首发抑郁症患者外周血CD4+CD25+CD127-调节性T细胞的研究

目的 探讨首发抑郁症患者治疗前后外周血中CD4⁺CD25⁺CD127^-占CD4⁺的比例及Treg细胞与抗抑郁治疗的相关性。方法 纳入在大庆市第三医院住院治疗的首发抑郁症患者共20例,均符合《精神障碍诊断与统计手册（第5版）》（DSM-5）关于抑郁障碍的诊断标准,汉密尔顿抑郁量表17项版（HAMD-17）总评分>20分;同期纳入在大庆市第三医院体检中心的健康成人作为对照组,共20例。对抑郁症患者治疗前及治疗12周后分别进行HAMD-17评定。对抑郁症患者治疗前、治疗12周后及健康对照组分别采取其外周血,利用流式细胞术（FCM）测定外周血CD4⁺CD25⁺CD127^-占淋巴细胞的百分比及CD4⁺CD25⁺CD127^-占CD4⁺T细胞的百分比,并作对照分析。结果 在CD4⁺CD25⁺CD127^-/CD4⁺的水平上,抑郁症患者治疗前、治疗后均高于健康对照组,差异均有统计学意义（P均<0.01）;抑郁症患者HAMD-17评分及血清CD4⁺CD25⁺CD127^-/CD4⁺的水平均下降,差异均有统计学意义（P均<0.05）,且抑郁症显效组CD4⁺CD25⁺CD127^-/CD4⁺的变化率与HAMD-17评分减分率呈正相关（r=0.716,P<0.05）。结论 抑郁症患者经SSRIs类药物治疗后,外周血CD4⁺CD25⁺CD127^-/CD4⁺水平的降低预示抗抑郁疗效较好,有效的抗抑郁治疗可能与调节T细胞有一定的相关性。     

实验性自身免疫性脑脊髓炎大鼠血CD4+CD25+T细胞的研究

目的探讨实验性自身免疫性脑脊髓炎(EAE)动物模型血CD4 CD25 T细胞的变化及其意义。方法以豚鼠全脊髓匀浆(GPSCH)为抗原免疫Wistar大鼠,建立EAE的动物模型,采用三色流式细胞仪检测EAE和正常大鼠外周血CD4 CD25 T细胞的细胞数并进行比较;通过观察大鼠行为学及脑和脊髓的病理改变确定EAE。结果EAE模型大鼠的成功率为48.9%,EAE大鼠外周血CD4 CD25 T淋巴细胞数(5.29±4.00)显著低于正常对照组(12.61±2.24)(P<0.01)。结论EAE大鼠血CD4 CD25 T细胞数明显减少,CD4 CD25 T淋巴细胞对神经系统脱髓鞘疾病是一种保护因子。     

Increased Fas-mediated cytotoxicity of CD4-positive T cells in patients with human T-lymphotropic virus type I-associated myelopathy

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release assay, we investigated Fas-mediated cytotoxicity of peripheral blood CD4⁺ T cells of patients with human T-lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) against T98G, a glioblastoma cell line which expresses Fas. Cytotoxic activity of CD4⁺ T cells against T98G was significantly higher in HAM patients than in controls. Moreover, when CD4⁺ T cells of HAM patients were preincubated with a monoclonal antibody to human Fas ligand (FasL), cytotoxic activity against T98G was significantly suppressed. These results suggest that damage to nervous tissues by the Fas/FasL system is involved in the pathogenesis of HAM.     

CD4+CD25+调节性T细胞、Foxp3与多发性硬化的关联

调节性T细胞(regulatoryT cell.Treg)是一类具有免疫调节功能的T细胞哑群,它们在维持外周免疫耐受、预防自身免疫性疾病的发牛过程中起着重要作用.     

