Foxp3

Foxp3: The Master Regulator of T Regulatory Cells

The discovery of Foxp3 as the key transcription factor controlling T regulatory cell (Treg) development and function represents one of the most significant advances in Treg immunobiology. While the complete function and differentiation of Tregs now appears to require the presence of additional transcription factors, including AhR and STAT-5, it remains clear that Foxp3 is the “master-regulator” for the Treg lineage. Induction of the Foxp3 gene in normal naïve T cells converts them to Treg-like cells with in vivo and in vitro suppressive function, indicating that Foxp3 is likely to play a key role in controlling expression of critical suppression-mediating molecules. Elucidation of the molecular targets of Foxp3 will be fundamental to a complete understanding of the suppressive functions of Tregs.

Reagents for Foxp3 Detection

Since the introduction of the first antibodies for the flow cytometric detection of Foxp3, eBioscience has been the industry leader in providing tools for the identification of Tregs. Our clones reactive with Foxp3 are conjugated to our leading offering of fluorophores to facilitate optimal multicolor panels for the analysis of Foxp3 and other important markers of Tregs. To develop our Foxp3 antibodies, eBioscience utilized protein deletion constructs to epitope-map our panel of Foxp3 antibodies. The figure below shows Foxp3 protein functional domains and the location of epitopes recognized by each of eBioscience's Foxp3 antibodies.

Foxp3 Protein Epitope Schematic

Schematic representation of the Foxp3 protein and the location of the epitopes reactive with eBioscience antibodies. Foxp3 contains a Zn finger/Leucine zipper and a conserved Forkhead Domain. In addition, in human Tregs, Exon 2 may be alternatively spliced (black box). Currently there is no evidence for alternative splicing in mouse Foxp3. The clone names of eBioscience Foxp3 antibodies are indicated next to the fragment of Foxp3 where they have been mapped

Identification of T Regulatory Cells with Foxp3

Mouse Tregs can be readily identified as CD4+CD25+Foxp3+ cells in mouse spleen and human PBMCs, respectively. There is a near 100% correlation in expression between the CD4+CD25 positive cells and the Foxp3+ Tregs. Under certain conditions, Foxp3+ Tregs can downregulate their expression of Foxp3, lose suppressor functions and manifest some of the functions of conventional effector Th1, Th2, Th17 and TFH cell subsets. The key causes of this loss of Foxp3 expression include inflammatory environments with high levels of cytokines that are normally involved in the induction of effector T cells, such as IL-6 and IFN gamma.

Foxp3 Expression Histogram

Foxp3 Expression in Tregs

Analysis of Foxp3 expression in the CD4+CD25+ Treg cell population reveals a complete overlap in staining of Foxp3 in Tregs.

Treg Identification using Foxp3 and CD4Foxp3 Identification using Foxp3 and CD25

Treg Identification Using Foxp3

BALB/c splenocytes were stained with Anti-Mouse CD4 FITC, Anti-Mouse CD25 APC, and Anti-Mouse/Rat Foxp3 PE. Tregs are identified in the upper-right quadrants as CD4+Foxp3+ cells (right) and CD25+Foxp3+ cells (left).

Detect Foxp3 in Whole Blood

eBioscience has simplified the analysis of Foxp3 in human whole blood. With the Foxp3 Whole Blood Staining Kit, it's now possible to analyze Foxp3 expression without isolating PBMCs from whole blood. The Foxp3 Whole Blood Staining Kit includes the PE-conjugated PCH101 clone for human Foxp3 and staning buffers specifically designed for fixation and permeabilization of cells in whole blood, resulting in robust detection of human Foxp3 expression.

Human Foxp3 Detection in Whole BloodHuman Foxp3 Detection in Whole Blood

Whole Blood Foxp3 Staining

Staining of normal human peripheral blood cells with FITC anti-human CD4 (OKT4) (left) or APC anti-human CD25 (BC96) (right) followed by RBC lysis and intracellular staining using the Whole Blood Foxp3 Staining kit. Cells in the lymphocyte gate were used for analysis.

Immunohistochemistry of Foxp3 in T Regulatory Cells

Until recently, Foxp3 expression has been thought to be restricted to the T-cell lineage. However, several lines of evidence indicate that Foxp3 is also expressed by tumor cells. It has been used recently as a biomarker and a prognostic factor for malignant human tumors. A direct link between the presence of Tregs and progression of ovarian carcinoma has been demonstrated where human tumor Foxp3+ Tregs were found to suppress tumor specific immunity and contribute to reduced survival of these patients. In breast cancer, an increase in the Treg population both in peripheral blood and tumor tissues was also reported and a recent study demonstrated a significant intratumoral infiltration of Foxp3+ Tregs in high-risk breast cancer patients and those at risk of late relapse. Given the central contribution of Foxp3 to Treg function, the expression of Foxp3 by tumor cells may represent a novel mechanism by which cancers suppress the immune system to escape destruction.

In addition to their use for flow cytometric detection of Foxp3, eBioscience Foxp3 antibodies have been reported useful for immunohistological staining (both frozen and paraffin sections) of endogenous protein in multiple tissues including spleen, breast, melanoma and others.

Foxp3 Mouse Spleen IHC Staining

C57BI/6 Mouse Spleen Staining

Staining of C57Bl/6 mouse spleen cryosections with anti-mouse/rat Foxp3 antibody (FJK-16s). Image courtesy of Cintia De Paiva.

Foxp3 Human Tonsillar IHC Staining

Human Tonsillar Tissue Staining

Staining of human tonsillar tissue section with anti-human Foxp3 (PCH101). Image courtesy of Roger Sutmuller.

Foxp3 Lymphocyte and Tumor Cell Staining

Lymphocyte and Tumor Cell Staining

IHC staining of human spleen sections (left) and tumor (right) using the Foxp3 antibody on FFPE sections.

References

Mouse Foxp3

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]

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]

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]

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)]

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]

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]

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]

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]

Human Foxp3 (PCH101)

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]

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]

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]

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)

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]

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]

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]

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]