What is Foxp3?
Foxp3 is the "master regulator of transcription" for CD4+regulatory T cells (Tregs)[1,2]. Naive and CD4 memory+T cells can differentiate into Tregs by induced expression of the Foxp3 gene . Mutation in the Foxp3 gene leads to hyperactive T cells and the production of proinflammatory cytokines [1,2]. The Foxp3 protein is used as the main identification marker for Treg cells .
Foxp3/Transcription Factor Staining Buffer Set(Cat. No. 00-5523-00) is recommended for the detection of Foxp3 proteins. This is a buffer kit containing Fix and Perm reagents that maximize detection of the Foxp3 protein and minimize the impact on surface staining. This kit can be used to stain other nuclear proteins.
Learn more about intracellular staining protocols
Identification of regulatory T cells with Foxp3
Tregs can be identified as CD4+zorrop3+yCD25+zorrop3+Cells in the spleen of mice and human PBMC . There is a correlation in the expression between the CD4+CD25+cells and the Foxp3+tregs Downregulation of Foxp3 expression causes Treg cells to lose suppressor functions and express some conventional effector functionsTh1,Th2,Th17, YTFHcell subsets. The main causes of this loss of Foxp3 expression are inflammatory environments with high levels of cytokines normally involved in the induction of effector T cells, such as IL-6 and IFN-gamma . Therefore, Foxp3 protein must be stained for functional Tregs.
Figure 1. Analysis of Foxp3 expression on CD4+The Treg cell population shows a positive population.C57BL/6 mouse splenocytes were stained intracellularly usingFoxp3/Transcription Factor Staining Buffer Set(Cat. No. 00-5523-00) and protocol, withCD4 monoclonal antibody, APC(Cat. No. 17-0042-82) and 0.5 µg ofRatten-IgG2a-kappa-Isotype Control, PE(cat. no. 12-4321-82) (left) or 0.5 µg Foxp3 monoclonal antibody, PE (right). Cells in the lymphocyte gate were used for analysis.
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Figure 2. Activation of Foxp3+Tregs con CD4, CD25 y Foxp3.(Top) BALB/c splenocytes were stained with anti-mouse CD4-FITC, anti-mouse CD25-APC, and anti-mouse/rat Foxp3-PE. Tregs are identified as CD4 in the upper right quadrant+zorrop3+cells (right) and CD25+zorrop3+cells (left). (Bottom) Staining of normal human peripheral blood cells with FITC anti-human CD4 (OKT4) (left) or APC anti-human CD25 (BC96) (right), followed by red blood cell lysis and intracellular staining usingFoxp3 staining kit for whole blood(Catalog No. 88-8996-40). Cells in the lymphocyte gate were used for analysis.
The Foxp3 protein is difficult to stain because it is not very abundant and is an intracellular protein. The following figure shows the functional domains of the Foxp3 protein and the position of the recognized epitopes.Foxp3 antibody. The choice of clone is important for the detection of the Foxp3 protein . For optimal detection of Foxp3 protein, it is important to have optimal antibody titers, optimized permanent and fixed buffers, and include cells known to be negative for the marker.
Figure 3. Schematic representation of the Foxp3 protein and location of the antibody-reactive epitopes.Foxp3 contains a Zn finger/leucine zipper and a conserved hairpin head domain. Furthermore, exon 2 can be alternatively spliced into human Tregs (black box). There is currently no evidence for alternative splicing in mouse Foxp3. The names of the Foxp3 antibody clones are provided next to the Foxp3 fragment to which they mapped.
Immunohistochemistry of Foxp3 in regulatory T cells
Foxp3 is also expressed in tumor cells. It has recently been used as a biomarker and prognostic factor for malignant human tumors . Immunohistologic staining for endogenous Foxp3 protein can be seen (both in frozen and paraffin sections) in multiple tissues, including spleen, breast, melanoma, and others.
Figure 4. Staining of C57BI/6 mouse spleen.Staining of C57Bl/6 mouse spleen cryosections with anti-mouse/rat Foxp3 antibody (FJK-16s). Image courtesy of Cintia De Paiva.
Figure 5. Staining of human tonsil tissue.Staining of human tonsil tissue sections with anti-human Foxp3 (PCH101). Image courtesy of Roger Sutmuller.
Figure 6. Staining of lymphocytes and tumor cells.IHC staining of human spleen (left) and tumor (right) sections with the Foxp3 antibody in FFPE sections.
Biller, BJ, et al. 2007. Using 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 with FJK-16s in dogs]
Sakaguchi, S. et al. 2005. Treatment of advanced tumors with agonist 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 with FJK-16s]
Fields, ML, et al. 2005. CD4+CD25+ regulatory T cells inhibit maturation but not initiation of an autoantibody response. J. Immunol. 175: 4255-4264. [Intracellular staining for flow cytometry with FJK-16s]
Beyersdorf, S., et al. 2005. Selective targeting of regulatory T cells with CD28 superagonists enables effective therapy of experimental autoimmune encephalomyelitis. J Exp. Med. 202: 445-55. [Intracellular staining for flow cytometry using FJK-16s (correction; not mFoxy)]
Anderson, MS, et al. 2005. The cellular mechanism of Air control of T-cell tolerance. Immunity. 28:227-39. [Intracellular staining for flow cytometry with FJK-16s]
Siegmund, R., et al. 2005. Migration issues: regulatory T cell compartmentalization determines silencing activity in vivo. Blood. 106: 3097-104. [Intracellular staining for flow cytometry with FJK-16s]
Wilson, MS, 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 with FJK-16s]
Kretschmer, K., et al. 2005. Induction and expansion of the population of regulatory T cells by foreign antigen. national immunol. 6(12): 1219-1227. [Intracellular staining for flow cytometry with FJK-16s]
Human Foxp3 (PCH101)
Ahmadzadeh, M., et al. 2005. Administration of IL-2 increases CD4+CD25hiFoxp3+ regulatory T cells in cancer patients. Blood (November epub). [Intracellular staining for flow cytometry with PCH101]
Crellin, NK., et al. 2005. Human CD4+ T cells express TLR5 and its ligand flagellin increases the silencing capacity and expression of FOXP3 on CD4+CD25+ regulatory T cells. J. Immunol. 175(12):8051-9. [Intracellular staining for flow cytometry with PCH101]
Hartwig, UF., et al. 2005. Depletion of alloreactive T cells via CD69: implications for antiviral, antileukemic, and immunoregulatory T cells. Bone Marrow Transplantation (5 Dec Epub ahead of publication). [Intracellular staining for flow cytometry with PCH101]
Lim, HW., et al. 2005. State-of-the-art technology: 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 with PCH101]
Zorro Humanop3 (236A/E7)
Alvaro, T., et al. 2005. Outcome in Hodgkin lymphoma can be predicted by the presence of concomitant regulatory and cytotoxic T cells. Clinical Cancer Res. 11(4):1467-73. [Paraffin IHC with 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 with 236A/E]
Roncador, G., et al. 2005. FOXP3, a selective marker for a subset of adult T-cell leukemia/lymphoma. Leukemia (Epub ahead of print). [IHC paraffin sections with 236A/E7]
Wolf, D., et al. 2005. Expression of the T cell-specific hairpin headbox transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clinical Cancer Res. 11(23): 8326-31. [Paraffin IHC with 236A/E7]
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