糖皮质激素对多发性硬化患者外周血淋巴细胞CD80和CD4+CD25+T细胞表达的影响

目的探讨糖皮质激素(GC)对多发性硬化(MS)患者外周血淋巴细胞CD80和CD4+CD25+T细胞表达的影响。方法利用流式细胞仪检测21例MS急性期患者GC治疗前后外周血淋巴细胞CD80和CD4+CD25+T细胞阳性率,并与正常对照组比较;比较MS患者治疗前后扩展功能障碍状况量表(EDSS)评分的变化。结果MS患者急性期外周血淋巴细胞CD80的阳性率[(5.031±1.782)%]较正常对照组[(6.436±2.035)%]明显下降(P<0.05),经GC治疗后CD80的阳性率[(6.467±1.882)%]明显增高(P<0.01);CD4+CD25+T细胞阳性率治疗前后与正常对照组间差异均无统计学意义;治疗后EDSS评分[(3.64±1.79)分]较治疗前[(4.26±1.68)分]明显下降(P<0.01)。结论GC可上调MS患者淋巴细胞CD80的表达,抑制细胞免疫,促进MS病情缓解。     

Impairment of circulating CD4+CD25+ regulatory T cells in patients with chronic inflammatory demyelinating polyradiculoneuropathy

Abstract   Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated peripheral nervous system disease. CD4⁺CD25⁺ T regulatory cells (Tregs) have been unequivocally shown to be critical in maintaining immune tolerance and preventing auto-immune diseases by suppressing self-reactive T cells. Thus, we hypothesized that the numbers and/or the function of Tregs would be deranged during the progressive or relapse phases of CIDP. The number of Tregs was determined by flow cytometry according to their characteristic CD4⁺CD25^high membrane phenotype. Functional characterization of Tregs was analyzed by suppression of proliferation and secretion of cytokines by co-cultured effector CD4⁺CD25⁻ T cells. FOXP3 message expression level was assessed by quantitative real-time polymerase chain reaction. The results showed significant reduction in both the number and the suppressive function of Tregs in the patients with CIDP compared with healthy controls. Also, Tregs isolated from CIDP patients expressed lower levels of FoxP3 mRNA. During the progressive or the relapsing phases of CIDP, the number of Tregs was reduced, and the suppressive function of them decreased. These findings may be helpful to our understanding of the possible role of Tregs in the pathogenesis of CIDP.     

EAE大鼠胸腺CD4~+ CD25~+ Foxp3~+ Treg细胞的动态变化及α-硫辛酸的干预作用

目的探讨实验性变态反应性脑脊髓炎(EAE)大鼠不同病程中胸腺CD4+CD25+Foxp3+Treg细胞变化情况及α-硫辛酸对EAE大鼠胸腺的干预作用。方法取不同时期对照组、自然病程EAE组及α-硫辛酸EAE组大鼠的胸腺组织做流式细胞学,动态检测CD4+CD25+Foxp3+Treg细胞的变化情况。结果 EAE组大鼠急性期、复发期CD4+CD25+Foxp3+Treg细胞较同时期对照组明显减少(P<0.05),缓解期有所上升;α-硫辛酸组与同期EAE组相比CD4+CD25+Foxp3+Treg细胞无明显变化;半年期三组大鼠胸腺CD4+CD25+Foxp3+Treg细胞都明显下降,各组间无统计学差异。结论 CD4+CD25+Foxp3+Treg细胞参与了EAE的发病,与病程的发展密切相关;α-硫辛酸对EAE大鼠的干预作用并非通过CD4+CD25+Foxp3+Treg细胞发挥其治疗作用;随着年龄的增长,胸腺不再是机体的主要免疫器官。     

Cytotoxic CD4+ T cells are correlated with better prognosis in Han Chinese grade II and grade III glioma subjects and are suppressed by PD-1 signaling

Purpose: Malignant gliomas are the most common tumors in the central nervous system with a poor prognosis. Recently, CD4⁺ cytotoxic T cells (CTLs) are being increasingly recognized as possessing antitumor capacity. However, their presence, activity and regulation in glioma have not been investigated in detail. Methods: To examine this, 72 grade II and grade III Han Chinese glioma patients and 30 Han Chinese healthy controls were investigated. Results: We found that compared to healthy controls, glioma patients had significantly upregulated frequencies of circulating CD4⁺ CTLs, identified by the expression of granzyme A (GzmA), granzyme B (GzmB) and/or perforin. The stimulated CD4⁺ CTLs in grade II and grade III glioma patients also had less proliferative ability than those in healthy controls, a feature of suppression that progressed with tumor grade. The frequencies of GzmB-expressing circulating CD4⁺ CTLs were directly associated with prognosis. We hypothesized that the programed death 1 (PD-1)/PD-ligand 1 (L1) interaction possibly contributed to the suppression of CD4⁺ CTLs in grade II and grade III glioma, since an upregulation of PD-1 was observed on CD4⁺ CTLs in glioma compared to those in the healthy individuals. Blockade of the PD-1/PD-L1 interaction with neutralizing antibodies significantly increased the proliferation and granzyme or perforin production by CD4⁺ CTLs in grade II and grade III glioma patients. Conclusions: These data suggest that the CD4⁺ CTLs in grade II and grade III glioma patients contribute to antitumor immunity and could be suppressed by PD-1 signal transduction.     

