Alterations of peripheral CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes

Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25- T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25- T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios-Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase iCD4+CD25+Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.     

Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level

CD4+ CD25(high) regulatory T cells (Tregs) of patients with relapsing-remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single-cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25(high) FOXP3+ T cells and lower FOXP3 protein expression per cell in RR-MS patients than in SP-MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)-beta-treated RR-MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25(high) FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS patients, expressed CD103 and CD49d, adhesion molecules involved in T-cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25(high) CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS patients. Taken together, these data show aberrant FOXP3 expression at the single-cell level correlated with Treg dysfunction in RR-MS patients. Our results also suggest that Tregs accumulate in the CSF of RR-MS patients, in an attempt to down-regulate local inflammation in the central nervous system.     

多参数分析多发性骨髓瘤患者外周血中CD4~+CD25~+调节性T细胞的表达

目的 通过分析CD4+ CD25+ 调节性T细胞(CD4+ CD25+ regulatory T cells,CD4+ CD25+ Tress)在不同病程阶段多发性骨髓瘤(multiple myeloma,MM)患者外周血中的变化和临床意义,初步探讨MM患者的免疫抑制机制.方法 流式细胞术检测56例MM患者及30例健康志愿者外周血CD4+ CD25+、CD4+ CD25high、CD4+ CD25+ CD127low、CD4+ CD25high CD127low T细胞的比例并分析比较.结果 1)56例MM患者:化疗后组CD4+ CD25+、CD4+ CD25high、CD4+ CD25+ CD127low、CD4+ CD25high CD127low T 细胞的比例均高于正常组,差异具有显著性;初诊组的CD4+ CD25+ CD127low T细胞与正常对照组比较没有差异,但CD4+CD25+、CD4+ CD25high、CD4+ CD25high CD127lwo T细胞与正常对照组比较差异具有显著性;初诊组CD4+ CD25+T细胞的比例与化疗后组相比没有差异,但其CD4+ CD25high 、CD4+ CD25+ CD127low、CD4+ CD25high CD127low T细胞的比例均低于化疗后组,差异具有统计学意义.2)37例化疗后MM患者:稳定期与活动期的CD4+ CD25+、CD4+CD25+CD127low T细胞相比没有差异,但稳定期的CD4+ CD25high、CD4+ CD25high CD127low T细胞明显低于活动期.结论 CD4+ CD25+ Tregs在MM患者外周血中比例明显升高,且化疗可能影响其在外周血的比例;活动期MM患者CD4+ CD25+ Tregs的比例明显高于稳定期.这些提示CD4+ CD25+ Tregs可能是MM免疫抑制的一个重要原因.     

多发性骨髓瘤患者外周血CD4~+CD25~+和CD8~+CD28~-调节性T细胞研究

目的:探讨CD4~+CD25~+和CD8~+CD28~-调节性T细胞(Tregs)在多发性骨髓瘤(MM)患者外周血中的变化及意义.方法:采用流式细胞术检测38例MM患者及20例健康对照外周血CD4~+CD25~+和CD8~+CD28~-Tregs水平.分别采用溴甲酚绿法、透射免疫比浊法测定患者血清白蛋白(Alb)、β2-微球蛋白(β2-MG).结果:初诊MM患者外周血CD4~+CD25~(+/high)、CD4~+CD25~(high)CD127~(low)及CD8~+CD28~-Tregs比率均明显升高;CD4~+CD25~(+/high)和CD4~+CD25~)(high)CD127~(low)Tregs比率在各临床分期均较对照组升高,随分期增高呈现增加趋势,且CD4~+CD25~(high)和CD4~+CD25~(high)CD127~(low)Tregs在Ⅲ期患者显著高于Ⅰ期患者;CD8~+CD28~(-Tregs)在Ⅱ、Ⅲ期显著高于正常对照,且Ⅱ期高于Ⅰ期,Ⅲ期高于Ⅱ期,逐期递增,而在Ⅰ期与对照组比较无显著差异;CD4~+CD25~(+/high)和CD4~+CD25~(high)CD127~(low)Tregs比率在进展期和稳定期均较对照组升高,但两期之间比较无明显差异,而CD8~+CD28~-Tregs在进展期高于稳定期及对照组,稳定期和对照组间比较无明显差异;CD4~+CD25~(high)Tregs和CD4~+CD25~(high)CD127~(low)Tregs比率与Alb水平均呈负相关.结论:MM患者体内存在CD4~+CD25~+Tregs和CD8~+ CD28~-Tregs异常增加,可能是MM免疫逃逸的一个重要机制,这些变化同临床分期、病情进展及预后存在一定程度相关性.     

