A Guide to Regulatory T Cell Markers

T regulatory cells, or Tregs, are a distinct population of T cells that suppress immune responses. As such, Tregs prevent self-directed immunity, or autoimmunity, regulate immunity during infection, resolve tissue inflammation, and mediate tissue repair, among other functions. Their immunosuppressive capabilities, however, can sometimes have negative consequences. For example, highly suppressive Tregs can restrict tumor-directed immune responses and curb sterilizing immune responses, or those that effectively prevent infection by a pathogen. This functional diversity reflects the tremendous plasticity of Tregs, which are capable of acquiring specialized tissue- and context-specific functional programs in response to local cues like tissue damage or cytokines. In this article, we outline the cellular markers of Tregs as documented in recent literature. 

Markers of Thymic Tregs

A majority of Tregs originate from the thymus, known as thymic Tregs (tTregs). Responsible for establishing immune tolerance, tTregs develop in two steps. First, activation of the T cell antigen receptors (TCR) upregulates markers GITR (TNFRSF18), OX40 (TNFRSF4), and TNFR2 (TNFRSF1B). CD25 (IL2RA) is also upregulated in some but not all tTregs. Then, IL-2 and STAT5 drive the differentiation of these progenitors into mature FOXP3-expressing tTregs. The genomic organizer and transcription factor SATB1 is a marker of tTreg progenitors but not mature tTregs, as SATB1 and FOXP3 are mutually repressive. 

Markers of Peripheral Lymphoid Tregs

tTregs that emigrate from the thymus to secondary lymphoid tissues are called central Tregs (cTregs). These cells represent a quiescent, or naive, subset of Tregs. cTregs navigate lymphoid organs using homing molecules such as CD62L (SELL) and CCR7. Recent thymic emigrants express CD31 (PECAM1). cTregs are sometimes referred to as Fr. I Tregs. 

Tregs can also originate in the peripheral lymphoid organs rather than the thymus. This occurs when naive CD4+ T cells emigrate from the thymus to secondary lymphoid tissues, where they differentiate into peripherally derived Tregs (pTregs) upon interaction with an antigen-presenting cell. Unlike tTregs, pTregs are negative for the markers HELIOS and NRP1. 

Both cTregs and pTregs can convert into effector Tregs (eTregs). eTregs express low levels of the homing molecules CD62L and CCR7 and high levels of IL10 and activation markers such as CD44, ICOS, and GITR. eTregs also express the transcription factor BLIMP1 (PRDM1). eTregs are also known as Fr. II Tregs. 

Finally, Tregs can exist in the B cell zones of the lymphoid organs, where they facilitate antibody maturation and production by B cells. These CXCR5⁺ CD45RA⁻ cells are referred to as T follicular regulatory cells, or Tfr cells. Tfr cells are occasionally called Fr. III Tregs.

Figure image: An overview of Treg subsets and key defining markers.

Markers of Tissue-Adapted Tregs

Tregs have been shown to adapt to non-lymphoid tissues, where they promote tissue homeostasis and repair. Skin, intestinal, muscle, adipose, and tumor-associated Treg populations have been described, each bearing its own complement of markers.  

Skin Tregs establish immune tolerance to self and commensal antigens, to which the skin is heavily exposed. Skin Tregs express the transcription factors GATA3, IRF4, and BATF, although GATA3 is not necessary for the skin Treg signature. Chemokine receptors CCR6 and CCR4 allow skin Tregs to home to inflamed sites in the skin. Skin Tregs produce IL-4 and IL-33.