Functional assay for human CD4+CD25+ Treg cells reveals an age-dependent loss of suppressive activity

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4+CD25+ T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4+CD25- indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25- indicator cells by both CD4+CD25(+high) and CD4+CD25(+low) T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO- (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA-4 and GITR than CD4+CD25- T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.     

多发性硬化患者外周血CD4+CD25+ T细胞变化及其机制探讨

目的探讨多发性硬化(MS)患者外周血CD4 CD25 T细胞数量及叉头样转录因子(FOXP3)表达水平与MS病情的关系。方法选择温州地区MS患者44例(男12例、女32例),均按Poser诊断标准诊断,结合头颅MRI增强扫描排除合并其他神经系统和免疫系统疾病,并统一行EDSS评分;对照组43例(男13例、女30例)为健康查体者。具体方法:流式细胞仪检测外周血CD4 CD25 T细胞数量;免疫磁珠法分离CD4 CD25 T细胞;RT-PCR法检测CD4 CD25 T细胞FOXP3 mRNA表达并进行半定量分析。结果MS患者外周血中CD4 CD25 调节性T细胞数量与对照组比较无明显变化(P>0.05);活化的效应性T细胞数量增加(P<0.05)且活动期增加更为显著(P<0.01)。同一个体疾病活动期外周血CD4 CD25 调节性T细胞数量较非活动期减少(P<0.05)。MS患者外周血中CD4 CD25 T细胞的FOXP3 mRNA表达降低(P<0.05),且活动期降低更明显(P<0.01)。结论此组MS患者外周血CD4 CD25 调节性T细胞抑制活性降低,FOXP3 mRNA表达减少,活化的效应性T细胞数量增加,且与MS疾病活动性有关。     

Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression

Accumulating evidence indicates an immunosuppressive role for CD4(+)CD25(+) regulatory T cells (Tregs) in autoimmune diseases. Although an impaired Treg function in patients with relapsing-remitting multiple sclerosis (RR-MS) has been reported recently, no information is available so far about Treg function in the progressive stage of the disease. In the present study, the phenotypic and functional characteristics of CD4(+)CD25(+) T cells isolated from the peripheral blood of patients with RR-MS and secondary progressive multiple sclerosis (SP-MS) were investigated. No significant quantitative or phenotypic abnormalities in CD4(+)CD25(+) T cells from RR- and SP-MS patients were detected. However, whereas a reduced suppressor function of CD4(+)CD25(+) T cells toward proliferation and interferon-gamma production of CD4(+)CD25(-) responder T cells was found in RR-MS patients, SP-MS patients showed a normal Treg function. The suppressive capacity of MS-derived CD4(+)CD25(+) T cells was correlated with disease duration but not with age, indicating that Treg function is more affected in the early phase of the disease process. Consistently with the suppressive capacity, CD4(+)CD25(+) T cells from SP-MS patients showed normal levels of FOXP3 mRNA in contrast to RR-MS patients that had a reduced FOXP3 expression. These data are the first to demonstrate differences in function and FOXP3 expression of CD4(+)CD25(+) T cells from patients with RR- and SP-MS.     

Vascular dementia in patients with immune thrombocytopenic purpura

Introduction: Platelets have been implicated in memory disorders but this has not been investigated in patients with immune or idiopathic thrombocytopenia (ITP). ITP is an autoimmune disorder in which autoantibodies bring about platelet destruction. We previously reported a group of ITP patients who manifested TIA-like syndrome and gradual memory loss leading to dementia: platelet microparticles (PMP), a marker of platelet activation, were often elevated, suggesting that procoagulant PMP released from stimulated platelets contributed to thrombosis in small vessels. We have expanded on those studies to better define the clinical, laboratory, and radiologic characteristics of this syndrome. Materials and methods: Twenty ITP patients with this syndrome were studied in comparison to twenty-three ITP patients without it (patient controls). Clinical and laboratory features were compared and radiologic images were analyzed. Factors influencing the rate of progression to advanced dementia were also investigated. Results and conclusion: Recurring dizzy or weak spells, TIA-like syndrome, recent memory loss, and cognitive impairment were common initial complaints. In some, these symptoms progressed rapidly to dementia but was indolent in others. Progression was faster in those with splenectomy and higher platelet counts. MRI showed enhanced signal in subcortical, periventricular areas, consistent with ischemic small vessel disease. Compared to patient controls, bleeding was less frequent and platelet activation (increased PMP, CD62p) was more frequent in the study group. Thrombotic complications may occur in ITP, manifested as TIA-like syndrome or memory loss due to ischemic small vessel disease, progressing to vascular dementia. Memory disturbances associated with platelet disorders warrants further investigation.     

