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.     

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。     

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.     

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。     

Specificity of regulatory CD4+CD25+ T cells for self-T cell receptor determinants

Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.     

CD4+CD25+FoxP3+ T lymphocytes fail to suppress myelin basic protein-induced proliferation in patients with multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disorder directed against self antigens of the central nervous system. CD4⁺CD25⁺FoxP3⁺ regulatory T cell (T_reg) mediated suppression is an essential mechanism of self-tolerance. We studied whether changes in the suppressive function of a mixture of CD25^high and CD25^intemediate expressing T_reg cells in myelin basic protein (MBP)-induced proliferation occurred in untreated MS patients. Suppression of MBP-induced proliferation was observed in 13 out of 29 (45%) MS patients; this was significantly (p < 0.05) less compared with 17 out of 19 (89%) healthy individuals. Relative T_reg counts was significantly increased in MS patients (mean ± S.D.; 20 ± 8%) compared with healthy individuals (15 ± 5%). These findings suggest that impaired T_reg function may be involved in pathogenesis of MS.     

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.     

Decreased FOXP3 levels in multiple sclerosis patients

Autoimmune diseases such as multiple sclerosis (MS) may result from the failure of tolerance mechanisms to prevent expansion of pathogenic T cells. Our study is the first to establish that MS patients have abnormalities in FOXP3 message and protein expression levels in peripheral CD4+ CD25+ T cells (Tregs) that are quantitatively related to a reduction in functional suppression induced during suboptimal T-cell receptor (TCR) ligation. Of importance, this observation links a defect in functional peripheral immunoregulation to an established genetic marker that has been unequivocally shown to be involved in maintaining immune tolerance and preventing autoimmune diseases. Diminished FOXP3 levels thus indicate impaired immunoregulation by Tregs that may contribute to MS. Future studies will evaluate the effects of therapies known to influence Treg cell function and FOXP3 expression, including TCR peptide vaccination and supplemental estrogen.     

Increased T cell expression of CD154 (CD40-ligand) in multiple sclerosis

CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients with secondary progressive MS (SPMS) had constitutive CD154 expression on CD4 and CD8 T cells in blood. Constitutive CD154 expression was not observed in patients with relapsing-remitting MS (RRMS) or clinically isolated syndromes (CIS) suggestive of demyelinating disease. After ex vivo activation CD154 was, however, expressed on a higher percentage of T cells from patients with CIS or RRMS than from healthy control subjects. These results suggest involvement of CD154 in the pathogenesis of MS, and the shift from a relapsing-remitting to a secondary progressive disease course may be associated with constitutive, systemic CD154 expression.     

重症肌无力患者外周血CD4+T细胞OX40的表达及作用机制研究

目的 分析重症肌无力(MG)患者外周血CD4+T细胞协同刺激分子OX40表达及其对FoxP3+CD4+CD25+调节性T细胞(Treg)的调控作用,初步探讨OX40在MG免疫学发病中的作用机制.方法 以流式细胞技术检测42例MG患者及38名健康对照的外周血OX40+CD4+T细胞、FoxP3+CD4+CD25+Treg表达水平,比较OX40表达在MG患者不同临床疾病状态、Osserman分型、临床绝对评分、胸腺病理类型等情况下的差异,并分析OX40对FoxP3+CD4+CD25+Treg细胞的影响.结果 (1) MG患者外周血OX40+CD4+T细胞占淋巴细胞百分比高于健康对照组(P<0.01).(2)MG患者OX40+CD4+T细胞百分比在发作或加重期高于缓解期(P＜0.05);在临床绝对评分呈中、重度患者OX40+CD4+T细胞百分比高于轻度患者(均P＜0.05);Osserman Ⅱ、Ⅳ型患者OX40+CD4+T细胞百分比高于Ⅰ型患者(均P＜0.05);胸腺增生及胸腺瘤患者OX40+CD4+T细胞百分比高于胸腺正常患者(P＜0.05,P＜0.01).(3)MG患者外周血OX40+CD4+T细胞百分比与FoxP3+CD4+CD25+Treg细胞百分比呈负相关(r=-0.843,P=0.01).结论 协同刺激分子OX40参与MG发病,可能通过抑制FoxP3+CD4+CD25+Treg细胞生成发挥作用.     

Increased T-lymphocyte interleukin-6 binding in patients with multiple sclerosis.

Multiple sclerosis (MS) represents a T-cell-mediated autoimmune demyelinating disease of the central nervous system associated with altered immunoregulation. Interleukin (IL)-6 is a cytokine that has several effects on the neuroimmune system. Specific IL-6 receptors have been found in human lymphocytes and neuroglial cells. The aim of the present study was to assay IL-6 binding on peripheral blood T lymphocytes in MS patients. We found that T cells from MS patients had significantly more IL-6 receptors [Bmax: 279 +/- 7 vs. 246 +/- 8 (mean +/- SEM) receptors/cell, in patients and controls, respectively], whereas Kd values were similar to those of healthy subjects [26.8 +/- 0.7 vs. 25.4 +/- 0.6 (mean +/- SEM) pM, in patients and controls, respectively]. Significant (P < 0.05) differences in IL-6 binding values were observed between stable patients and those relapsing (272 +/- 9 vs. 300 +/- 12 (mean +/- SEM) receptors/cell, respectively). We found significantly (P < 0. 001) higher amounts of IL-6 receptors on CD4+ T cells from MS patients than on CD4+ lymphocytes from controls (434 +/- 11 vs. 363 +/- 9 (mean +/- SEM) receptors/cell, respectively); CD8+ T cells showed very few IL-6 receptors in both patients and controls. These data are discussed in terms of MS immune pathogenesis and pathophysiology, because T-cell activation seems to be linked to increased IL-6 binding. The upregulated IL-6 system might be involved in antibody-mediated demyelinating pathways, because IL-6 is well known to enhance humoral immune response.     

