• New Regulatory Reagents: Folate Receptor 4 (FR4), CD39, CD73
• Ordering Information: Foxp3, Human, Mouse, Non-Human Primate
• Foxp3 Antibody-Specific References
• Foxp3 Antibody Data: FJK-16s, PCH101, NRRF-30, eBio7979, 236A/E7
• CD127 (IL-7Rα)
• GITR: Regulator of Treg Suppressor Functions

Tregs & Foxp3: the Suppression Phenomenon

Until recently, definitive flow cytometric analysis of CD4+CD25+ regulatory T cells, expressing Foxp3, has been hindered due to lack of suitable Foxp3 antibodies. Using eBioscience Foxp3 antibodies PCH101, FJK-16s and NRRF-30, Foxp3 can now be identified at the single-cell level (see figures and table below).

Since the discovery of regulatory suppressor cells by Gershon in 1970, great controversies have ensued. These cells have broad implications ranging from immune suppression to cancer. Consequently, the interest in the transcription factor Foxp3, identified by S. Sakaguchi (2003) as a hallmark of naturally arising CD4+CD25+ regulatory T cells (Tregs), has intensified.

Recently, one splice variant of Foxp3, lacking amino acids 71-105, has been reported in human T cells. This variant is expressed in CD8+ T cells from some donors, while both forms are present in CD4+CD25+ cells. The western blot data on page two reveals two bands in human tissue; presumably from each splice variant, while only a single band is detected in mouse. The presence of this splice variant has potentially complicated the complete characterization of Foxp3 expression.

Mouse Foxp3

BALB/c splenocytes were stained with CD4-FITC (clone RM4-5; cat. 11-0042), CD25-APC (clone PC61.5; cat. 17-0251), and mouse/rat Foxp3-PE (clone FJK-16s) using the PE anti-mouse/rat Foxp3 Staining Set (cat. 72-5775). Quadrant lines demarcate the isotype control. Left: FJK-16s vs CD4 Right: FJK-16s vs CD25			The histogram demonstrates FJK-16s-FITC (cat. 71-5775) staining (blue histogram) and isotype control (yellow histogram) on CD4+CD25+ mouse spleen cells.

Human Foxp3

Human PBMCs were stained with CD4-FITC (clone RPA-T4; cat. 11-0049), CD25-APC (clone BC96; cat. 17-0259), and human Foxp3-PE (clone PCH101) using the PE anti-human Foxp3 Staining Set (cat. 72-5776). Quadrant lines demarcate the isotype control. Cells in the lymphocyte gate were used for analysis. Left: PCH101 vs CD4 Right: PCH101 vs CD25			The histogram demonstrates PCH101-FITC (cat. 71-5776) staining (blue histogram) and isotype control (yellow histogram) on CD4+CD25bright human PBMC.

Immunological Identity and Specificity of Foxp3 Antibodies

As has been done with many cell surface and intracellular (e.g., cytokine) proteins, corroboration of staining pattern using antibodies to distinct epitopes from the same protein allows researchers to confirm staining patterns and hence specificity. To this end, eBioscience utilized protein deletion constructs to epitope-map a panel of Foxp3 antibodies. The Figure below shows the schematic representation of the Foxp3 protein with functional domains and also localizes the regions recognized by each of eBioscience's Foxp3 antibodies.

Schematic representation of the Foxp3 protein and the location of the epitopes for eBioscience antibodies. Foxp3 contains a Zn finger/Leucine zipper and a conserved Forkhead Domain. In addition, in human Exon 2 may be alternatively spliced in some cells (black box). Currently there is not evidence for alternative splicing in the mouse. The clone names for each of eBioscience antibodies are shown in the location of their individual epitopes.

In the figures below, co-staining experiments indicate that several Foxp3 antibodies stain the same cells. The anti-mouse Foxp3 antibodies, FJK-16s and NRRF-30 recognize different epitopes in the amino terminus, with the former recognizing the region corresponding to the human splice variant. These two antibodies identify the same population of lymphocytes, and co-staining experiments demonstrate 100% correlation (see figure below). These data suggest that these two antibodies, which recognize different regions of recombinant Foxp3, bind the same protein expressed in the same cells. Additionally, the same approach was taken for the human Foxp3 antibodies.