Circulating CD4+CD8+ cells in myasthenia gravis: supplementary immunological parameter for long-term prognosis

Summary Twenty patients with myasthenia gravis (MG) were studied prospectively for up to 5 years after thymectomy, in order to clarify the relationships between disease severity, anti-acetylcholine receptor antibody (anti-AChR) titres, proportions of circulating CD4⁺CD8⁺ cells (CD4⁺CD8⁺ cell level) and major lymphocyte subsets. The CD4⁺CD8⁺ cell levels were closely related to the clinical change within 1 year after surgery in 8 patients who showed a preoperative elevation in the cell levels. This group of patients consisted of six thymomatous and two non-thymomatous patients; the latter were both negative for anti-AChR. The anti-AChR titres generally changed in parallel with the clinical state in 9 of the 16 patients who were followed up for more than a year after thymectomy, and the CD4⁺CD8⁺ cell levels were useful in predicting the clinical course in 6 of the above 9 patients and 3 other patients, including antibody-negative cases. The present study suggests that the CD4⁺CD8⁺ cell levels may serve as an indicator for long-term prognosis of MG.     

Glucocorticoids increase CD4CD25 cell percentage and Foxp3 expression in patients with multiple sclerosis

Objectives – To determine whether percentages of CD4⁺CD25^high T cells (a group of regulatory T cells, Treg) differ in patients with multiple sclerosis (MS) in relapse vs remission after glucocorticoid treatment and whether treatment for relapses changes Treg population and the expression of Foxp3, a key Treg‐associated molecule. Materials and methods – Peripheral blood mononuclear cells (PBMC) were obtained from 20 patients with MS during relapse, just before and 2 days after starting steroid treatment (i.v. methylprednisolone 1 g/day for 3 days) and then 6 weeks after treatment. CD4⁺CD25^hi cells were analysed by using flow cytometry. Cytokines were measured by using an ELISA and Foxp3, CD3 and CD25 expression by using quantitative real‐time PCR. Results – The percentage of CD4⁺CD25^hi cells, plasma IL‐10 and Foxp3/CD3 ratio increased 48 h after methylprednisolone initiation and returned to baseline values by 6 weeks post‐treatment. Conclusions – Results suggest that glucocorticoids increase Treg cell functional molecules and percentages. This may be a mechanism whereby steroids expedite recovery from MS relapses.     

Immunotherapy of rat glioma without accumulation of CD4~+CD25~+FOXP3~+ regulatory T cells

Immunotherapy may be used for the treatment of glioblastoma multiforme;however,the induced immune response is inadequate when either T cells or dendritic cells are used alone.In this study,we established a novel vaccine procedure in rats,using dendritic cells pulsed with C6 tumor cell lysates in combination with adoptive transfer of T lymphocytes from syngenic donors.On day 21 after tumor inoculation,all the rats were sacrificed,the brains were harvested for calculation of glioma volume,cytolytic T lymphocyte responses were measured by cytotoxic assay,and the frequency of regulatory T lymphocytes(CD4+CD25+FOXP3+) in the peripheral blood was investigated by flow cytometric analysis.The survival rate of rats bearing C6 glioma was observed.Results showed that the co-immunization strategy had significant anti-tumor potential against the pre-established C6 glioma,and induced a strong cytolytic T lymphocyte response in rats.The frequency of peripheral blood CD4+CD25+FOXP3+ regulatory T lymphocytes was significantly decreased following the combination therapy,and the rats survived for a longer period.Experimental findings indicate that the combined immunotherapy of glioma cell lysate-pulsed dendritic cell vaccination following adoptive transfer of T cells can effectively inhibit the growth of gliomas in rats,boost anti-tumor immunity and produce a sustained immune response while avoiding the accumulation of CD4+CD25+FOXP3+ regulatory T lymphocytes.     