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。     

Effect of high-dose methylprednisolone administration on immune functions in multiple sclerosis patients

Objectives – To investigate the in vivo effect of corticosteroid pulse therapy on immunocompetent cells in 18 patients given methylprednisolone to treat an acute episode of MS. Material and methods – Blood was sampled before and after 3 days of methylprednisolone administration at doses of 1 g/day. Lymphocyte subtyping was performed and whole blood cell cultures were used to measure the cytokine producing capacity for interleukin-1 (IL-1), interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-a (TNF-α) and interferon-α (IFN-α). In addition, serum levels of the immunoglobulin classes IgG, IgA and IgM were determined. Results – Before treatment, production of IL-1 was significantly increased in MS patients as compared to healthy controls. After therapy, production of all cytokines was significantly decreased, whereas there were significant increases in the numbers of monocytes, neutrophils and T and B lymphocytes. Treatment had no effect on serum immunoglobulin levels. Conclusion – An important mechanism for the antiinflammatory effect of corticosteroids in MS results from a suppression of the activation of the peripheral immune compartment through inhibition of cytokine production and lymphocyte endothelial adhesiveness.     

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.     

Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis

OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring their migration into the CNS.     

T cell subsets in multiple sclerosis: a serial study

The relevance of abnormalities in the distribution of peripheral blood T lymphocyte subsets to the clinical manifestations of multiple sclerosis is not firmly established. A clinical and immunological follow-up of relapsing-remitting multiple sclerosis patients was performed in order to study the relationship of immune changes with the clinical course of the disease. Twenty patients were monitored monthly during a mean time of nine months for peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD19), including the immunoregulatory subsets CD4CD29 (helper-inducer), and CD4CD45RA (suppressor-inducer) and activated T helper cells (CD4CD25) by flow cytometry. A total of 14 untreated relapses was included. The most significant observations were a decrease in T suppressor-inducer CD4⁺ CD45RA⁺ subset during clinical relapses ( P = 0.028) that was also detectable one month before ( P = 0.020) and the lack of changes in CD4⁺ CD29⁺ and CD8⁺ T cells. In addition, variations in the percentage of CD4⁺CD25⁺ activated T helper cells were not associated with clinical exacerbations. These results indicate the existence of a temporal association of immune changes in peripheral blood, but not activation, with the clinical manifestations of multiple sclerosis.     

Expression of costimulatory molecules on peripheral blood mononuclear cells in multiple sclerosis

OBJECTIVE: To determine whether there was aberrant expression of costimulatory molecules and their receptors on peripheral blood mononuclear cells of MS patients and healthy individuals and whether expression correlated with disease status. METHODS: Forty-six patients with MS and 29 healthy individuals were analyzed by direct 2-color immunofluorescence flow cytometry for expression of CD80, CD86, CD28 and CTLA-4 on T and B cells and monocytes. RESULTS: Expression of CD80 on CD4+ T cells was upregulated in progressing MS patients compared to stable MS patients and controls. Marked increase in the expression of CD80 and CD86 was seen on both CD4+ and CD8+ T cells from an MS patient with rapidly progressing disease. No difference in the expression of the costimulatory molecules or their ligands was seen between IFN-beta treated and non-treated MS patients. CONCLUSIONS: Aberrant expression of costimulatory molecules occurs on T lymphocytes in MS patients with progressing disease.     

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。     

CD4 and CD8 lymphocyte subsets in cerebrospinal fluid and peripheral blood from patients with multiple sclerosis, meningitis and normal controls

Objectives - To study the distribution of CD4⁺ and CD8⁺ T-cell subsets in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (MS), meningitis, other neurological diseases and healthy controls.
Material and methods - The expression of markers for naive and memory cells (CD45RA⁺ and CD45RO⁺), and helper/inducer cells (CD29⁺) on CD4⁺ cells as well as CD45RO⁺ and killer/effector (S6F1⁺) on CD8⁺ cells was investigated in cerebrospinal fluid (CSF) and peripheral blood from patients with multiple sclerosis (n=28), meningitis (n = 13), other neurological diseases (n = 16), and healthy controls (n = 16) by 2-color flow cytometry.
Results - The majority of T cells in the CSF of the 4 groups exhibited the phenotype of memory cells (CD45RO⁺) on both CD4⁺ and CD8⁺ cells. The proportion of helper/inducer (CD29TD4⁺ in CD4⁺) cells was also larger in the CSF compared to peripheral blood in the 3 patient groups and controls investigated. In contrast, CD8⁺ cells with killer/effector (S6F1+) phenotype were fewer in CSF compared to peripheral blood in all 4 groups. There were no significant differences between patients and controls regarding the distribution of these activation markers in the CSF or peripheral blood.
Conclusion - Our observations support the notion that activated T cells of both CD4⁺ and CD8⁺ phenotype selectively pass the blood-brain barrier under both pathological and normal conditions.