Transcript Blimp-1 + 和Blimp-1

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The transcription factors Blimp-1 and IRF4
jointly control the differentiation and
function of effector regulatory T cells
Cretney E, Xin A, Shi W, Minnich M, Masson F, Miasari M, Belz GT,
Smyth GK, Busslinger M, Nutt SL, Kallies A.
Honglin Wang
Shanghai Institute of Immunology
Institute of Medicine Science
Shanghai Jiao Tong University School of Medicine
Shanghai
China
Nature Immunology 12, Pages: 304–311 Year
published: (2011)
Published online: 06 March 2011
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简历
王宏林
1972.03.15 生于安徽省马鞍山市
主要学习、工作经历
 2009.01 - ：上海交通大学医学院 研究员 博士生导师 课题组长；
 2009.01 -：上海市特聘教授（东方学者）；
 2002.10 – 2009.10： 瑞典隆德大学（Lund Uni.） 合作课题研究；
 2006.10 – 2009.03：德国乌尔姆大学（Ulm Uni.）医学院 博士后；
 2002.10 – 2006.10：德国乌尔姆大学（Ulm Uni.）医学院 攻读博士学位；主修免
疫学、皮肤免疫性疾病；
 2001.07 – 2002.10：南京农业大学 教师；
 1998.09 – 2001.07： 南京农业大学 攻读硕士学位 主修遗传学。
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Introduction
Regulatory T cells (Treg cells) – 调节性T细胞
Regulatory T cells: (sometimes known as suppressor T cells) are a specialized subpopulation
of T-cells that act to suppress activation of the immune system and thereby maintain immune
system homeostasis and tolerance to self-antigens.
Commonly, natural Treg cells are generated in the thymus and display a defined CD4+CD25+
phenotype accompanied by expression of the forkhead/winged helix transcription factor, Foxp3.
Fontenot, J.D. et al. 2003. Nat Immunol 4:330-336.
Treg cells have been identified as key players in the control of T-cell-mediated autoimmune
processes.
Sakaguchi, S. 2000. Cell 101:455-458.
Treg cells suppress tumor immunity.
Chen M.L., 2005. PNAS. 419-24.
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Treg cells常用的标记
CD4
CD4
CD127
Co-receptor for TCR recognition of
MHC II/Ag
CD25
CD25 IL-2Ra
IL-2R component, confers high affinity
binding to IL-2Rbg
Key TReg growth factor
Foxp3
Treg
FoxP3
Forkhead/winged-helix TF critical for TR
activity and development
Unlike surface markers / receptors
CD127  chain of the IL-7 receptor
Treg cells are CD127-. Activated T
cells/memory T cells express high
levels of CD127
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FoxP3 （forkhead box P3 ） – 细胞内转录因子
Autoimmune Diseases in Mice and Human Carrying Deficient FoxP3
Fontenot, J.D., Gavin, M.A. & Rudensky, A.Y. Foxp3 programs the development and
function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003).
Bennett, C.L., et al. 2001. The immune dysregulation, polyendocrinopathy, enteropathy, Xlinked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27:20-21.
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Treg cells的分化和功能
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Blimp-1 - PR锌指蛋白1
Blimp-1/Prdm1: Positively regulatory domain zinc finger protein 1调节性T细胞关键因子FoxP3的靶基因
(a, b) Foxp3-binding regions for several genes visualized using the Affymetrix Integrated
Genome Browser. Each blue bar represents the signal intensity of an individual oligonucleotide
probe. Structure, chromosomal location, and mouse–human homology VISTA plot of the given
gene are shown below each peak.
Zheng, Y. et al. Nature 2007
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Results
Figure 1
Expression of Blimp-1 in IL-10-producing Treg cells
IL-10 （Interleukin 10，白介素10）
It is capable of inhibiting synthesis of pro-inflammatory cytokines like IFNγ, IL-2, IL-3, TNFα and GM-CSF made by cells such as macrophages and
regulatory T-cells.
IL-10 also displays potent abilities to suppress the antigen presentation
capacity of antigen presenting cells.
It is mainly expressed in monocytes and Type 2 T helper cells (TH2),
mast cells, CD4+CD25+Foxp3+ regulatory T cells, and also in a certain
subset of activated T cells and B cells.
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Figure 1: Con.
Blimp-1（PR锌指蛋白1）在CD4+CD25+调节性T细胞中的表达
Blimp-1-GFP（绿色荧光报告基因）小鼠
GFP是从一种生活在北太平洋寒冷水域的
水母体内发现的，这种水母体内含有一种生
物发光蛋白质；
日本科学家下村修、美国科学家马丁•查尔
非和钱永健因发现和改造绿色荧光蛋白
（GFP）而获得2008年诺贝尔化学奖。1962
年被发现，1992年被克隆，中间隔了30年。
Splenocytes
FACS
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Figure 1: Con.
