CD4+CD25+CD127low/-T淋巴细胞在类风湿关节炎患者外周血中的表达及意义

目的 检测调节性T淋巴细胞新旧标志在活动性类风湿关节炎(RA)患者外周血中的表达,探讨用CD127代替FOXP3对调节性T淋巴细胞进行研究的可能性.方法 选取活动性RA患者32例,其中未经缓解病情抗风湿药(DMARDs)治疗者20例,经DMARDs治疗效果不佳者12例,以25例原发性干燥综合征(pSS)患者和24名健康人作为对照,采用流式细胞术检测外周血中CD4+CD25high、CD4+CD25+CD127low/-及CD4*CD25+FOXP3+T淋巴细胞的比例,同时检测外周血C反应蛋白(CRP)、红细胞沉降率(ESR)、免疫球蛋白、补体等水平并进行相关性分析.结果 RA患者CD4+CD25highT淋巴细胞百分率与健康对照组相比差异无统计学意义(P>0.05).其CD4+CD25+FOXP3+T淋巴细胞与CD+CD25+CD127low/-T淋巴细胞的百分率均低于健康对照组(P<0.05),但CD4+D25+FOXP3+、CD4+CD25+CD127low/-T淋巴细胞在RA患者与pSS患者中的表达差异无统计学意义(P>0.05);这三群细胞的表达水平在RA未经治疗组和治疗效果不佳组之间差异均无统计学意义(P>0.05). RA患者CD4+CD25+FOXP3+T淋巴细胞与CD4+CD25+CD127low/-T淋巴细胞之间呈明显正相关(r=0.698,P=0.001),但这两群细胞与患者的CRP、ESR等水平及抗环瓜氨酸肽(CCP)抗体、类风湿因子(RF)均无相关性(P>0.05).结论 活动性RA患者外周血中,CD4+CD25+CD127low/-T淋巴细胞的百分率降低,并且与CD4+CD25+FOXP3+T淋巴细胞呈显著正相关性,提示CD127可能被作为Treg细胞中FOXP3的替代标记.     

CD4+CD25+调节性T细胞及其分子标记物与支气管哮喘

CD4+CD25+调节性T细胞是一类以免疫抑制和免疫无能为特征的淋巴细胞群,FOXP3是CD4+CD25+调节性T细胞一个特征性的分子标志物,并且对CD4+CD25+调节性T细胞的发育、外周表达和功能维持有着关键性的作用.近年来,多项研究显示CD4+CD25+调节性T细胞参与并影响了支气管哮喘的发生、发展过程,对调节性T细胞或其相关基因的干预也许会成为支气管哮喘治疗的新方向.     

FOXP3在炎症性肠病患者外周血和肠黏膜中的表达特点

背景：炎症性肠病（IBD）的发病与一系列免疫异常有关。免疫反应受特定的免疫调节细胞调控，包括CD4^＋CD25^＋T细胞．而CD4^＋CD25^＋T细胞的发育和功能可能受FOXP3基因调控。目的：研究FOXP3的表达特点以及IBD时FOXP3表达的改变。方法：以免疫磁珠分离CD4^＋、CD4^＋CD25^＋和CD4^＋CD25^-T细胞。以逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测IBD患者和非IBD对照外周血单个核细胞（PBMC）、肠固有层单个核细胞（LPMC）和肠黏膜组织中FOXP3的表达。结果：CD4^＋CD25^＋T细胞中FOXP3 mRNA的表达明显强于CD4^＋T细胞和未分离的PBMC或LPMC。T细胞受体（TCR）激活后，或经白细胞介素（IL）-2、IL-15作用后，PBMC或LPMC FOXP3 mRNA或蛋白表达明显增强。12例IBD中9例（75．0％）肠黏膜LPMC中检测到FOXP3 mRNA表达，高于对照肠黏膜的47．1％（8／17．P〉0．05）。未受刺激的IBD和对照肠黏膜组织中未检测到FOXP3 mRNA表达。结论：人类FOXP3的表达受T细胞活化影响。IBD患者肠黏膜LPMC中FOXP3 mRNA的表达率有增高的趋势．但与对照肠黏膜相比无显著差异．     