Interferon-beta-1a treatment increases CD56 natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis

Disease modifying effects of interferon (IFN)-β therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56^bright subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-β-1a(IM) treatment of relapsing–remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56^bright NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-β-1a in MS include control of pernicious autoimmunity.     

Virus-activated T cells regulate expression of adhesion molecules on endothelial cells in sites of infection

To study the role of cell adhesion molecules in the fatal CD8⁺ T-cell mediated meningitis which is induced by intracerebral infection with lymphocytic choriomenmgitis virus, the expression of relevant molecules on inflammatory cells and local endothelium was analyzed immunohistochemically. Most inflammatory cells were strongly positive for LFA-1, VLA-4, Pgp-1 and ICAM-1. Expression of ICAM-1 and VCAM-1 was upregulated on the endothelial cells in immunocompetent mice, but hot in T-cell deficient nude mice. analysis of mice deficient in either CD4⁺ or CD8⁺ T cells, revealed that not only was the inflammatory reaction dependent on the presence of CD8⁺ cells, but these cells also appeared to be required for maximal upregulation of ICAM-1 and VCAM-1 on the endothelial cells. These results indicate that virus-specific CD8⁺ T cells are crucially involved in regulating the inflammatory reaction through effects on endothelial expression of adhesion molecules.     

Cytokine production by naive and primary effector CD4+ T cells exposed to norepinephrine

We recently showed that clones of Th1 cells, but not Th2 cells, expressed a functional beta-2-adrenergic receptor (beta2AR) and that either norepinephrine or the beta2AR agonist terbutaline stimulated this receptor to modulate the level of Th1 cytokines produced. In the present study, we show that norepinephrine and terbutaline stimulate the beta2AR to decrease the level of IL-2 produced by freshly isolated murine splenic naive CD4+ T cells from either Balb/C or DO11.10 transgenic mice and activated polyclonally with anti-CD3 and anti-CD28 mAbs. In contrast, the level of cytokines produced by primary effector Th1 and Th2 cells were unaffected when norepinephrine, terbutaline, or cAMP analogs were added at the time of restimulation. These results suggest that a diversity exists among CD4+ T-cell subsets with respect to the level of adrenergic receptor expression, responsiveness to cAMP, stage of cell differentiation, or a combination of the above.     

Identification of a novel T cell epitope of human proteolipid protein (residues 40–60) recognized by proliferative and cytolytic CD4 T cells from multiple sclerosis patients

Research into the pathogenesis of multipe sclerosis (MS) has focused on myelin antigens as potential targets of autoimmune attack. Proteolipid protein (PLP), which makes up more than 50% of central nervous system myelin, is a hydrophilic membrane protein with many properties that historically have made it difficult to study. The use of synthetic peptides based on the PLP sequence provides an alternative method for studying the immunological properties of PLP. Using peripheral blood lymphocytes from MS patients, long-term TCL established in the presence of PLP reacted weakly to PLP in proliferation assays; however, these same lines were much more reactive to synthetic peptides of PLP. Thus, we established short-term T cell lines (TCL) from the peripheral blood lymphocytes (PBL) of MS patients in the presence of five separate synthetic PLP peptides. In six out of seven MS patients, proliferative responses were elicited most often to PLP 40–60 compared to four other PLP peptides (PLP 89–106, 103–120, 125–143, and 139–154). Characterization of PLP 40–60-responsive TCL from a single MS patient, MS1, indicated that six out of seven TCL proliferating to the peptide also lysed PLP 40–60 pulsed autologous targets. All cytolytic PLP 40–60 TCL were CD4⁺ and MHC class II restricted and further analysis of MS1 TCL showed that the PLP 40–60 TCL were restricted by DR4 whereas the MBP TCL from MS1 were restricted by DR6. These findings suggest that difficulties in examining the immune response to PLP have been due to the poor response generated in vitro using the whole molecule and that the use of synthetic peptides may represent an alternative approach to the study of PLP. These results also suggest that MS PBL recognize several PLP peptides, with the predominant response to PLP 40–60. Since these cells phenotypically resemble T cells known to mediate experimental autoimmune encephalomyelitis, it is possible that they may play a role in the pathogenesis of MS.