© 2004 Rockefeller University Press
构建Blimp-1绿色荧光报告基因小鼠
Kallies A et al. J Exp Med 2004;200:967-977
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Figure 1: Con.
Blimp-1在CD4+CD25+调节性T细胞中的表达
(a) Blimp-1-GFP expression (right) in CD4+ CD25+ splenic T cells isolated from a 51-week-old Blimp1+/GFP mouse
(b) Quantitative RT-PCR analysis of Blimp1 mRNA expression in sorted CD4+ T cells pooled from the spleens and
lymph nodes of Blimp1+/GFP mouse
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Figure 1: Con.
Blimp-1+ Treg cells和Blimp-1-Treg cells
均表达FoxP3
Blimp-1在脾脏、淋巴和胸腺中Treg Cells的表达量
Blimp-1+ Treg cells和Blimp-1-Treg cells
体外免疫抑制能力相同
(c) Blimp-1-GFP expression on CD4+ T cells from the spleen, mesenteric lymph nodes (mesLN) and thymus
of an 8-week-old Blimp1+/GFP mouse
(d) Foxp3 expression in splenic CD4+ CD25+ T cells and CD4+ CD25- T cells
(e) Proliferation of CD4+ CD25- responder T cells (CD25- ) cultured in vitro at various ratios with GFP- or
GFP+ CD4+ CD25+ T cells (CD25+ )
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Figure 1的结论：
Blimp-1不是被所有的Treg cells表达，而是被其中一个亚群表达，这个亚
群是什么？
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Figure 2
Blimp-1-expressing Treg cells have an effector
phenotype, produce IL-10 and localize to mucosal sites
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Blimp-1的表达与Treg cells效应性标记的相关性分析
Inducible Co-stimulatory Molecule
整合素E, 外周Treg
cells的标记
T细胞活化的标记
T细胞活化的标记
T细胞是否参与抗原提呈的标记
CD127，Treg cells的标记
Glucocorticoid-induced TNFR
family-related protein
Treg cells的标记
(a) Flow cytometry of CD4+ CD25 + T cells from the mesenteric lymph nodes of Blimp1 +/GFP mice
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Figure 2: Con.
Blimp-1+ Treg cells高分泌IL-10，几乎无IFN-的分泌
(b) Cytokine production by pooled Blimp1 +/GFP splenic and lymph node CD4+ T cells
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Figure 2: Con.
粘膜部位（Mucosal Sites） Treg cells高表达Blimp-1
（如肠道和肺部，黏膜组织丰富，此类Treg cells参与肠道和肺的炎症反应）
（肠）派亚氏腺（peyer’s patches）
（肠）内皮中淋巴细胞
(c) Blimp-1-GFP expression in CD4+ CD25+ T cells from Peyer’s patches (PP) and among intraepithelial lymphocytes
(IEL) of Blimp1 +/GFP mice
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Figure 2: Con.
Blimp-1+ Treg cells高表达IRF4和T-bet
Tbx21: 编码T-bet的基因
Interferon regulatory factor 4
T-bet is a STAT1-induced regulator of IL-12R
expression in naïve CD4+ T cells
(d) Quantitative RT-PCR analysis of the expression of Irf4 and Tbx21 in Blimp1 +/GFP Treg cells and conventional
CD4+ T cell populations
T-bet的发现：A novel transcription factor, T-bet, directs Th1 lineage commitment
Cell. 2000 Mar 17;100(6):655-69. 被应用1477次。
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Figure 2的结论：
Blimp-1+Treg cells具有效应性表型，此类细胞属于效应性Treg cells分
泌IL10且富集在器官黏膜部位。
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Figure 3
Blimp-1 is dispensable for the generation of effector
Treg cells but is required for their IL-10 production and
tissue homeostasis
 尽管有FoxP3的存在, Blimp-1基因KO小鼠一样发自身免疫性疾病，重组Blimp-1基因WT小
鼠的骨髓可以明显减缓发病，这说明了Blimp-1在自身免疫性疾病发病过程中有重要作用。
Kallies, A. et al. Transcriptional repressor Blimp-1 is essential for T cell homeostasis and
self-tolerance. Nat. Immunol. 7, 466–474 (2006).
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Ly5.1 / Ly5.2 / Mixed chimeric mice
 CD45: Protein tyrosine phosphatase（蛋白酪氨酸磷酸酶）, leukocyte
common antigen, highly expressed by T cells.