FOXP3与自身免疫性甲状腺疾病

叉状头／翅膀状螺旋转录因子（FOXP3）作为CD4^＋CD25^＋调节T（Treg）细胞表面的特征性标志,是Treg细胞发育和功能维持的关键因素,在维持机体免疫耐受和免疫应答稳态方面具有非常重要的作用。FOXP3基因突变可引发多种自身免疫性疾病,目前研究发现,FOXP3基因表达异常在自身免疫性甲状腺疾病的发病中发挥重要作用。     

小鼠FOXP3-siRNA慢病毒载体的构建及其对小鼠动脉粥样硬化的影响

目的:构建小鼠FOXP3基因特异性siRNA慢病毒载体,并对其进行功能性研究.方法:根据GeneBank提供的小鼠FOXP3cDNA序列,设计4条RNA干扰靶点序列,制备双链DNA oligo,与制备好的双酶切慢病毒载体连接,再转入细菌感受态细胞DH-5a,行PCR鉴定出阳性克隆并测序,制备成FOXP3-siRNA慢病毒载体,利用Western-blot方法对构建的载体进行功能性研究;将载体通过尾静脉注入小鼠体内,观察其对小鼠动脉粥样硬化形成的影响.结果:构建的小鼠FOXP3基因siRNA慢病毒载体,经PCR和DNA测序证实与设计完全一致,并对Foxp3+CD4+CD25+调节性T细胞有显著敲减效应;其能显著促进动脉粥样硬化形成.结论:体内外实验表明,成功构建了小鼠FOXP3基因的特异性siRNA慢病毒载体.     

强直性脊柱炎患者外周血CD4^＋CD25^＋CD127^low/-T细胞检测及其意义△

目的检测CD4^＋CD25^＋CD127^low/-T细胞在强直性脊柱炎（ankylosing spondylitis，AS）患者外周血的表达，并分析其与CD4^＋CD25^＋FOXP3^＋T细胞的相关性。方法 以21例AS患者外周血为研究组，20例类风湿关节炎（rheumatoid arthritis，RA）患者和24名正常人外周血作为对照组，采用三色直接荧光素标记法和多参数流式细胞仪检测外周血CD4^＋CD25^＋CD127^low/-T细胞、CD4^＋CD25^high细胞及CD4^＋CD25^＋FOXP3^＋T细胞的比例，同时检测外周血C反应蛋白（C-reactive protein，CRP）、血沉（ESR）、免疫球蛋白、补体等水平以及HLA—B27并进行相关性分析。结果与正常对照组相比，AS患者外周血CD4^＋CD25^＋CD127^low/-T及CD4^＋CD25^＋FOXP3＋T细胞百分率降低（均P〈0．05）。CD4^＋CD25^＋细胞百分率升高（P〈0．05）；CD4^＋CD25^＋CD127^low/-T细胞与CD4^＋CD25^＋FOXP3^＋T细胞呈正相关（r=0．589，P=0．021），与CRP、ESR、血小板及免疫球蛋白（IgG、IgA、IgM）和补体（C3、C4）、BASDAI、HLA-B27等均无明显相关性（均P〉0．05）。AS患者外周血CD4^＋CD25^＋CD127^low/-T细胞、CD4^＋CD25^＋FOXP3^＋T细胞和CD4＋CD25^highT细胞的百分率与RA患者外周血比较差异均无统计学意义（P〉0．05）。结论AS患者外周血CD4^＋CD25^＋CD127^low/-T细胞降低，且与CD4^＋CD25^＋FOXP3^＋T细胞呈正相关，提示在调节性T细胞研究中CD127具有替代FOXP3的可能性。     

Quantification of hepatic FOXP3+ T‐lymphocytes in HIV/hepatitis C coinfection

Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T‐regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P = 0.031) and CD4+ cells (P = 0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P = 0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P < 0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients.     

