Regulatory T Cells

Tools for the identification, isolation, and multicolor analysis of human regulatory T cells

As published data on the immunosuppressive potential of Regulatory T Cells (Tregs) has accumulated, enthusiasm for their potential application has intensified. Thus Treg research is very active, and new publications emerge almost daily. Today, commonly used markers for Treg identification, isolation, and characterization are CD4, CD25, CD127, and FoxP3. However, new targets with functional significance such as CD39, CD45RA, CTLA-4, and others are rapidly emerging.

About Tregs Identifying Tregs Treg Enrichment

Different subsets, similar functions

Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis1. Tregs suppress the function of other T cells to limit the immune response. Alterations in the number and function of Tregs has been implicated in several autoimmune diseases including multiple sclerosis, active rheumatoid arthritis, and type 1 diabetes. High levels of Tregs have been found in many malignant disorders including lung, pancreas, and breast cancers. Tregs may also prevent antitumor immune responses, leading to increased mortality.

CD4 and CD8 Tregs

Two major classes of Tregs have been identified to date: CD4 and CD8 Tregs. CD4 Tregs consist of two types, “natural” Tregs (nTregs) that constitutively express CD25 and FoxP3, and so-called adaptive or inducible Tregs (iTregs).

Natural Tregs originate from the thymus as CD4+ cells expressing high levels of CD25 together with the transcription factor (and lineage marker) FoxP3. nTregs represent approximately 5–10% of the total CD4+ T cell population, and can first be seen at the single-positive stage of T lymphocyte development.2 They are positively selected thymocytes with a relatively high avidity for self-antigens. The signal to develop into Treg cells is thought to come from interactions between the T cell receptor and the complex of MHC II with self peptide expressed on the thymic stroma.3 nTregs are essentially cytokine independent.

Adaptive or inducible Tregs originate from the thymus as single-positive CD4 cells. They differentiate into CD25 and FoxP3 expressing Tregs (iTregs) following adequate antigenic stimulation in the presence of cognate antigen and specialized immunoregulatory cytokines such as TGF-β, IL-10, and IL-4.4

FoxP3 is currently the most accepted marker for Tregs, although there have been reports of small populations of FoxP3- Tregs. The discovery of transcription factor FoxP3 as a marker for Tregs has allowed scientists to better define Treg populations leading to the discovery of additional Treg markers including CD127.

CD127 discovery

Research from the laboratories of Barbara Fazekas de St. Groth (Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia) and Jeffrey Bluestone (UCSF Diabetes Center, San Francisco, CA, USA) and confirmed in laboratories at BD Biosciences demonstrated that CD127 expression is down-modulated on the Treg cells, inversely correlating with the expression of Treg marker FoxP3. It was also demonstrated that Tregs are also present (at varying levels) in populations of cells expressing high and low levels of CD25. These findings provided a new cell surface marker for Tregs,5,6 enabling isolation of viable cells by flow cytometric sorting for further downstream analysis.

CD127 is part of the heterodimeric IL-7 receptor that is composed of CD127 and the common g chain, which is shared by other cytokine receptors (IL-2R, IL-4R, IL-9R, IL-15R, and IL-21R). CD127 is expressed on thymocytes, T- and B-cell progenitors, mature T cells, monocytes, and some other lymphoid and myeloid cells. Studies have shown that IL-7R plays an important role in the proliferation and differentiation of mature T cells, and in vitro experiments show that the expression of CD127 is down-regulated following T cell activation. 7-9 It is believed that FoxP3 interacts with the CD127 promoter and might contribute to reduced expression of CD127 in Tregs. 6

Treg subsets

  Natural Treg (nTregs) Induced Tregs (iTregs)
  nTregs Tr1 Th3
Phenotype CD4+CD25 int/high, CD127 low CD4 +CD25 - CD4 +CD25 + from CD25 - precursors
Other associated markers CTLA4 +, GITR +, FoxP3 +, CD127 low CD25 low-variable, CD45RB low, FoxP3 - CD25 low-variable, CD45RB low, FoxP3 +
Suppression Contact dependent, granzyme B-dependent, makes TGF-β Through cytokines produces IL-10 Through cytokines, produces TGF-β
Target cells APC and T effector cells T effector cells Not yet Identified
CD28 Involvement Thymic development and maintenance in periphery Not for development or function Not involved
In vivo Role Suppression of autoreactive T cells Mucosal immunity, inflammatory response Mucosal immunity, inflammatory response
In vitro Expansion Expandable using TCR/CD28 stimulation and IL-2 CD3, IL-10, rehnoic acid CD3, TGF-β

FoxP3: The classic Treg marker

Although several surface markers were defined for Treg identification, a classic marker specific and unique to Tregs remained undiscovered until FoxP3 was identified as a Treg marker in mice in simultaneously reported studies by Sakaguchi and Rudensky.

