A Guide to Breg Markers

Regulatory B cells (Bregs) are a unique subset of B cells with immunosuppressive functions resembling those of regulatory T cells (Tregs). As such, Bregs limit excessive inflammation during infection, allergies, autoimmunity, and transplantation. Like that of Tregs, the immunosuppressive activity of Bregs can promote tumor immune escape. 

Bregs are thought to arise at multiple junctures throughout B cell development: immature B cells can differentiate into IL-10+ Bregs, an abundant population comprising several subsets; immature and mature B cells can differentiate into IL-10+ plasmablasts; and memory B cells can differentiate into B10 Bregs. Unlike for Tregs, which all express Foxp3, there is no single marker that identifies all Bregs. Beyond normal B cell markers, the most inclusive Breg markers are IL-10 and TIM-1, which are concomitantly expressed on roughly 70% of all Bregs. CD5, CD21, and CD1d are also common but not completely inclusive markers of Breg subsets. CD19, which can label all Bregs, also labels all B cells.

Mouse and human Bregs differ somewhat in their marker profiles. This article reviews the cellular markers of mouse and human Bregs as well as markers of Breg function.

Figure: This table highlights notable Breg subsets and associated markers in mice and humans.

Markers of Mouse Breg Subsets

Largely isolated from spleens and lymph nodes, mouse Bregs are more extensively phenotyped than human Bregs. Mouse Breg subsets include marginal-zone precursor (T2-MZP) cells, B10 cells, marginal-zone (MZ) B cells, TIM-1+ B cells, CD138+ plasma cells, plasmablasts, killer B cells, and GITRL+ B cells. 

T2-MZP Bregs, defined as CD19⁺CD21^hiCD23^hiCD24^hiIgM^hiIgD^hiCD1d^hi, were the first Bregs to be discovered in mice. Although they were initially discovered in the spleen of a mouse model of arthritis where they slowed disease progression, similar splenic IL-10-producing Breg populations have since been discovered in mouse models of infection, allergy, autoimmunity, transplantation, and cancer. Descended from T2-MZP Bregs, MZ Bregs also express high levels of CD1d but do not express CD23.

B10 cells are Bregs that exclusively produce IL-10. This is in contrast to other Breg subsets that produce additional cytokines. These splenic Bregs can be identified as CD1d^hiCD5⁺.

Plasmacytic B cell populations can acquire immunosuppressive functions, forming new Breg subsets. Primarily identified as CD138+, Breg plasma cells can be found in the spleen and Breg plasmablast in the draining lymph nodes of mice. It is unclear whether different CD138+ Bregs reflect distinct subsets or the same population adapted to different environments.    

GIFT15 Bregs, a subset of plasmacytic Bregs induced by a chimeric granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-15 protein, share the same cell surface markers as T2-MZP Bregs except for CD19, which they do not express, and CD138, which they do. CD138 is a key marker of plasmacytic differentiation.

Unlike other Breg subsets, regulatory B-1a cells derive from the B-1 lineage and arise in the fetus as opposed to B-2 lineage that arises after birth and from which conventional B cells differentiate. Regulatory B-1a cells express CD5 but not CD1d, allowing them to be distinguished from B10 cells using these two markers. A subset of regulatory B-1a cells, called killer B cells, also express FasL, which they use to trigger T cell apoptosis. 

Most mouse Breg subsets express the cell surface marker TIM-1. 

Markers of Human Breg Subsets

Comparatively fewer Breg subsets have been discovered in humans. Those that have been discovered were found in the blood. Human Bregs can be found among many B cell populations, including immature cells, B10 cells, plasmablasts, Br1 cells, and GZMB+ B cells. 

Immature human Bregs express high levels of CD24 and CD38 and are the highest producers of IL-10 in humans. These cells participate in autoimmunity, where their exhaustion by excessive inflammation leads to impaired immunosuppressive function. They have also been found in HIV-1 patients, where they suppress CD8+ cytotoxic T cell responses, and transplant patients, where they promote transplant tolerance. 

B regulatory 1 cells are another IL-10-producing subset of human Bregs that participate in allergy immune responses. These CD25^hiCD71^hiCD73^lo cells maintain immune tolerance to allergens through antigen-specific interactions with T cells and the production of allergen-specific IgG4 antibodies.

A unique population of human Bregs expressing the cytotoxic protease granzyme B has been detected in solid tumors. These CD19⁺CD38⁺CD1d⁺IgM⁺ cells, called GZMB+ Bregs, interfere with antitumor immunity. 

Other human Breg subsets, including B10 cells and plasmablasts, resemble their mouse counterparts in function more than marker expression. Although both subsets can be found among the CD19⁺CD24^hiCD27⁺ B cell population, human Breg plasmablasts also express high levels of CD38, distinguishing them from human B10 cells. 

Markers of Breg Function

Breg cells use a variety of mechanisms to suppress innate and adaptive immune responses. Most actions are antigen-specific and involve multiple effectors (cytokines and receptor interactions, for example). Many of the mechanisms are similar to those used by regulatory T cells.

Breg cells produce immunoregulatory cytokines that exert diametric effects on pro-inflammatory and immunosuppressive immune cell types. For example, Bregs halt the differentiation of pro-inflammatory immune cell populations like TNFα-producing monocytes, IL-12-producing dendritic cells, Th17 cells, Th1 cells, and cytotoxic CD8+ T cells by secreting IL-10, IL-35, and TGF-beta. The same cytokines also stimulate the differentiation of immunosuppressive regulatory T cells. IL-10 is the most commonly secreted cytokine across Breg subsets.  

In addition to secreting immunoregulatory cytokines, Bregs secrete immunoregulatory enzymes such as indoleamine 2,3-dioxygenase (IDO/IDO1) and granzyme B. Secretion of IDO dampens T cell activation by catabolizing the essential amino acid tryptophan, whereas secretion of the granzyme B kills T cells.  

Bregs also use contact-dependent mechanisms involving membrane proteins and/or receptors to promote immune tolerance. Bregs suppress effector T cells via CD80/CD86 and induce their apoptosis via FasL and PD-L1. GITRL+ Bregs promote Treg expansion via GITRL. CD1d+ T2-MZP Bregs support the development and function of immunosuppressive invariant natural killer (iNKT) cells via the presentation of lipids on CD1D. Engagement of TIM-1 on TIM-1+ Bregs induces their expansion. 

Lastly, like Tregs, CD73⁺CD39⁺ Bregs can convert immunostimulatory ATP and ADP into immunosuppressive adenosine via the ectoenzymes CD73 and CD39 (ENTPD1, NT5E). 

Table of Breg Markers

The table below lists human and mouse proteins characterizing various subsets of Breg cells as described by recent review literature. The majority of proteins listed are membrane markers expressed on the cell surface, but also included are other defining proteins, including transcription factors and signaling proteins, such as cytokines. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for Breg cell immunophenotyping.

Note: *Some markers are multi-subunit protein complexes composed of several distinct genes. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org for human genes. 

References

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