Co-staining of eBioscience foxp3 antibodies that recognize different epitopes. Left: Mouse splenocytes were co-stained with FJK-16s and eBio7979. Right: NRRF-30 co-staining with FJK-16s.			Existence of splice variant in human, not mouse: immunoblotting of PBMC (Lane A), BALB/c splenocyte (Lane B), or recombinant human Foxp3 fusion protein (Lane C); probed with eBio7979 (cat. 14-7979).

Co-staining of eBioscience Foxp3 antibodies that recognize different epitopes. Left: Human PBMCs were co-stained with PCH101 and 236A/E7. Right: Co-staining of PCH101 and eBio7979.

The expanding panel of antibodies reactive with distinct epitopes of Foxp3 is useful for investigating the complete Foxp3 protein expression profile at the single-cell level in both human and mouse, particularly with regards to its relationship to functionally-defined Regulatory T cells. In addition, the presence of an alternatively spliced transcript in human adds another layer of complexity to the Foxp3 expression pattern and functional significance. Currently this phenomenon appears to be unique to human since both co-staining at the single-cell level using several different combinations of antibodies to different domains and immunoblotting data suggests that only one isoform exists in the mouse. (See flow analysis and immunoblot data above.) In the human, two isoforms can be present; the anti-mouse/rat Foxp3 antibody, FJK-16s, crossreacts to human Foxp3 but recognizes the region that can be alternatively spliced in some transcripts. Therefore, the FJK-16s antibody labels the Foxp3 protein isoform that has not been spliced, while PCH101 and 236A/E7 antibodies recognize both isoforms of the protein. Using these reagents together will allow researchers to investigate the function of these two isoforms of human Foxp3 protein in the same cell population.

Foxp3 Reagents Validated for Intracellular Staining and Flow Cytometric Analysis, Immunohistological Staining and Immunoblotting

The Foxp3 antibodies have been validated for intracellular staining with flow cytometric analysis. The protocols and staining buffers have been developed for optimal and consistent staining Foxp3 staining while preserving surface antigen staining. Furthermore, the protocol was designed for robustness, reproducibility, ease-of-use, as well as flexibility, to allow researchers to stain at their convenience. In addition, the antibodies have been reported useful for immunohistological staining (both frozen and paraffin sections) and western blotting of endogenous protein from total human PBMCs or mouse splenocytes, without the need for enrichment of Tregs.

C57BL6 spleen cryosections were stained with anti-mouse/rat Foxp3 antibody, FJK-16s (cat. 14-5773) and revealed with colorimetric methods. Image courtesy of Cintia De Paiva.		Staining of human tonsillar tissue section with PCH101 (cat. 14-4776). Image courtesy of Roger Sutmuller.

New! For Non-Human Primate Studies and Beyond

The anti-human Foxp3 antibodies, PCH101 and 236A/E7, crossreact to several non-human primate species in intracellular staining studies. Additionally, antibodies to CD4 (OKT4) and CD25 (BC96) also stain rhesus and cynomolgus lymphocytes, making characterization of natural Tregs in non-human primates more comprehensive.

It has been confirmed that FJK-16s crossreacts with canine Foxp3 protein (Biller BJ, Elmslie RE, Burnett RC, Avery AC, Dow SW. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol. 2007 Mar 15;116(1-2):69-78, pubmed).

As Researchers Learn, the Partnership with eBioscience Grows

As researchers learn more about the importance of Foxp3 as a master control of immune regulation, the commitment of eBioscience to providing critically-important, well-validated reagents becomes clear. The Foxp3 antibodies are available in a variety of fluorochromes (including tandem dyes and Alexa Fluors^®*) that is constantly expanding to meet your needs. In combination with our vast offering of fluorochrome-conjugated antibodies reactive with cell surface antigens, Foxp3 staining can be analyzed against more than 6 colors allowing for the intricate analysis of cell populations and subsets of populations.