 Mixed chimeric mice （混合嵌合体 小鼠）: mixed chimeric bone marrow
repopulation
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Ly5.1
Ly5.2-WT-Blimp-1GFP/GFP (Prdm1GFP/GFP，Blimp1基因缺陷小鼠)
Lethally Irradiated
Blimp-1的存在与否对脾脏Treg cells的数量无影响
效应性Treg cells的产生不需要Blimp-1
Blimp-1 缺陷
Blimp-1 缺陷
Blimp-1 缺陷
Blimp-1 缺陷
(a,b) Flow cytometry of splenic CD4+ T cells from wild-type (Ly5.1 +)-Blimp1GFP/GFP (Ly5.2 +) mixed bone marrow
chimeras(WT-Blimp1GFP/GFP)
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Figure 3: Con.
Ly5.1
Blimp-1GFP/GFP
Lethally Irradiated
Blimp-1缺陷的Treg cells的不产生IL-10
分离出Blimp-1 缺陷的Treg cells
Blimp-1
正常
Blimp-1
缺陷
(c) Bead assay of cytokines in the supernatants of GFP -and GFP +CD4+ CD25+ T cells sorted from
Blimp1 mice +/GFPmice (+/GFP) or from the Blimp1 GFP/GFP compartment of mixed bone marrow
chimeras (GFP/GFP)
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Figure 3a、3b和3c的结论：
Blimp-1对效应性Treg cells的产生可有可无，但对IL-10的分泌非常关键。
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Figure 3: Con.
 Blimp-1存在与否不影响淋巴节中的Treg cells的数量，表明Blimp-1存在与否对淋巴中未致敏Treg cells的产生
无影响，但对组织中效应性Treg cells的数量有影响。
淋巴
（肠）派亚氏腺（peyer’s patches）
（肠）内皮中淋巴细胞
肺
Flow cytometry analysis of Foxp3 expression
(d) and frequency of Foxp3+ cells among total CD4+ T cells
(e) for cells from or among the peripheral lymph nodes (pLN), spleen, Peyer ’s patches,
intraepithelial lymphocytes and
lungs of chimeras generated with a mixture of wild-type (WT; Ly5.1+ ) and Blimp1 GFP/GFP (GFP/GFP; Ly5.2+ ) bone marrow
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Figure 3: Con.
After infection, both wild-type and Blimp-1-deficient Treg cells were efficiently recruited into the
lungs, but only wild-type Treg cells upregulated ICOS expression, whereas Blimp-1-deficient
Treg cells failed to substantially increase their expression of ICOS (Fig. 3f,g). This suggests
that Blimp-1 deletion impairs effector Treg cell activation.
Blimp1正常
Blimp1正常
Blimp1缺陷
Blimp1缺陷
(f) ICOS expression on Treg cells from the lungs and lung-draining mediastinal lymph nodes (mLN) of wild-typeBlimp1GFP/GFP mixed chimeras before (Naive) 10 d after (HKx31) infection with influenza virus (strain HKx31)
(g) ICOS expression on wild-type and Blimp1GFP/GFP Foxp3 +CD4+ cells in the lungs of naive mice (open
histograms) and influenza-infected mice (filled histograms)
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Figure 3d、3e、3f和3g的结论：
 Blimp-1存在与否对淋巴中未致敏Treg cells的产生无影响，但对组织中
效应性Treg cells的数量有影响；
 Blimp-1的缺失对效应性Treg cells的活化很关键。
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Figure 4
Blimp-1 limits numbers of Treg cells and is induced by
IL-2 and inflammatory signals
_
+
Colitogenic CD4 CD25 T cells
Blimp-1+ Treg cells
Blimp-1- Treg cells
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Figure 4: Blimp-1 limits numbers of Treg cells and is induced by IL-2 and
inflammatory signals
Blimp-1- Treg cells可以成熟成Blimp-1+Treg cells
Blimp-1- Treg cells比Blimp-1+Treg
cells更容易扩增
GFP expression in CD4+ T cells
(a) and number of Ly5.2 +CD4+ T cells
(b) in the spleens of Rag1-/- Ly5.1+ mice 8 weeks after transfer of CD4+ CD25- CD62L + T cells
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Figure 4: Con.
