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Foxp3: Treg的主要调节因子

日期:2012年10月16日 浏览次数:20,304

Foxp3是控制Treg细胞发育和功能的关键转录因子之一,它的发现是Treg免疫生物学重要的进步,为人们进一步了解Treg功能和作用机制打开了一扇“门”。虽然人们也发现Treg发育和功能也需要其他转录因子如AhR和STAT5的参与,但是Foxp3仍旧是Treg细胞系的主要调节因子。实验表明,在体外或体内诱导初始T细胞表达FoxP3后可以出现Treg样的免疫抑制作用,表明Foxp3是控制免疫抑制分子表达的关键因素;因此,阐明Foxp3的分子靶点是透彻理解Treg免疫抑制作用的必要条件之一。

Reagents for Foxp3 Detection

检测Foxp3的试剂

EBioscience利用蛋白表位图谱构建抗体来检测Foxp3,下图就是不同克隆抗体针对的Foxp3蛋白的表位。

图示:eBioscience公司Foxp3抗体针对Foxp3蛋白上不同的抗原表位。

 

Foxp3鉴定Treg细胞

通过流式抗体标记CD4+CD25+Foxp3+细胞可以容易的鉴定小鼠脾脏细胞和人PBMC中Treg。一般情况下,CD4+CD25+细胞和Foxp3+细胞相关性接近100%。在特定条件下Foxp3+Treg细胞会下调Foxp3表达,从而丧失免疫抑制功能,表现出Th1、Th2和TFH等具有传统功能的细胞亚群。导致Foxp3下调的关键因素包括高水平的炎症因子内环境,如可诱导效应性T细胞产生的细胞因子:IL-6和IFN-gamma。

Tregs 细胞中Foxp3的表达

分析CD4+CD25+ Treg 细胞中Foxp3 +细胞群表明与Foxp3+Treg完全重合。

使用Foxp3鉴定Treg

用Anti-Mouse CD4 FITC、Anti-Mouse CD25 APC和Anti-Mouse/Rat Foxp3 PE 标记BALB/c小鼠 脾细胞. 右上象限是Tregs 细胞:CD4+Foxp3+ cells (左) and CD25+Foxp3+ cells (右).

 

在全血中检测Foxp3

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 staining buffers specifically designed for fixation and permeabilization of cells in whole blood, resulting in robust detection of human Foxp3 expression.

eBioscience公司将全血Treg染色方法简化为一个试剂盒——人全血Treg染色试剂盒(cat#88-8996-40-),科研人员可以使用该试剂盒直接检测人类全血中的Treg,而无需分离PBMC。此外,该试剂盒成分齐全,完全满足Treg染色需要。

试剂盒组成:

eBioscience 1X 红细胞裂解液:200 ml
eBioscience 流式染色液:600 ml
eBioscience 固定破膜浓缩液:30 ml
eBioscience 固定破膜稀释液:100 ml
Anti-human Foxp3 PE (PCH101):25 tests

全血Foxp3染色结果

Foxp3免疫组化鉴定

到目前为止,一般认为Foxp3仅限于T细胞系表达。然而,也有证据表明肿瘤细胞也可以表达Foxp3,因此Foxp3也被作为人类恶性肿瘤生物标志物和预后判断参考因素。有文献报道, FoxP3+ Treg细胞存在与否与卵巢癌预后有直接的关系,人们发现肿瘤部位的Foxp3+Treg 可以抑制机体抗肿瘤免疫反应从而减低病人的存活率。此外,不管是外周血还是肿瘤浸润部位Foxp3+Treg数目都与乳腺癌病人预后好坏有着高度的相关性。由Foxp3在Treg抑制功能中的核心作用来看,一些肿瘤细胞表达Foxp3似乎可以抑制机体对肿瘤细胞的免疫反应,从而导致肿瘤细胞的免疫逃逸。

除了用流式细胞技术检测Foxp3之外,有报道称可以用eBioscience Foxp3抗体对脾脏、乳腺、黑素瘤及其他组织进行免疫组化染色(冰冻切片和石蜡切片)。

C57BI/6 小鼠脾脏染色

使用anti-mouse/rat Foxp3 抗体 (克隆号:FJK-16s)标记C57BL/6小鼠脾脏冰冻切片,用Cintia De Paiva照相。

人类扁桃体组织染色

用anti-human Foxp3 (PCH101)标记人扁桃体组织, 使用 Roger Sutmuller照相

淋巴细胞和肿瘤细胞染色

人类脾脏切片(左图)和肿瘤切片(右图)福尔马林固定石蜡包埋切片,用Foxp3抗体标记后免疫组化结果

参考文献

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]

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