Publications using eBioscience Foxp3 Clones for IC Staining & Flow Cytometric Analysis

Mouse Foxp3

1. Biller, BJ., et al. 2007. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol. 116(1-2):69-78. [Intracellular staining for flow cytometry using FJK-16s in canine]
2. Sakaguchi, S. et al. 2005. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J. Exp. Med. 202: 885-891. [Intracellular staining for flow cytometry using FJK-16s]
3. McGeachy, MJ., et al. 2005. Natural Recovery and Protection from Autoimmune Encephalomyelitis: Contribution of CD4+CD25+ Regulatory Cells within the Central Nervous System. J. Immunol. 175: 3025 - 3032. [Intracellular staining for flow cytometry using FJK-16s]
4. Fields, ML., et al. 2005. CD4+CD25+Regulatory T Cells Inhibit the Maturation but Not the Initiation of an Autoantibody Response. J. Immunol. 175: 4255 - 4264. [Intracellular staining for flow cytometry using FJK-16s]
5. Lu, LF., et al. 2005. NFB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation. J. Immunol. 175: 1651 - 1657. [Intracellular staining for flow cytometry using FJK-16s]
6. Beyersdorf, S., et al. 2005. Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis. J Exp Med. 202: 445-55. [Intracellular staining for flow cytometry using FJK-16s (correction; not mFoxy)]
7. Anderson, M.S., et al. 2005. The cellular mechanism of Aire control of T cell tolerance. Immunity. 28: 227-39. [Intracellular staining for flow cytometry using FJK-16s]
8. Chang, X., et al. 2005. The Scurfy mutation of FoxP3 in the thymus stroma leads to defective thymopoiesis. 202:1141-51. [Intracellular staining for flow cytometry using FJK-16s]
9. Aswad, F., et al. 2005. High sensitivity of CD4+CD25+ regulatory T cells to extracellular metabolites nicotinamide adenine dinucleotide and ATP: a role for P2X7 receptors. J. Immunol. 175:3075-83. [Intracellular staining for flow cytometry using FJK-16s]
10. Siegmund, R., et al. 2005. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo. Blood. 106: 3097-104. [Intracellular staining for flow cytometry using FJK-16s]
11. Romagnoli, S., et al. 2005. Genetic control of thymic development of CD4+CD25+FoxP3+ regulatory T lymphocytes. Eur J Immunol. 35: 1-8. [Intracellular staining for flow cytometry using FJK-16s]
12. Wilson, M.S., et al. 2005. Suppression of allergic airway inflammation by helminth-induced regulatory T cells. J Exp Med. 202: 1199-1212. [Intracellular staining for flow cytometry using FJK-16s]
13. Knoechel, B., et al. 2005. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen J. Exp. Med. Nov (Epub ahead of print). [Intracellular staining for flow cytometry using FJK-16s]
14. Kretschmer, K., et al. 2005. Inducing and expanding regulatory T cells population by foreign antigen. Nat. Immunol. 6(12): 1219-1227. [Intracellular staining for flow cytometry using FJK-16s]
15. Krupnick, AS., et al. 2005. Cutting Edge: Murine Vascular Endothelium Activates and Induces the Generation of Allogeneic CD4+25+Foxp3+ Regulatory T Cells. J. Immunol. 175: 6265-6270. [Intracellular staining for flow cytometry using FJK-16s]
16. Liu, R., et al. 2005. Cooperation of Invariant NKT Cells and CD4+CD25+ T Regulatory Cells in the Prevention of Autoimmune Myasthenia. J. Immunol. 175(12):7898-7904. [Intracellular staining for flow cytometry using FJK-16s]
17. Moore, AC., et al. 2005. Anti-CD25 Antibody Enhancement of Vaccine-Induced Immunogenicity: Increased Durable Cellular Immunity with Reduced Immunodominance. J. Immunol. 175: 7264-7273.
18. Stephens, LA., et al. 2005. CD4+CD25+ regulatory T cells limit the risk of autoimmune disease arising from T cell receptor crossreactivity. PNAS 102:17418-17423.

Human Foxp3 (PCH101):

1. Ahmadzadeh, M., et al. 2005. IL-2 Administration Increases CD4+CD25hiFoxp3+ Regulatory T Cells in Cancer Patients. Blood (Nov Epub). [Intracellular staining for flow cytometry using PCH101]
2. Crellin, NK., et al. 2005. Human CD4+ T Cells Express TLR5 and Its Ligand Flagellin Enhances the Suppressive Capacity and Expression of FOXP3 in CD4+CD25+ T Regulatory Cells. J. Immunol. 175(12): 8051-9. [Intracellular staining for flow cytometry using PCH101]
3. Hartwig, UF., et al. 2005. Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes. Bone Marrow Transplant (Dec 5 Epub ahead of print). [Intracellular staining for flow cytometry using PCH101]
4. Lim, HW., et al. 2005. Cutting Edge: Direct Suppression of B Cells by CD4+CD25+ Regulatory T Cells. J. Immunol. 175: 4180 - 4183. [IHC of frozen sections using 236A/E; Intracellular staining for flow cytometry using PCH101]

