FOXP3~+调节性T细胞

调节性T细胞是一类可以调节其他多种免疫细胞功能的T细胞亚型,其正常生理功能对体内免疫稳态维持必不可少。调节性T细胞功能失调与人类多种重大疾病,如自身免疫性疾病、感染性疾病、过敏性疾病、恶性肿瘤、移植排斥的发生、发展及治疗都密切相关。调节性T细胞可分为多种亚型,其中最重要也是目前研究最多的为表达叉头状家族转录因子FOXP3的天然调节性T细胞及诱导调节性T细胞。深入研究FOXP3+调节性T细胞的发育及功能的分子及细胞免疫学机制,将为重大免疫性疾病的临床治疗提供创新性线索。     

FOXP3 在食管癌中表达规律研究

目的:研究FOXP3在食管癌中表达规律。方法:选择2010年1月~2010年10月在我院接受手术治疗的食管鳞癌患者48例,选取所有患者肿瘤组织及正常食管黏膜组织进行免疫组化分析,观察FOXP3在食管癌细胞系Eca-109、正常食管黏膜细胞系Eca-109、人食管癌组织、正常食管黏膜组织的表达,FOXP3在不同年龄、性别、肿瘤分化程度、肿瘤直径、淋巴结转移上的表达。结果:FOXP3在食管癌组织中阳性表达率(85.42%)显著高于正常食管黏膜组织(14.58%),P0.05。FOXP3在正常细胞系表达为阴性,在食管癌细胞系Eca-109中的表达阳性,FOXP3在正常食管黏膜组织中表达阴性,在食管癌组织中表达阳性。在食管癌组织中,FOXP3阳性表达率和食管癌患者的性别、年龄等不同临床病理特征均无关联(P0.05),和T分期、TNM分期、肿瘤直径、淋巴结转移、远处转移等存在关联(P0.05)。结论:食管癌细胞能够表达FOXP3,且其表达水平和食管癌的进展关系密切,可以作为临床诊断及治疗食管癌的重要参考指标。     

FOXP3+调节性T细胞与非感染性炎症疾病

FOXP3~+调节性T细胞(FOXP3~+Tregs)是一类具有免疫抑制等调节作用的CD4~+T淋巴细胞亚群,其正常生理功能对人体免疫稳态的维持不可或缺。基于免疫疗法的临床需求,越来越多研究着眼于深入理解FOXP3~+Treg在局部组织炎症条件下的功能紊乱及其相关分子机理。现将系统阐述近年来有关FOXP3~+Treg生理功能及其调控机制的研究新进展,重点聚焦在多种非感染性炎症疾病,如自身免疫性疾病、免疫代谢性疾病、神经退行性疾病中,FOXP3~+Treg功能稳定性及组织特异性的分子机理及其潜在干预新靶点和新策略。     

B7-H4和FOXP3在乳腺癌中的表达及临床意义

目的:研究B7-H4和FOXP3在乳腺癌中的表达及其相互关系和临床意义.方法:应用S-P免疫组化法检测B7-H4蛋白和FOXP3蛋白在11例正常乳腺、25例乳腺良性病变、272例乳腺癌组织中的表达情况;运用原位杂交检查B7-H4 mRNA在10例正常乳腺、10例良性病变、20例乳腺癌组织中的表达情况.结果:B7-H4蛋白在乳腺癌中的阳性表达率(83.39%)高于乳腺良性病变组(64.00%)和乳腺正常组(54.55%),差异有统计学意义(p＜0.05);FOXP3在乳腺癌中的阳性表达率(82.67%)高于乳腺良性病变组(60.00%)和乳腺正常组(54.55%),差异有统计学意义(p＜0.05);B7-H4和FOXP3在乳腺癌中的阳性表达率与患者年龄、肿块大小和组织学分型无关(p＞0.05),而与组织学分级和淋巴结转移相关(p＜0.05);B7-H4和FOXP3在乳腺癌组织中的表达呈正相关(r=0.306,p＜0.001);B7-H4 mRNA在正常乳腺组、乳腺良性病变组、乳腺癌组中的阳性表达率差异没有统计学意义(p＞0.05);B7-H4 mRNA在乳腺癌中的阳性表达率与年龄、肿块大小、组织学分级、淋巴结转移无关(p＞0.05).结论:B7-H4和FOXP3在乳腺癌中表达上调,可能与乳腺癌的转移和预后相关.     

