rim zone progenitors) were identified by expression of IgM, IgD, CD21 and CD23. Different levels of IgM and CD23 expression help distinguish the stages of the transitional subset (T1, T2 and T3). Measurement of Pax-5 expression in different subpopulations revealed differences in Pax-5 expression in follicular and marginal zone B cells.

Combine metrics to maximize results

Advances in buffer systems and methods are now facilitating the simultaneous measurement of surface and intracellular markers, helping researchers move to information-rich combined measurements. For example, a combination of cell surface and intracellular markers can be used to monitor multiple cell types in a heterogeneous sample of differentiated B cells, thereby more efficiently extracting more relevant data from each sample. Antibodies against B-cell surface markers and specific transcription factors are available in various conjugates, including the new BD Horizon™ dyes, increasing flexibility in designing multicolor experiments.

base plate

Depending on the type of sample to be analyzed, different markers can be chosen to define particular B cell subsets of interest. The example shown analyzes human peripheral blood with CD19, CD20, IgD, CD27, CD38 and CD24. This six color panel allows for the identification of transitional B cells (CD24).^HochCD38^Hoch), distinguish naive cells from memory cells (naive cells are CD27).^–Immunoglobulin D⁺, memory cells CD27⁺Immunoglobulin D^–) and identified plasmablasts (CD38⁺）。¹

In-depth phenotype analysis

Because the phenotypes of some subgroups are very similar to others, a wider range of phenotypes is often required. For example, one group can distinguish the many stages of B cell development present in bone marrow B cells. Adding markers can also allow for more detailed analysis, for example by defining memory cells or different types of plasma cells. Other molecules (chemokine receptor, CD62L) gave us insight into the potential of cells to home and localize in vivo. Adding activation markers (CD69, CD25, CD80, CD86) can shed light on which subsets are being activated and provide important clues about the body's immune response.

Building panel tool

antigen density.Researchers can use antigen density information to optimize antigen-fluor selection when adding markers to the panel. In general, we recommend choosing bright fluorochromes for markers with low antigen density and less bright fluorochromes for markers with high expression. To assist the researchers, BD generates information about the antigen density of many markers on peripheral blood cells.Bright conjugates for fog tokens.Certain B cell populations are very rare or are defined by poorly expressed antigens. Therefore, achieving sufficient resolution is crucial for obtaining good data in staining experiments. Especially with such rare or faint populations, the use of very bright fluorescent dyes can help improve resolution. A range of B cell marker antibodies can be conjugated to highly transparent BD Horizon Brilliant Violet™ (BV) dyes such as BV421, BV510 and BV605.

Extension panel to define additional subsets

Antigen density information helps optimize fluorochrome distribution when designing panels for the 10 color assay experiments shown. Bright fluorescent dyes were chosen for the low-density antigen CD138, which allowed the resolution of populations expressing different levels of IgD and CD38. An extremely bright BV dye was used for optimal detection of CD27 and IgM. Analysis with this panel provides additional information on the expression of Ig subclasses and allows the identification of two IgGs⁺and immunoglobulin G^–After school change hukommelsesceller (IgM^–Immunoglobulin D^–CD38^–CD27^{+/- darken}) and plasma cells (IgM^–Immunoglobulin D^–CD20^–CD38⁺⁺）。

1. Finak G, Langweiler M, Jaimes M, et al. Standardization of flow cytometry immunophenotyping by the Human Immunphenotyping Consortium.representatives of science。 2016；6：20686。 doi:10.1038/srep20686