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B cells immunophenotyping

B cells are responsible for producing and releasing antibodies to specific antigens, and hence, are an essential component of the humoral immune response.

B cells are mediators of the humoral response, or antibody-mediated immunity. By studying this particular cell group we learn more about the inner workings of the immune system, which consequently increases our awareness of the possible causes behind a variety of autoimmune disorders and cancers. Broad immunological research unlocks valuable insight of what future steps might be taken to treat these pathologies.

Development from stem cell to B cell

Generation of the B cell begins in the bone marrow where stem cells give rise to lymphoid cells. Throughout each stage of development the antibody locus— a site where an antigen interacts with the cell— undergoes genetic recombination. This recombination is specific to the developmental stage of the B cell. Development starts with the pro-B cell, which expresses Igα and Igβ. The cell matures further into the pre-B cell that expresses the pre-B cell receptor (Igμ) on its surface. Maturation in the bone marrow ends with the naïve B cell that expresses the B cell receptor (containing IgM and IgD) capable of recognizing an antigen. These cells then leave the bone marrow and enter the periphery (Cambier JC, et al. Nat Rev Immunol. 2007).

Subtypes of conventional B cells

Conventional B cells, also referred to as B-2 cells, terminally differentiate into one of two common subtypes upon activation:

  • Plasma B cells: a plasma cell is the sentry of the immune system. The naïve B cell circulates throughout the body. When it encounters a unique antigen, the plasma cell takes in the antigen through receptor-mediated endocytosis. Antigenic particles are transferred to the cell surface, loaded onto MHC II molecules and presented to a helper T cell. The binding of the helper T cell to the MHC II-antigen complex activates the B cell. The activated B cell goes through a period of rapid proliferation and somatic hypermutation. Selection occurs for those cells that produce antibodies with a high affinity for that particular antigen. Once terminally differentiated, the plasma B cell only secretes antibodies specific for that antigen and can no longer generate antibodies to other antigens.
  • Memory B cells: memory cells are held in reserve, in the germinal centers of the lymphatic system, for when the immune system re-encounters a specific antigen. During any repeat exposure the follicular helper T cell causes the memory cell to differentiate into a plasma B cell that has a greater sensitivity to that specific antigen. This jump-starts the immune system to mount a quicker, more powerful response than was possible previously.    

Other B cell subtypes include:

  • B-1 cells: a minor subtype, only about 5% in humans, of self-renewing fetal B cells that act in a similar fashion to plasma cells. B-1 cells are primarily present during fetal and neonatal life.
  • Marginal zone (MZ) B cells: mature memory B cells that are found only in the marginal zone of the spleen. These cells can be activated through toll-like receptor-ligation and not necessarily through the B cell receptor.
  • Follicular (FO) B cells: these are mature, but inactive, B cells. This subset of B cells is primarily found in the follicles of the spleen and lymph nodes. Activation of these cells requires the aid of T cells. FO B cells can differentiate into either plasma or memory B cells.
  • Regulatory B (Breg) cells: Breg cells negatively regulate the strength of the immune response and inflammation by secreting chemical messages called cytokines, such as IL-10. Although these cells make up a small portion of the B cell population (~0.5% in humans), it is thought that loss of functional Breg cells contributes to autoimmune disorders.

Immunophenotyping of B cells through flow cytometry

Immature B cells express CD19, CD 20, CD34, CD38, and CD45R, but not IgM. For most mature B cells the key markers include IgM and CD19, a protein receptor for antigens (Kaminski DA. Front Immunol. 2012). Activated B cells express CD30, a regulator of apoptosis. Plasma B cells lose CD19 expression, but gain CD78, which is used to quantify these cells. Memory B cells can be immunophenotyped using CD20 and CD40 expression. The cells can be further categorized using CD80 and PDL-2 regardless of the type of immunoglobulin present on the cell surface (Zuccarino-Catania GV et al. Nat Immunol. 2014.). Globally, cytokines (such as interlukein-10) and chemokines involved with chemokine receptor 3 play an important role in transmitting the biological messages to drive the immune response.

A table of common B cell subtypes with some cell markers which can be useful for flow cytometry:

B Cell TypeMarkerCellular localization
B cell (all except plasma cell)IgM, CD19Secreted
Activated B cellCD19, CD25, CD30Secreted
Plasma cellIgG, CD27, CD38, CD78, CD138, CD319Secreted
Plasma cellIL-6Secreted (cytokine)
Plasma cellCD138Cell membrane
Memory cellIgA, IgG, IgE, CD20, CD27, CD40, CD80, PDL-2Secreted
Memory cellCXCR3, CXCR4, CXCR5, CXCR6Secreted (chemokines)
Marginal zone B cellsCD1, CD21, CD27Secreted
Marginal zone B cellsNotch2Cell membrane
Follicular B cellsIgD,  CD21,  CD22, CD23Secreted
Regulatory B cellsIgD,  CD1, CD5, CD21, CD24, TLR4Secreted
Regulatory B cellsIL-10, TGFβSecreted (cytokines)


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