Intestinal Tregs are responsible for limiting immune responses to food and commensal antigens. They can be divided into three populations: FOXP3⁺RORγt⁻HELIOS⁻NRP1⁻, FOXP3⁺RORγt⁺HELIOS⁻NRP1⁻, and FOXP3⁺GATA3⁺HELIOS⁺ST2⁺. Whereas FOXP3⁺RORγt⁻HELIOS⁻NRP1⁻ intestinal Tregs mediate tolerance to food antigens, FOXP3⁺RORγt⁺HELIOS⁻NRP1⁻ intestinal Tregs mediate tolerance to antigens derived from gut microbiota. The third population of intestinal Tregs (FOXP3⁺GATA3⁺HELIOS⁺ST2⁺) facilitates tissue repair via secretion of amphiregulin (AREG), IL-10, and TGF-β.    

Like FOXP3⁺GATA3⁺HELIOS⁺ST2⁺ intestinal Tregs, muscle, and lung Tregs express ST2 (IL1RL1) and produce amphiregulin, indicating roles in tissue repair. Muscle and lung Tregs can be identified using CCR2 and IL18R, respectively. Visceral Adipose Tissue (VAT) Tregs maintain metabolic homeostasis by mitigating adipose inflammation. VAT Tregs can be identified using GATA3, CD69, ST2, and CCR2.

Tumor-infiltrated Tregs represent a unique population among tissue-adapted Tregs because tumors can exist in any tissue. Nevertheless, BATF, CCR8, and IL2RA are commonly observed in tumor-infiltrated Tregs across a variety of tissues, including breast and gastric cancers as well as brain and liver metastases of lung and colorectal cancers, respectively. Higher expression of FOXP3 in tumor-infiltrating macrophages portends a worse prognosis, but more research is needed to fully characterize the functions of Tregs in the tumor microenvironment.

Markers of Treg Function

Tregs primarily target two broad classes of immune cells to suppress immune responses: dendritic cells and effector T cells. 

Tregs directly suppress effector Th cells using three primary modes of action: cytokines, cytolysis, and metabolic disruption. Tregs secrete immunosuppressive cytokines such as interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and IL-35.  Tregs also secrete serine proteases such as granzyme B (GZMB) to trigger effector Th cell apoptosis. Finally, Tregs use CD25 and CD73 to generate pericellular adenosine, which suppresses effector Th cells via activation of adenosine receptor 2A. 

Tregs suppress effector Th cells indirectly by regulating the maturation and function of dendritic cells. Tregs primarily use receptor molecules to do this, including CTLA4 and LAG3. The former binds CD80/CD86 on dendritic cells, causing them to produce the immunoregulatory molecule indoleamine 2,3-dioxygenase (IDO). The latter binds MHC class II molecules on dendritic cells, inhibiting their maturation and function.

Finally, Tregs support Th cell responses by mirroring the functions of different Th cell subsets. Th1-like Tregs produce IFN-γ like Th1 cells, Th17-like Tregs produce IL-17 like Th17 cells, Th22-like Tregs produce IL-22 like Th22 cells, and Th2-like Tregs produce IL-4 like Th2 cells. Th-like Tregs and Th cells develop in parallel; for example, Th1-like Tregs differentiate alongside Th1 cells. These functional Treg subsets are further defined by transcription factor and chemokine receptor expression: Th1-like Tregs are T-bet⁺CXCR3⁺, Th17-like Tregs are RORγt⁺CXCR3⁻CCR6⁺CCR4⁺CCR10⁻, Th22-like Tregs are CXCR3⁻CCR6⁺CCR4⁺CCR10⁺, and Th2-like Tregs are GATA3⁺CCR6⁻CXCR3⁻CCR4⁺. These combinations of chemokine receptors, transcription factors, and cytokines allow each Treg subset to coordinate with and amplify the immune responses of the Th subset they resemble.  

Markers of Treg Markers

The table below lists human and mouse proteins characterizing various subsets of Treg 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 Treg cell immunophenotyping.

Note: *Some markers are protein isoforms, multi-subunit protein complexes, or protein families composed of several distinct genes. **This marker is a carbohydrate attached to a glycan. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

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Hero image: Scanning electron microscope image of T regulatory cells (red) interacting with antigen-presenting cells (blue). Source: NIH / NIAID (flickr).