小鼠CD4＋ CD25＋免疫调节性T细胞体外扩增及功能检测的研究

目的 探讨小鼠CD4＋ CD25＋调节性T细胞（Tregs）的体外扩增方法及扩增后Tregs的免疫功能.方法 利用免疫磁珠法分离小鼠Tregs;采用anti-CD3mAb＋rIL-2和Allo-APC＋rIL-2两种方法进行体外扩增,通过TRANSWELL培养系统、细胞因子表达及CFSE染色标记等方法检测Tregs免疫抑制作用的机制.结果 体外分离的Tregs纯度为89.5％;活细胞率约为98％.Tregs扩增的倍数在两组各时间点差异无统计学意义.扩增后CD4＋ CD25＋T细胞、CD4＋T细胞和CD4＋ CD25-T细胞的每分钟计数（CPM）分别为1 470、12 700和14 300.混合淋巴细胞反应（MLR）中当CD4＋：CD25＋T细胞的比例为1∶1时,抑制率为79％,比例为8∶1时,抑制率为33％.CFSE标记后,进入分裂周期的CD4＋T细胞的百分比在未加入CD4＋ CD25＋T细胞组为83.7％,加入CD4＋ CD25＋T细胞组为31.7％,差异具有统计学意义（P=0.0006）.CD4＋ CD25＋T细胞对CD4＋T细胞的增殖抑制率在Transwell和Non-Transwell的培养系统中分别为＜5％和95％.Transwell培养组中IL-2含量高于Non-Transwell组,差异具有统计学意义[Transwell培养组为（158.33±2.08）pg/mL,Non-Transwell组为（23.00±2.00） pg/mL,P ＜0.0001].结论 采用免疫磁珠法分离小鼠Tregs可行,Tregs可有效扩增;Tregs的作用机制为抑制CD4＋T细胞的增殖及抑制CD4＋T细胞分泌IL-2;其抑制作用需要细胞-细胞间接触;体外扩增后的Tregs仍具有免疫特性,其体外免疫抑制作用增强.     

CD4-mediated functional activation of human CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells (CD25(+) Tregs) constitute a specialized population of T cells that is essential for the maintenance of peripheral self-tolerance. The immune regulatory function of CD25(+) Tregs depends upon their activation. We found that anti-CD4 antibodies activate the suppressive function of human CD25(+) Tregs in a dose-dependent manner. We demonstrate that CD4-activated CD25(+) Tregs suppress the proliferation of CD4(+) and CD8(+) T cells, their IL-2 and IFN-gamma production as well as the capacity of CD8(+) T cells to re-express CD25. By contrast, anti-CD4 stimulation did not induce suppressive activity in conventional CD4(+) T cells. These results identify CD4 as a trigger for the suppressive function of CD25(+) Tregs and suggest a possible CD4-mediated exploitation of these cells.     

T regulatory cells lacking CD25 are increased in MS during relapse

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4⁺CD25⁺FoxP3⁺ Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25^? Tregs in human autoimmunity. We analyzed relapsing–remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25⁺CD4⁺ and CD25⁺CD8⁺ effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25^? FoxP3⁺ Tregs. With time, the proportion of Tregs decrease while effector T cells expand.     