活体内注射IL-2可以诱导更多的Blimp-1表达
Anti-CD40诱导T细胞炎症反应，也可增加Blimp-1的表达
(c) Blimp-1-GFP expression in CD4+ CD25+ T cells (left) and number of GFP+ and GFP- CD4+ CD25+ T
cells (right) in the spleen 7 d after injection of PBS or IL-2-anti-IL-2 complexes (IL-2c) into Blimp1+/GFP
mice
(d) Flow cytometry of CD4 + T cells on day 5 after injection of PBS or monoclonal anti-CD40 or PBS into
Blimp1 +/GFP mice on days 0, 2 and 4
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Figure 4的结论：
 Blimp-1限制Treg cell的数量的扩增，IL-2和炎症环境可以诱导Blimp-1
的表达。
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Figure 5
IRF4 (Interferon Regulatory Factor 4) is required for
the generation of Blimp-1-expressing effector Treg
cells, but T-bet is not
IRF4 is required during an immune response for lymphocyte activation
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Tbx21-/-Blimp-1+/GFP
IRF4-/-Blimp-1+/GFP
Tbx21/T-bet
Blimp-1的表达不依赖于T-bet
Blimp-1的表达依赖于IRF4
Blimp-1的活化依赖于IRF4
(a,b) Flow cytometry of CD4+ T cells from the spleens and mesenteric lymph nodes of Tbx21 -/- Blimp1 +/GFP mice
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Figure 5: Con.
染色质免疫沉淀分析ChIP 技术介绍
（Chromatin Immunoprecipitation Assay, ChiP）
ChiP（染色质免疫沉淀分析）分析证实了IRF4
通过绑定Blimp-1调控其表达
是研究体内DNA 与蛋白质相互作用的重要工具。它可
以灵敏地检测目标蛋白与特异DNA 片段的结合情况，还
可以用来研究组蛋白与基因表达的关系。
(c) ChIP analysis of purified Treg cells with an IRF4-specific antibody and PCR primers specific for the following
regulatory regions of Blimp1: promoter (Prom), conserved noncoding sequence (CNS) and region 3’ to
Blimp1 (3’)
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Figure 5: Con.
IRF4的缺失阻止了效应性Treg cells的分化
(d-f) Flow cytometry of Treg cells and CD4+ T cells from the mesenteric lymph nodes (d,e) and
lungs (f) of wild-type (Ly5.1+ )-Irf4-/- (Ly5.2+ ) mixed-bone marrow chimeras (WT-Irf4-/- )
mediastinal lymph nodes and
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Figure 5: Con.
IRF4的缺失阻止了黏膜组织中Treg cells的归槽和分化
(g) Contribution of IRF4-deficient Treg cells to various tissues in wild-type Irf4-/- chimeric mice
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Figure 5: Con.
IRF4的缺失阻止了炎症条件下Treg cells向黏膜组织中的归槽和活化
(h) Flow cytometry of CD4+ Foxp3+ T cells from mediastinal lymph nodes and lungs before and 10
d after infection of wild-type-Irf4-/- chimeric mice with influenza virus (strain HKx31)
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Figure 5的结论：
 几乎所有的效应性Treg cells的分化需要IRF4。
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Figure 6
Blimp-1 and IRF4 jointly regulate the effector Treg
cell differentiation program
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Blimp-1+/GFP：Blimp-1 GFP报告基因小鼠
Blimp-1GFP/GFP : Blimp-1基因缺失小鼠
Blimp-1+ 和Blimp-1-的Treg cells基因
比较（差别最大的30个基因）
(a) Microarray analysis of Blimp-1-GFP+ Treg cells
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Figure 6: Con.
Blimp-1+ 和Blimp-1-的Treg cells差异基因的验证
(b) Quantitative RT-PCR analysis of gene expression in Treg cell populations from Blimp1+/GFP and
Blimp1GFP/GFP mice
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Figure 6: Con.
Blimp-1的缺失导致了Bcl2和CCR6的表达上调
(c,d) Flow cytometry of Treg cells from wild-type (Ly5.1 +)-Blimp1GFP/GFP (Ly5.2 +) or wild-type (Ly5.1+ )-Blimp1+/GFP (Ly5.2+ )
mixed-bone marrow chimeras before or after infection with influenza virus (strain HKx31)
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Figure 6的结论：
 Blimp-1和IRF4共同调节效应性Treg cells的功能。
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Figure 7
Binding of IRF4 and Blimp-1 to regulatory regions
in the IL10 and CCR6 loci
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IRF4+/+ Treg cells和IRF4-/-Treg cells
ChiP分析得出IRF4绑定区域是IL-10和CCR6基因位点
(a) ChIP analysis of purified Irf4-/- and Irf4+/+ Treg cells, assessed with an IRF4-specific antibody and PCR
primers specific for regulatory regions of Il10 and Ccr6
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Figure 7（Figure 7b、7c略）的结论：
 Blimp-1和IRF4的绑定区域是IL-10和CCR6的位点。
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Summary
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2011-02-03