基于密码子优化策略的斑马鱼FOXP3A分子的原核表达及多克隆抗血清制备

目的:构建斑马鱼FOXP3A融合蛋白的原核表达系统,诱导表达并制备多克隆抗血清。方法:选取斑马鱼foxp3a基因cDNA的894 bp特异区段(s-foxp3a),对该片段进行密码子优化后构建原核表达载体pET-32a-s-foxp3a,转化大肠杆菌BL21(DE3),诱导表达斑马鱼S-FOXP3A-His融合蛋白并纯化,纯化后的蛋白免疫家兔,制备斑马鱼FOXP3A蛋白的多克隆抗血清,4次免疫后取血清,采用间接ELISA法检测抗血清效价。结果:在16℃经0.5 mmol/LIPTG诱导10 h的条件下,SDS-PAGE分析表明斑马鱼S-FOXP3A-His融合蛋白以包涵体形式产生;间接ELISA检测结果显示,FOXP3A蛋白多克隆抗血清的效价在1.6×10~5以上。结论:制备了高效价的斑马鱼FOXP3A多克隆抗血清,为进一步解析斑马鱼FOXP3A蛋白的功能提供了基础工具。     

A rare polyadenylation signal mutation of the FOXP3 gene (AAUAAA→AAUGAA) leads to the IPEX syndrome

The mouse scurfy gene, Foxp3, and its human orthologue, FOXP3, which maps to Xp11.23-Xq13.3, were recently identified by positional cloning. Point mutations and microdeletions of the FOXP3 gene were found in the affected members of eight of nine families with IPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked; OMIM 304930). We evaluated a pedigree with clinically typical IPEX in which mutations of the coding exons of FOXP3 were not detected. Our reevaluation of this pedigree identified an A-->G transition within the first polyadenylation signal (AAUAAA-->AAUGAA) after the stop codon. The next polyadenylation signal is not encountered for a further 5.1 kb. This transition was not detected in over 212 normal individuals (approximately 318 X chromosomes), excluding the possibility of a rare polymorphism. We suggest that this mutation is causal of IPEX in this family by a mechanism of nonspecific degradation of the FOXP3 gene message.     

转录因子FOXP1在胃癌组织中的表达及意义

目的:检测FOXP1蛋白在胃癌组织及癌旁正常组织中的表达情况,并探讨其与胃癌患者病理参数及预后的关系。方法:采用免疫组织化学染色方法检测90例胃癌组织和相应癌旁正常组织中FOXP1的表达情况并进行评分,进一步统计分析FOXP1表达与胃癌患者临床病理参数以及预后的关系。结果:FOXP1在胃癌组织中的表达显著低于癌旁正常组织(P0.01);对临床资料进行统计分析表明,在临床III期和IV期患者的胃癌组织中FOXP1的表达显著低于临床I期和II期的患者(P0.05);FOXP1在低分化胃癌中的表达显著低于高、中分化胃癌(P0.01);FOXP1表达阳性者术后总体生存率较表达阴性者高(P=0.145),中位生存期长。结论:FOXP1表达的减少可能参与了胃癌的发生发展,FOXP1在胃癌中可能扮演抑癌基因的角色,FOXP1可作为胃癌治疗的潜在靶点。     

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.

Methodology/Principle Findings

Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.

Conclusions/Significance

Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.     

