重症肌无力患者胸腺和外周血CD4～+CD25～+ Treg细胞数量和表型变化的研究
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摘要
背景调节性T细胞的概念首先是由Gershon等在1971年提出来的,但直到1995年Sakaguchi等在小鼠脾脏和淋巴结中发现了具有无能性和免疫抑制特性的CD4+CD25+ T细胞以后,调节性T细胞才引起了人们的重视。目前人们已经发现了多种具有调节作用的T细胞亚群,如Tr1细胞、Th3细胞、CD8+CD25+ T细胞、γδT细胞以及NKT细胞等,它们共同参与了外周自身免疫耐受的维持。越来越多的证据表明CD4+CD25+ Treg细胞在外周免疫耐受的维持中发挥着主要的作用,它的缺乏或功能缺陷与动物自身免疫性疾病的发生和发展密切相关。由于人类效应性T细胞(Responsive T cell, Tresp)在活化的早期也可中低水平表达CD25,因此目前人类Treg细胞的研究大都采用CD25hi作为细胞的标记物。现已发现人类外周血CD4+CD25hi细胞与多种自身免疫性疾病如系统性红斑狼疮、类风湿性关节炎及多发性硬化症等关系密切,但同时也有许多研究得出了相反的结论。重症肌无力(Myathenia gravis,MG)是一种典型的自身免疫性疾病,但目前关于人类MG Treg细胞的研究还比较少,并且结论也不一致。
最近的研究发现一些CD25呈中低表达(CD25low-int)的CD4+细胞也具有抑制功能,因此以CD25hi作为标记物就会遗漏许多CD4+CD25lo-int Treg细胞。在Treg细胞的研究中最重要的进展就是发现了Foxp3基因,它是目前最特异的nTreg细胞标记物。但由于Foxp3表达于细胞核内,其检测需要固定和破膜,因此不能用于鉴别和分选活性Treg细胞以进行体外功能检测。最近有研究发现CD25与CD27联合表达可以很好地区分风湿性关节炎滑液中的Treg细胞和活化的CD25+效应性T细胞,但目前尚未见用于其他人类自身免疫性疾病研究的报道。
胸腺切除是目前治疗MG的主要方法,虽然其疗效已经得到了公认,但确切机制尚不完全清楚。深入研究胸腺切除前后外周血Treg细胞和活化的Tresp细胞的变化,也有助于我们了解胸腺切除对MG外周免疫耐受的影响。
研究已发现IL-6可以通过阻断Treg细胞的抑制作用来促进Tresp细胞的活化和功能,并且MG外周血中IL-6、IL-2及TNF-a等细胞因子水平升高。因此分析它们在胸腺切除前后外周血中的变化,可能有助于了解手术前后MG Treg细胞和自身反应性T细胞的活动状态。
方法1、采用免疫组化染色方法同时检测MG胸腺组织中nTreg细胞标分子CD25和Foxp3的阳性表达及分布情况。2、通过流式细胞技术检测MG患者和正常人胸腺组织中CD4+ CD25+、CD4+ CD25hi、CD4+ CD25+ CD27+和CD4+ CD25+CD27- T细胞的数量及其功能表型Foxp3、CTLA-4、GITR和CD62L的表达。3、通过流式细胞技术检测正常对照和MG患者术前外周血中CD4+ CD25+、CD4+ CD25hi、CD4+ CD25+ CD27+ T细胞的表型特点和数量变化以及CD4+ CD25+CD27-T细胞的数量变化,同时分析上述细胞亚群数量变化与手术前后QMG评分的关系;通过CBA流式蛋白分析系统检测胸腺切除前后外周血IL-6、IL-2及TNF-a水平,并分析它们与各细胞亚群和QMG的关系。
结果1、CD25+和Foxp3+细胞在正常胸腺中主要分布于胸腺的髓质和血管周围纤维间隔区,皮质中也有少量分布;在增生胸腺的淋巴滤泡内也有分布并围绕在生发中心的周围,髓质胸腺小体的周围也有CD25+和Foxp3+细胞环绕。2、MG胸腺CD25阳性表达明显高于正常胸腺(P<0.05),Foxp3表达明显低于正常胸腺(P<0.05),同时各MGFA分型中CD25和Foxp3的表达也明显不同(P<0.05,P<0.01)。3、与对照相比,MG胸腺中CD4+CD25+细胞的比例明显增加(P<0.05),CD4+CD25hi细胞的比例无明显变化(P>0.05)。4、MG胸腺CD4+CD25+、CD4+CD25hi细胞亚群中Foxp3+和CTLA-4+T细胞的比例与对照相比均明显降低(P<0.01,P<0.05),CD62L+和GITR+细胞的比例无明显差异(P>0.05) ;MG和正常胸腺CD4+CD25+T细胞中的Foxp3+和CTLA-4+ T细胞的比例都显著低于CD4+CD25hi T细胞中的比例(P<0.01),GITR+T细胞的比例高于CD4+CD25hi细胞(P<0.01),CD62L+T细胞的比例未见显著性差异。