FOXP3 positive regulatory T-cells in cutaneous and systemic CD30 positive T-cell lymphoproliferations

The CD30-positive lymphoproliferations encompass a spectrum of disorders that share histological and phenotypic similarities but differ markedly in clinical behaviour. The basis for this diversity is not known, but it has been proposed that immune suppression by cytokines and/or regulatory T-cells (Tregs) may be implicated. In this study, skin biopsies from lymphomatoid papulosis (LyP) (n = 14), primary cutaneous anaplastic large cells lymphoma (C-ALCL) (n = 13) and systemic anaplastic large cells lymphoma (S-ALCL) with (n = 9) or without (n = 6) ALK expression were examined by immunohistology for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30-positive lymphoproliferations is generally confined to tumour infiltrating Tregs. These cells may have influence upon the clinical behaviour, possibly depending upon the net degree of Treg mediated immune suppression of tumour cells relative to tumour infiltrating, cytotoxic effector cells, thereby implicating the more favourable outcome of LyP compared to C-ALCL.     

Abnormal DNA methylation in CD4+ T cells from people with latent autoimmune diabetes in adults

Aberrant DNA methylation in T cells has been linked to pathogenesis of autoimmune diseases. To investigate genomic and gene-specific DNA methylation levels in CD4⁺ T cells from patients with latent autoimmune diabetes in adults (LADA), and to investigate changes in the expression of genes that regulate methylation as well as the autoimmune-related gene FOXP3 in these patients. Global CD4⁺ T cell DNA methylation was measured in 15 LADA patients and 11 healthy controls using a methylation quantification kit. mRNA levels of DNA methytransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and FOXP3 were measured by real time PCR. Methylation of a FOXP3 regulatory element region was determined by bisulphite genomic sequencing. Genomic DNA methylation in CD4⁺ T cells from LADA patients was significantly increased compared to controls. DNMT3b mRNA levels were higher in CD4⁺ T cells from LADA patients than in controls. DNMT3b expression positively correlated with global DNA methylation in LADA CD4⁺ T cells. FOXP3 expression was decreased, and the FOXP3 promoter region was hypermethylated in CD4⁺ T cells from LADA patients compared with controls. DNA methylation levels are altered in CD4⁺ T cells from LADA patients, which may contribute to disease onset and progression by affecting the expression of autoimmune-related genes.     

Regulatory T-cell phenotype in association with large cell transformation of mycosis fungoides

INTRODUCTION: Tumor cells of primary cutaneous T-cell lymphomas are able to adopt a regulatory T-cell phenotype in vitro. The significance of this finding in vivo is matter of debate. METHODS: We stained five cases with transformed mycosis fungoides (MF) with an antibody against FOXP3, which is a sensitive and specific marker for the regulatory T-cell phenotype. RESULTS: Transformed T cells in four of five patients with MF stained positive for FOXP3. One patient who showed no CD30 expression of large transformed T cells was also negative for FOXP3. Comparison of plaques and tumors in one patient showed that FOXP3 and CD30 expression was exclusively observed in large transformed tumor cells whereas malignant T cells without large cell transformation were negative. CONCLUSION: Transformation of MF to high grade lymphoma may be associated with the adoption of a regulatory T-cell phenotype. FOXP3 expression may contribute to aggressive behavior of MF after large cell transformation via immune escape mechanism. The significance of this observation is limited by the low case number in this study.     

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.     

Expression of CD4+ forkhead box P3 (FOXP3)+ regulatory T cells in inflammatory bowel disease

OBJECTIVE: Forkhead box P3 (FOXP3) plays an important role in the development and function of CD4⁺ regulatory T (Treg) cells. In this study the percentage of CD4⁺ FOXP3⁺ Treg cells in peripheral blood mononuclear cells (PBMC) and the frequency of Treg cells in the colonic mucosa of patients with inflammatory bowel disease (IBD) were investigated. METHODS: The percentage of CD4⁺FOXP3⁺ Treg cells in PBMC was analyzed by flow cytometry. Immunohistochemistry was used to examine the FOXP3⁺ cells in the inflamed mucosa. Real‐time polymerase chain reaction and Western blot were used to detect the expressions of FOXP3 mRNA and protein in PBMC and mucosal biopsy specimens of IBD patients, respectively. RESULTS: Together with the decrease of percentage of Treg cells in PBMC, we found that the frequency of Treg cells increased significantly in inflamed mucosa of active or inactive Crohn's disease (CD) and ulcerative colitis (UC). The expressions of FOXP3 mRNA and protein increased in inflamed mucosa when compared with those in healthy controls, especially the FOXP3 mRNA in patients with active CD or UC. Interestingly, the expression of FOXP3 protein in active UC was higher than that in active CD. CONCLUSIONS: There was a decrease of CD4⁺FOXP3⁺ Treg cells in peripheral blood and an accumulation of Treg cells in inflamed mucosa. These data suggested that the suppressive function of Treg cells may be partially inhibited and this could be an important factor in the recurrence of disease, especially in UC.     