TcR-induced regulated secretion leads to surface expression of CTLA-4 in CD4+CD25+ T cells

In this study we show that CD4+ T cells develop a functional regulated secretory compartment after differentiation into effector cells, as shown by their increased expression and T-cell receptor-induced exocytosis of lysosomal and cytotoxic effector proteins. We tested the hypothesis that activation-induced surface cytotoxic T-lymphocyte-associated antigen (CTLA-4) expression in CD4+CD25+ regulatory T cells occurs via a similar regulated secretory pathway. Fluorescence microscopy showed that internal CTLA-4 in these cells was stored in a vesicular compartment distinct from lysosomal vesicles. Rapid activation-induced CTLA-4 surface expression in mouse CD4+CD25+ T cells is independent of protein synthesis and Rab-27a. When antigen-dependent T-cell-antigen-presenting cell (APC) conjugates were analysed for surface distribution of CD86 on APC, a higher concentration of CD86 molecules was observed in the synapse of APC conjugated to CD4+CD25+ cells than APC conjugated to CD4+CD25- cells. These results demonstrate that fast delivery of mediators by the regulated secretory pathway in CD4+ T cells can be used to perform other functions that are not involved in cytotoxic function but that can influence/regulate other cells.     

Interleukin-4 suppresses the cytotoxic potential of in vitro generated,adaptive regulatory CD4+ T cells by down-regulation of granzyme B

Regulatory CD4⁺ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4⁺ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.     

Inverse correlation between CD4+ regulatory T-cell population and autoantibody levels in paediatric patients with systemic lupus erythematosus

CD4(+) CD25(+) regulatory T cells (Tregs) are critical in maintaining self-tolerance and preventing organ-specific autoimmunity. Their role in paediatric systemic lupus erythematosus (SLE), an autoimmune disease characterized by inappropriate regulation of hyperactivated B and T cells, has not been clearly defined. Using flow cytometry to determine cell populations and real-time polymerase chain reaction to assay mRNA expression for FOXP3, CTLA-4, and GITR, we characterized CD4(+) CD25(+) T cells in paediatric SLE patients and healthy subjects. The frequency of CD4(+) CD25(+) Tregs was significantly decreased in patients with active SLE compared with patients with inactive SLE and with controls (7.27% +/- 2.50%, 9.59% +/- 2.80% and 9.78% +/- 2.11%, respectively; P = 0.027 and P < 0.001, respectively), and was inversely correlated with disease activity, as assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 scores (r = -0.59, P = 0.001) and serum anti-double-stranded DNA levels (r = -0.65, P < 0.001). Our preliminary investigations found elevated surface expression of GITR in CD4(+) CD25(+) T cells, elevated mRNA expression of CTLA-4 in CD4(+) T cells and higher amounts of mRNA expression for FOXP3 in CD4(+) cells in patients with active SLE compared with patients with inactive disease and controls. We demonstrated reduced CD4(+) CD25(+) Treg levels were inversely correlated with disease activity, indicating a defective Treg population in paediatric SLE patients. The differences in the expression of FOXP3, CTLA-4 and GITR imply the possible role of CD4(+) Tregs in the pathogenesis of SLE.     

Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis

Immunoregulatory T cells of (CD4+)CD25+ phenotype suppress T cell function and protect rodents from organ-specific autoimmune disease. The human counterpart of this subset of T cells expresses high levels of CD25 and its role in human autoimmune disorders is currently under intense investigation. In multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), the activation of circulating self-reactive T cells with specificity for myelin components is considered to be an important disease initiating event. Here, we investigated whether MS is associated with an altered ability of (CD4+)CD25high regulatory T cells (Treg) to confer suppression of myelin-specific immune responses. Whereas Treg frequencies were equally distributed in blood and cerebrospinal fluid of MS patients and did not differ compared to healthy controls, the suppressive potency of patient-derived (CD4+)CD25high T lymphocytes was impaired. Their inhibitory effect on antigen-specific T cell proliferation induced by human recombinant myelin oligodendrocyte protein as well as on immune responses elicited by polyclonal and allogeneic stimuli was significantly reduced compared to healthy individuals. The effect was persistent and not due to responder cell resistance or altered survival of Treg, suggesting that a defective immunoregulation of peripheral T cells mediated by (CD4+)CD25high T lymphocytes promotes CNS autoimmunity in MS.     