5、MG胸腺与正常胸腺相比,CD4+CD25+CD27+T细胞的比例显著下降(P<0.05),CD4+CD25+CD27-细胞的比例显著增加(P<0.01);在对照胸腺和MG胸腺中,CD4+CD25+亚群中CD27+的比例都明显低于CD4+ CD25hi亚群(P<0.001),CD27-细胞的比例都明显高于CD4+ CD25hi亚群(P<0.001)。6、MG胸腺CD4+ CD25+ CD27+、CD4+ CD25+ CD27- T细胞亚群中Foxp3+、CTLA-4+、GITR+和CD62L+ T细胞的比例与对照胸腺相比,均无明显差异(P>0.05);对照胸腺和MG胸腺CD4+CD25+ CD27+T细胞中Foxp3+、CTLA-4+T细胞的比例均明显高于CD4+ CD25+ CD27- T细胞中的比例(P<0.001),GITR+T细胞和CD62L+T细胞的比例在上述各组别之间未见显著性差异(P>0.05)。7、MG患者术前外周血CD4+CD25+和CD4+CD25hi T细胞的比例与健康对照相比均无明显差异(P>0.05),胸腺切除前后也无明显改变(P>0.05)。8、MG患者术前外周血CD4+CD25+细胞中Foxp3+的比例明显低于正常对照(P<0.05),CTLA-4+、GITR+及CD62L+的比例无明显差异(P>0.05); CD4+CD25hi细胞中Foxp3+、CTLA-4+、GITR+及CD62L+的比例与正常对照相比都无显著性差异(P>0.05)。9、MG患者术前外周血CD4+CD25+CD27+细胞中Foxp3+、CTLA-4+、GITR+、CD62L+的比例与正常对照相比无显著差异(P>0.05)。10、MG外周血CD4+CD25+CD27+细胞的比例与正常对照相比无明显差异(P>0.05),手术前后也无明显变化(P>0.05);CD4+ CD25+ CD27- T细胞的比例与对照相比明显升高(P<0.01),术后较术前稍有下降(P>0.05);CD25+ CD27+ /CD25+ CD27-比值(RCD27)与对照相比明显降低(P<0.01),术后较术前明显升高(P<0.05)。11、MG患者术前外周血IL-2、IL-6和TNF-a的水平与正常对照相比明显升高(P<0.05, P<0.01,P<0.05)。术后IL-6水平与术前相比下降明显(P<0.05),但与对照相比仍明显升高(P<0.05);术后IL-2和TNF-a与术前相比下降都不明显(P>0.05),IL-2仍明显高于对照(P<0.05),但TNF-a与对照相比无明显差异(P>0.05)。12、术后溴吡啶斯的明用量较术前明显减少(P<0.05), QMG评分明显下降(P<0.01)。13、术前CD25+CD27-细胞的比例与QMG评分呈正相关(R=0.723,P<0.01),与血中IL-6水平呈正相关(R=0.582,P<0.05)。术前RCD27值与QMG评分、手术前后QMG评分下降幅度及IL-6水平均呈负相关(R=-0.896, P<0.001; R=-0.786,P<0.001;R=-0.975,P<0.001),术后Rcd27值与QMG呈显著负相关(r=-0.939,p<0.01),但与手术前后QMG评分下降幅度无关(P>0.05)。外周血中CD4+CD25+CD27+细胞的比例与QMG评分无显著相关性(P>0.05)。
结论1、在胸腺中,并不是所有的CD4+CD25+T细胞都是调节性T细胞。MG胸腺内nTreg细胞的数量下降,活化的自身反应性T细胞大量增加,在MG胸腺内确实存在着免疫调节的失控。2、采用CD25hi作为Treg细胞标记物是导致目前MG患者外周血Treg细胞研究中结论相互矛盾的主要原因。3、CD27与CD25联合表达可以很好地区分CD4+CD25+Treg细胞和活化早期的CD25+自身反应性T细胞,可用于MG胸腺和外周血Treg细胞群整体抑制功能体外评价研究中的细胞分选。4、MG患者外周血Treg细胞的数量没有明显的改变,Treg细胞对自身反应性T细胞的抑制功能的下降是导致外周血中免疫调节失控的原因。5、外周血IL-6的水平与CD25+ CD27-的比例、RCD27值及QMG评分明显相关,IL-6水平的升高是外周血Treg细胞抑制作用下降的重要原因之一。6、胸腺切除对外周血Treg细胞的比例无影响,但可以降低外周血中活化的自身反应性T细胞的数量,降低IL-6水平,恢复Treg细胞与自身反应性T细胞的动态平衡。7、MG患者术前外周血RCD27值与手术前后QMG评分下降的幅度高度相关,术前检测外周血RCD27值对胸腺切除的预后判断有一定的意义。