CD4^＋CD25^＋调节性T细胞及其分子标记物与支气管哮喘

CD4^＋CD25^＋调节性T细胞是一类以免疫抑制和免疫无能为特征的淋巴细胞群,FOXP3是CD4^＋CD25^＋调节性T细胞一个特征性的分子标志物,并且对CD4^＋CD25^＋调节性T细胞的发育、外周表达和功能维持有着关键性的作用。近年来,多项研究显示CD4^＋CD25^＋调节性T细胞参与并影响了支气管哮喘的发生、发展过程,对调节性T细胞或其相关基因的干预也许会成为支气管哮喘治疗的新方向。     

Resting and Activated Natural Tregs Decrease in the Peripheral Blood of Patients with Atherosclerosis

Background: Atherosclerosis is a chronic inflammatory disease affecting large and medium arteries. CD4+ T cells are known to play a role in the progression of the disease. CD4+CD25+Foxp3+ natural Treg (nTreg) cells seem to have a protective role in the disease and their reduction in acute coronary syndrome is recently shown. Objective: To investigate the frequency of nTreg subsets in the peripheral blood of patients with atherosclerosis. Methods: Confirmation of atherosclerosis was done by angiography and 15 ml heparinized blood was obtained from each of the 13 nondiabetic patients and 13 non-diabetic, non-smoker individuals with normal/insignificant coronary artery disease which was also confirmed by angiography. Lipid profiles of the patients and controls were measured at the time of sampling. Mononuclear cells were used for both RNA extraction and immunophenotyping by real-time PCR and flowcytometry techniques, respectively. Results: In natural Treg subsets, the frequency of CD4+CD45RO-CD25+Foxp3lo T-cells (resting nTregs) was greater in controls than patients (p=0.02). The frequency of CD4+CD45RO+CD25hiFoxp3hi T-cells (activated nTregs) was significantly higher in controls compared with patients (p=0.02). However, the frequency of CD4+CD25+CD45RO+Foxp3- T-cells (effector/memory) increased in patients compared with controls (p=0.01). Both the MFI and gene expression of Foxp3 were higher in control group than in patients (p=0.015 and p=0.017, respectively). Moreover, the TGF-β gene expression showed a decrease in the peripheral blood mononuclear cells of patients compared with controls (p=0.03). Conclusion: Decrease in both subsets of resting and activated nTregs along with a decrease in the expression of Foxp3 and TGF-β genes in patients with atherosclerosis suggests phenotypic changes in these subsets, which may as well be correlated with a more inflammatory profile in their lymphocytes.     

Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype

Thymic-derived natural T-regulatory cells (nTregs) are important for the induction of self-tolerance and the control of autoimmunity. Murine CD4+CD25(-)Foxp3(-) cells can be induced to express Foxp3 after T-cell receptor (TCR) activation in the presence of transforming growth factor beta (TGFbeta) and are phenotypically similar to nTregs. Some studies have suggested that TCR stimulation of human CD4+CD25(-) cells results in the induction of transient expression of FOXP3, but that the induced cells lack a regulatory phenotype. We demonstrate here that TCR stimulation alone was insufficient to induce FOXP3 expression in the absence of TGFbeta, whereas high levels of FOXP3 expression could be induced in the presence of TGFbeta. Although FOXP3 expression was stable, the TGFbeta-induced FOXP3+ T cells were neither anergic nor suppressive and produced high levels of effector cytokines. These results suggest that even high levels of FOXP3 expression are insufficient to define a human CD4+ T cell as a T-regulatory cell.