FoxP3 (also known as Scurfin, IPEX, and JM2) is a transcriptional repression factor of the forkhead or winged helix family of transcription factors.13 FoxP3 has been found to be expressed in all CD4+ Treg cells that have regulatory activity. Mutations in FoxP3 are associated with the inherited auto-immune diseases Scurfy in mice and its human counterpart, IPEX (immune dysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome).14

FoxP3 is useful for confirming purity and yield of isolated Tregs or for characterizing fixed Treg cells. However, it is not suitable for use in isolating viable Treg cells, since FoxP3 staining requires fixation and permeablization of the cells. In these cases CD127- is a better solution.

FoxP3 staining

Human FoxP3 monoclonal antibody clone 259D/C7 from BD Biosciences reacts with all currently identified isoforms of the human FoxP3 transcription factor and is cross-reactive with cynomolgus, rhesus, and baboon.

The BD Pharmingen™ human FoxP3 antibody, available in multiple sizes and conjugates is a high performance reagent system for the detection of FoxP3 positive Tregs. An easy-to-use buffer system allows researchers to fix and permeabilize cells in just a few simple steps, with the option of freezing samples up to 72 hours.

Supporting an emerging list of target markers

While FoxP3 is a commonly used marker for Treg identification, isolation, and characterization, Tregs are a very active area of research, and an emerging list of targets has been published in the literature.

To support these emerging discoveries, the BD Biosciences portfolio of new high quality reagents and solutions continues to grow.

CD39: enhanced characterization of Tregs

Previously localized primarily on B cells, dendritic cells, and certain subsets of T cells, CD39 has recently been shown to be coexpressed with FoxP3 in CD4 + Tregs in humans and mice. 15 This discovery is adding to the growing list of cell surface markers such as CD25, CD45RA, HLA-DR, and CTLA-4, that are important in the identification and functional characterization of CD4 + Tregs.

Extracellular ATP and its metabolites are potent regulatory molecules modulating a broad range of cell and organ functions. Cellular ATP release is an indicator of tissue destruction and a danger “signal” that activates the immune response. CD39 hydrolyzes extracellular ATP (or other triphosphates) into its respective nucleotides such as AMP. Extracellular nucleoside monophosphates are, in turn, rapidly degraded to nucleosides (eg, adenosine) by soluble or membrane bound ecto-5′ nucleotidases (CD73). Peri-cellular adenosine then mediates anti-inflammatory T cell responses. Coexpression of CD39 and CD73 is thought to be one of the key mechanisms of immunosuppression mediated by Tregs. 16,17

The BD Pharmingen brand anti-human CD39 (clone TÜ66) monoclonal antibody is a novel marker for human Tregs and is available as PE and APC conjugates in ready-to-use reagent kits for flow cytometry. TÜ66 recognizes ENTPD1, an ectoenzyme that belongs to the family of ectonucleoside triphosphate diphospho-hydrolases (E-NTPDases). The members of this family are involved in extracellular nucleotide catabolism, controlling the extracellular nucleoside triphosphate pool (NTPs).

CD152 (CTLA-4)

Cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) is considered to be critical for Treg suppressive function. 18 In a study by Zheng et al, CD152 was transfected into CD25 -FoxP3 - T cells. The resulting cells had suppressive activity but did not express FoxP3. 19

Studies from other groups have demonstrated that blockage of CD152 impairs the suppressive activities of Tregs. Abnormalities in CD152 expression have been reported to play a role in autoimmune diseases such as rheumatoid arthritis. 19

CD152 may mediate suppressive activities through the down-regulation of CD80 and CD86 expression on dendritic cells, affecting the potency of antigen-presenting cells to activate other T cells. 20

Studies such as those described for CD152 further our understanding of Treg function. The existence of defined populations, existing markers, and emerging markers will greatly contribute to exciting new discoveries in Treg biology.

Reported markers of human Tregs

This table highlights research reagents that are most relevant for human Regulatory T cell research. Our reagent portfolio is constantly expanding.