乳腺癌组织FOXP3、HMGA2、RASAL2的表达及与临床病理特征和预后的关系

摘要 目的：探讨乳腺癌组织叉头框蛋白P3（FOXP3）、高迁移率族蛋白A2（HMGA2）、大鼠肉瘤蛋白活化因子2（RASAL2）的表达及其与临床病理特征和预后的关系。方法：选取2014年6月至2015年6月期间我院收治的102例乳腺癌患者作为研究对象。检测乳腺癌组织以及癌旁组织中FOXP3、HMGA2、RASAL2表达,分析FOXP3、HMGA2、RASAL2表达与临床病理参数的相关性。Kaplan-Meier生存曲线分析不同FOXP3、HMGA2、RASAL2表达患者总生存率的差异。Cox比例风险回归模型分析乳腺癌患者预后的影响因素。结果：与癌旁组织相比,乳腺癌组织中FOXP3、HMGA2阳性表达率升高,而RASAL2阳性表达率降低（P<0.05）。FOXP3、HMGA2、RASAL2表达均与TNM分期和淋巴结转移相关（P<0.05）。FOXP3、HMGA2阳性患者的生存率明显低于FOXP3、HMGA2阴性患者,RASAL2阳性患者的生存率明显高于RASAL2阴性患者（P<0.05）。TNM分期Ⅲ期、FOXP3阳性表达、HMGA2阳性表达是影响乳腺癌患者预后的危险因素（P<0.05）,而RASAL2阳性表达是乳腺癌患者预后的保护因素（P<0.05）。结论：乳腺癌组织中FOXP3和HMGA2阳性表达率升高,而RASAL2阳性表达率降低,FOXP3和HMGA2阳性表达以及RASAL2阴性表达与乳腺癌患者TNM分期、淋巴结转移相关,并且是患者预后不良的影响因素。     

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-β and Increase in IL-6

Background

50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4⁺ subsets of Th17 cells and CD25⁺FOXP3⁺ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions.

Methodology and Principle Findings

Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3⁺ Treg cells as well as reduction in TGF-β and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients.

Conclusions

Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-β and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.     

CD4+CD25+FOXP3+ T cells,Foxp3 gene and protein expression contribute to antiasthmatic effects of San’ao decoction in mice model of asthma

ObjectiveSan’ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San’ao decoction's immunomodulatory effects on Tregs.MethodsUPLC–PDA–TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks’ treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4⁺CD25⁺Foxp3⁺ Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well.Results22 Peaks signal chemical components in SAD were identified by UPLC–QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p < 0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells and promoted Foxp3 expression in a mouse model of asthma.ConclusionThese results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4⁺CD25⁺Foxp3⁺ Treg cells.     

特异性蛋白1(SP1)上调FOXP4-AS1促进结直肠癌细胞增殖

大量的证据表明,长链非编码RNAs (long non-coding RNAs)在结直肠癌发生发展中发挥重要作用。有迹象表明,lncRNA FOXP4-AS1推动结直肠癌的进程。本文通过数据库信息分析发现,FOXP4-AS1在结直肠癌中高表达且与患者较差的预后正相关。实时荧光定量PCR方法也证实,FOXP4-AS1在结直肠癌细胞和组织中的表达量均高于正常细胞和组织。其中,FOXP4-AS1在结肠癌细胞LOVO中表达量最高,是正常结直肠细胞的13倍。生物信息学预测结合双荧光素酶报告基因实验和染色质沉淀研究结果表明,特异性蛋白1（specificity protein 1,SP1）可直接结合在FOXP4-AS1启动子上,上调其活性。过表达FOXP4-AS1,可下调p16和p18表达,同时上调CDK4、CDK6和细胞周期蛋白 D1表达,最终促进结直肠癌细胞增殖。相反,敲低FOXP4-AS1将上调p16和p18表达,抑制CDK4、CDK6和细胞周期蛋白 D1,获得相反的结果。总之,特异性蛋白1(SP1)上调FOXP4-AS1,促进结直肠癌细胞增殖。     

Selective depletion of FOXP3high cells by Fas–Fas-L–induced apoptosis occurs in CD4+CD25+-enriched populations during repeated expansion

Background aimsExpansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined.MethodsMagnetic-bead isolated human CD25⁺ cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas–Fas-L–mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L.ResultsRepeated expansion of bead-enriched CD4⁺CD25⁺ cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8⁺ T cells and CD4⁺ T cells producing interferon-γ and/or interleukin-2. We showed that Fas–Fas-L–mediated apoptosis of CD4⁺FOXP3^high cells and rapid cell-cycling of CD8⁺ T cells were collectively responsible for the reduced proportion of CD4⁺FOXP3^high cells in expanded cultures. The depletion of CD4⁺FOXP3^high cells and activation of Caspase 8 in CD4⁺FOXP3^high cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4⁺FOXP3^high cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies.ConclusionsTaken together, the data show that Fas–Fas-L–mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.