Circulating CD4+CD25+ T Regulatory Cells Are Not Altered in Multiple Sclerosis and Unaffected by Disease-Modulating Drugs

Experimental animal models for autoimmunity have demonstrated the existence and crucial role of CD4(+)CD25(+) T regulatory (Tr) cells in suppressing autoreactive T cells and promoting peripheral tolerance. Recent in vitro functional studies showed that Tr cells are enriched in the CD25(high) cell population among CD4(+) T cells, and that they totally inhibit proliferation and cytokine secretion by CD4(+) T cells. It is not yet known if circulating Tr cells are involved in multiple sclerosis (MS). This study was done firstly to determine whether alterations of the CD4 (+) CD25(high) T cells occur in MS, examining their frequencies. As it was reported that the suppressive activity of CD4(+)CD25(+) Tr cells is mainly through cell surface contact pathway, we secondly analyzed the expression of the functionally important cell surface molecules of CD4(+)CD25(high) Tr cells. Two- or three-colour flow cytometry was used to identify and quantify CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells among blood CD4(+) T cells in MS patients without treatment vs. patients treated with either interferon-beta (IFN-beta) or glatiramer acetate (GA) or IFN-beta + GA in combination vs. healthy controls (HC). Expression of functionally important surface molecules CD45RO, CD69, CD95, HLA-DR, and intracellular CTLA-4 and IL-10 production by CD4(+)CD25(high) Tr cells were investigated. CD4(+)CD25(+) T cells constituted around 6% of CD4(+)T cells in all MS patient groups, and 7% in HC. There were also no changes in the proportions of CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells in a longitudinal follow-up of MS patients before and during IFN-beta treatment. Frequencies of circulating CD4(+)CD25(high)Tr cells among CD4(+) T cells were also similar and their surface or intracellular molecular expression did not vary in MS patients, irrespective of treatment, compared to HC. This study suggests that levels of circulating CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells are not altered in MS, and are unaffected by substances currently used to modulate the disease.     

CD4+ CD25+调节性T细胞在流产中的研究进展

CD4+CD25+调节性T细胞(Tr)是一个具有独特免疫调节功能的T细胞亚群.Tr免疫学特性主要在于抑制自身反应性T细胞的活化,并参与外周免疫耐受,对维持机体内环境的稳定起重要作用.Tr在流产中发挥重要的免疫调节作用.     

CD4(+)CD25(high)CD127(low) regulatory T cells in patients with stable angina and their dynamics after intracoronary sirolimus-eluting stent implantation

Rapamycin contributes to the expansion of regulatory T cells (Tregs) in vitro. We investigated CD4(+)CD25(high)CD127(low) Treg level dynamics as well as the major parameters of cell immunity and sCD25 and highly sensitive C-reactive protein (hsCRP) concentrations in the blood of patients after coronary stenting (CS) with sirolimus (rapamycin)-eluting stents (SES; n = 43). The relation between initial Treg values and the severity of coronary atherosclerosis was observed. Treg and sCD25 levels were increased 1 month after CS versus baseline values and versus data in the control group (coronary angiography [CA], n = 20). A positive correlation between Treg and sCD25 levels was reported, whereas no relation was observed with the length of SES implanted. HsCRP level was increased during the first 7 days and returned to baseline values 1 month after CS/CA. Treg content is lower in patients with multivessel CAD. Elevated levels of Tregs and sCD25 after SES implantation might occur because of the immunomodulating effect of rapamycin.     

Detectable expression of IL-35 in CD4+ T cells from peripheral blood of chronic hepatitis B patients

Epstein-Barr virus-induced gene 3 (Ebi3) and the p35 subunit of IL-12 have been reported to form a heterodimeric cytokine, named IL-35, in human and mouse. In mice, IL-35 has been shown to be constitutively expressed by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) and suggested to contribute to their suppressive activity. However, human CD4(+)CD25(+)Foxp3(+) Tregs do not constitutively express detectable amounts of IL-35 in both mRNA and protein levels. Circulating CD4(+)CD25(+) Treg frequency of chronic Hepatitis B patients significantly correlates with serum viral load. In this study, we investigated whether IL-35 expression could be detected in CD4(+) T cells from peripheral blood of chronic Hepatitis B patients. Using both RT-PCR and immunoprecipitation plus Western blot analysis, we demonstrated that IL-35 expression could be detected in the CD4(+) T cells from peripheral blood of Chronic Hepatitis B patients.     

Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25− T cells from atopic and nonatopic donors

Background:  Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.
Objective:  To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25− T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro .
Methods:  Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25− T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.
Results:  CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25− effector cells, which downregulated CCR7 and upregulated CCR4.
Conclusions:  CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25− effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.     

小鼠CD4~+ CD25~+ Foxp3~+调节性T细胞体外扩增

目的:本研究旨在探讨CD4+CD25+Foxp3+调节性T细胞体外扩增的方法。方法:采用磁珠分选小鼠CD4+T细胞,αCD3单克隆抗体包被24孔板,加入αCD28单克隆抗体、雷帕霉素、rhIL-2,培养3周后,流式细胞仪测定培养细胞中CD4+CD25+T细胞的含量,实时定量PCR检测CD4+CD25+T细胞Foxp3 mRNA的表达;单向混合淋巴细胞反应和增殖抑制试验测定扩增的CD4+CD25+T细胞的增殖及其抑制功能;ELISA检测培养上清中IL-10和TGF-β1的含量。结果:小鼠CD4+T细胞培养3周后,CD4+CD25+T细胞达(76.05±2.73)%,高于未加雷帕霉素组(52.17±1.36)%(P<0.001),磁珠分选的CD4+CD25+T细胞Foxp3 mRNA的表达是未加雷帕霉素组的5倍(P<0.001),增殖能力是未加雷帕霉素组的0.29倍(P<0.001),对CD4+T细胞增殖抑制能力是未加雷帕霉素组的3.6倍(P<0.001),培养上清中IL-10和TGF-β1分别是对照组的1.8倍和1.6倍(P<0.001)。结论:小鼠CD4+T细胞在含有1μg/ml的αCD28、rhIL-2 100 U/ml和终浓度为10 nmol/L雷帕霉素的培养体系中培养3周后能有效扩增CD4+CD25+Foxp3+调节性T细胞。     

Reduction of forkhead box P3 levels in CD4+CD25high T cells in patients with new-onset systemic lupus erythematosus

The aim of this study was to quantify and evaluate the forkhead box P3 (FoxP3) expression regulatory T cells in new-onset systemic lupus erythematosus (SLE) patients before and after treatment. Forty-four newly diagnosed and untreated SLE patients, including 24 with active disease (SLEDAI > or = 10) and 20 with inactive disease (SLEDAI < 5), were enrolled in this study. Twenty-one age- and sex-matched healthy volunteers were also included as controls. Peripheral blood samples were collected and mononuclear cells isolated. The expression of CD25 and FoxP3 in CD4(+) T cells were analysed with flow cytometry. CD4(+)CD25(+) (3.95-13.04%) and CD4(+)CD25(high) (0.04-1.34%) T cells in peripheral blood in untreated patients with new-onset active lupus were significantly lower than that in the patients with inactive lupus (7.27-24.48%, P < 0.05 and 0.14-3.07% P < 0.01 respectively) and that in healthy controls (5.84-14.84%, P < 0.05). Interestingly, the decrease in CD4(+)CD25(high) T cells was restored significantly in patients with active lupus after corticosteroid treatment. There was, however, a significantly higher percentage of CD4(+)FoxP3(+) T cells in patients with active (5.30-23.00%) and inactive (7.46-17.38%) new-onset lupus patients compared with healthy control subjects (2.51-12.94%) (P < 0.01). Intriguingly, CD25 expression in CD4(+)FoxP3(+) T cells in patients with active lupus (25.24-62.47%) was significantly lower than that in those patients with inactive lupus (30.35-75.25%, P < 0.05) and healthy controls (54.83-86.38%, P < 0.01). Most strikingly, the levels of FoxP3 expression determined by mean fluorescence intensity in CD4(+)CD25(high) cells in patients with active SLE were significantly down-regulated compared with healthy subjects (130 +/- 22 versus 162 +/- 21, P = 0.012). CD4(+)CD25(high) T cells are low in new-onset patients with active SLE and restored after treatment. Despite that the percentage of CD4(+)FoxP3(+) T cells appear high, the levels of FoxP3 expression in CD4(+)CD25(high) T cells are down-regulated in untreated lupus patients. There is a disproportional expression between CD25(high) and FoxP3(+) in new-onset patients with active SLE.