Antigen Human Expression Reference Number
CCR4 positive 21, 22
CCR6 positive 22
CCR7 positive 23
CD4 positive 12, 24, 25
CD11a high 26, 27
CD25 positive 28
CD39 positive and negative subsets 29
CD45RA positive 30
CD45R0 positive 31
CD46 positive 10
CD54 (ICAM-1) high  
CD62L low and high subsets 32
CD69 positive 33
CD103 positive 34
CD122 (IL-2Rβ) positive  
CD127 low 5, 6
CD134 (OX-40) positive and negative subsets 35
CD152 (CTLA-4) high 36, 37
CD154 (CD40L) positive 38
CD223/LAG-3 positive 39, 40, 46
CXCR4 positive 33
CXCR5 positive 33
CXCR6 positive 33
FoxP3 positive 41, 42
GITR high 43
GPR83 positive 44
Granzyme B positive 45
IL-10 positive 46
IL-35 positive 47
MHCII positive 48
Neuropilin 1 positive 49
Perforin positive 50
TGF-β positive 51

Enrichment of Tregs with CD4, CD25, and CD127

Tregs represent a small population of cells; enrichment is often necessary for downstream analysis. Several methods exist for the enrichment of whole or subpopulations of Tregs. While FoxP3 currently is considered the most accepted marker for Tregs, its intracellular localization prohibits its use for the isolation of viable Tregs. Other markers used for enrichment are either negative, positive, or used in combination. One reported method of negative selection is the removal of cells expressing CD127 and CD49d.10 Cells expressing CD4+ and the highest levels of CD25 are used for positive selection. Combination methods can include the use of magnetic beads to remove contaminating populations prior to cell sorting. One of the best characterized methods enriches for CD4+, CD25+, CD127- cells.

Enrichment of Tregs with CD4 and CD25

In humans, initial analysis of Treg populations revealed that only those ex vivo cells that express the highest levels of CD25, which represent approximately 2-3% of total CD4 T cells, demonstrate an in vitro suppressive activity11,12 in contrast to mouse cells in which all CD25 cells are considered Tregs.4,5 Furthermore, cells expressing low to intermediate levels of CD25 were thought not to exhibit any suppressive activity directly ex vivo.

Additionally, the definition of high and low levels of CD25 expression lacks consensus and has limited its use for obtaining viable human Tregs via flow cytometric cell sorting. As a result, many researchers only select cells with the highest expression of CD25, dramatically reducing the yield of isolated Tregs. These results intensified research to identify cells surface markers other than CD4 and CD25 that are exclusive to human Tregs.

Cell surface characterization and isolation of viable human Tregs using CD127

Staining human CD4 T lymphocytes with CD4, CD25, and CD127 enables the enrichment of viable regulatory T cell fractions for further culture and/or use in other in vitro assays. Using this method, viable cells can be quickly isolated through cell sorting. The BD Pharmingen™ brand Human Regulatory T Cell Cocktail, a pre-mixed three-color reagent, simplifies enrichment of viable T cells using a gating strategy that relies on CD4+CD25int/high CD127low cells. This strategy enhances the recovery of CD25int/high sorted Treg cells by 2 to 4 times compared to gating on CD25high cells alone, while eliminating contaminating CD25-/int effector cells.

Magnetic based pre-enrichment of CD4+ T cells to increase yield of human Tregs

Human Tregs represent a small population of total cells. One method to increase the yield of highly enriched human Treg cells combines magnetic pre-enrichment of CD4+ cells with BD IMag™ technology with CD127-based cell sorting.

The BD IMag Human CD4 T Lymphocyte Enrichment Set (Cat. No. 557939) offers a convenient way to pre-enrich CD4+ T cells. Purities of greater than 90% were routinely attained for the enrichment. The procedure captures leucocytes that are not CD4 T lymphocytes, by magnetically removing them from the sample to create an enriched CD4 population. Enriched cells are then stained and shown in the figure on the left.

Cells were stained with anti-human CD4 PerCP (Cat. No. 345770), anti-human CD25 FITC (Cat. No. 555431), and anti-human CD127 PE (Cat. No. 557938). The enriched cells were sorted using a BD FACSAria™ cell sorter. A detailed protocol and recommended gating strategy can be found at

Correlation and regression analysis

Enrichment of Tregs with CD4, CD25, and CD127

The BD Pharmingen brand Human Regulatory T Cell Cocktail

Profile of anti-CD152 (CTLA-4) staining on fixed PBMCs

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