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