Human Foxp3 (236A/E7):

1. Alvaro, T., et al. 2005. Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin. Cancer Res. 11(4):1467-73. [IHC paraffin using 236A/E7]
2. Roncador, G., et al. 2005. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur. J. Immunol. 35: 1681-91. [IHC of paraffin sections using 236A/E; Intracellular staining for flow cytometry using 236A/E]
3. Roncador, G., et al. 2005. FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma. Leukemia (Epub ahead of print). [IHC paraffin sections using 236A/E7]
4. Wolf, D., et al. 2005. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin. Cancer Res. 11(23): 8326-31. [IHC paraffin using 236A/E7]

CD127 (IL-7Rα)

CD127 (IL-7Rα) complexes with CD132, also known as the common γ chain (γc), to form the multi-functional IL-7 receptor (IL-7R). CD127 is a type I glycoprotein with a molecular weight of 75-80 kDa and is expressed by immature B cells through the early pre-B stage, by thymocytes during several stages of their development, and on most mature T cells, with transient down-regulation upon activation. Binding of IL-7 results in signal tansduction which occurs through several tyrosine kinase pathways including the Jak/STAT pathway. IL-7 is indispensible for the development of lymphocytes, and the control of homeostatic proliferation of T-cells in the periphery. In addition, IL-7R signaling is known to be involved in the regulation of T cell receptor (TCR) locus rearrangment in γδ T cells.

Interestingly, it has recently been demonstrated that CD127 expression is down-regulated on CD4+CD25+ regulatory T cells (T regs). While the co-expression of CD4 and CD25 has become widely used as an indicator of T regs, this method of identification may also include cells without suppressive activity. It has clearly been shown that CD4+CD25+ cells that have down-regulated the expression of CD127 are significantly more highly-enriched for the regulatory T population, as defined by expression of the T reg-specific transcription factor Foxp3 and the suppressive activity of these cells, in vitro.

Staining of normal human peripheral blood cells with FITC anti-human CD3 (OKT3, cat. 11-0037) and Biotin Mouse IgG1, K Iso Cntrl (cat. 13-4714) (left) or Biotin anti-human CD127 (eBioRDR5, cat. 13-1278) (right) followed by SAv-PE (cat. 12-4317). Cells in the lymphocyte gate were used for analysis.

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GITR: Regulator of Treg Suppressor Functions

The mouse glucocorticoid-induced TNF-related receptor, GITR, is expressed by activated conventional CD4+ and CD8+ T cells as well as by regulatory T cells, generally defined as CD4+/CD25+ T cells. The expression of GITR is rapidly upregulated on naive CD4+/CD25- upon stimulation through TCR. AITR, the human counterpart to mouse GITR, is expressed by activated peripheral T cells. The ligand for GITR, also known as TL6 in human, is expressed by several antigen-presenting cell types including mature dendritic cells, macrophages and B cells, as well as by cultured vascular endothelial cells, and retinal pigment epithelium (RPE).

GITR provides a potent costimulatory signal for the antigen-driven proliferation of naive T cells, and polarized T helper 1 and T helper 2 clones. The interaction of GITR with its ligand augments T cell activation, enhanced cell cycle progression; induction of several activation molecules such as CD69 and CD25; cytokine secretion including IL-2, IFN-γ, IL-4, and IL-10, and anti-CD3-induced redirected cytotoxicity. In addition, GITR plays a key role in regulating the suppressive function of and the peripheral tolerance mediated by regulatory T cells, inhibiting TCR induced apoptosis, and upregulating inducible nitric oxide synthase (iNOS).

Removing GITR-expressing regulatory T cells in vivo by administration of specific monoclonal antibody results in organ-specific autoimmune disease, highlighting the evidence that in addition to its involvement as a potent and unique costimulator for an early CD4+ T cell activation, signals delivered by GITR abrogate regulatory T cell-mediated suppression.

Expression of mouse GITR on the surface of CD4+/CD25+ splenic T cells. Mouse spleen cells were simultaneously stained with anti-CD4-FITC, anti-CD25-APC, and anti-GITR-PE. Viable cells were gated for analysis. Plot above shows staining of CD4 vs. CD25, revealing a small subset of resting splenic CD4+ cells that express CD25. The GITR is expressed by this double-positive subset only (green population).

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