Backgroud The concept of regulatory T cells(Tregs) was first proposed by Gershon in 1971. No attention was payed on it till 1995, when CD4+CD25+ T cells with anergy and immunoregulatory function were found in the spleen and lymphonodes of rats by Sakaguchi and his colleagues. Up to now, researchers have founded many kinds of Tregs, such as Tr1, Th3,CD8+ CD25+ T cell,γδT cell , NKT cell, etc. These Tregs together take part in the maintance of peripheral immune tolerance. More and more evidences shows that CD4+CD25+ Tregs are the key controllers in the peripheral immunological self-tolerance, and their quantitative deficiency or the functional down-regulation had significant correlations with the onset and persistance of autoimmune diseases in animal models. Now the CD4+CD25+ Tregs are called natural regulatory T cells (nTregs) so as to be differentiated from other Tregs. Because the human responsive T cells could also express CD25 in the early stage of activation ,CD25hi is used as the marker molecule in most human Treg cell Researches now. Unfortunately, while some researches got the conclusion that the deficiency of peripheral CD4+CD25hi Treg cells had close relationships with the onsets of many kinds of human autoimmune diseases , such as systemic lupus erythematous , rehumatoid arthritis and multiple sclerosis etc, there are still some other researches with contradictory conclusions exist. Although myasthenia gravis (MG) is a typical autoimmune disease, few studies have been down on the roles of CD4+CD25hi Treg cells in the onset and progression of MG so far and the conclusions of them are not in accordance either. As the CD4+ T cells with low-intermediate expression of CD25 (CD25low-int) also have immuno-suppressive function in recent researches, a lot of CD4+CD25low-int Treg cells might be lost in the studies using CD25hi as the marker of Treg cells. Then, the usage of CD25hi as the marker molecule might be the main reason of the inconsistent conclusions in the studies of human Tregs. The most important progress in the research of Tregs is the discovery of Foxp3, which is the most specific marker molecule for Tregs up to date. Hower, Foxp3 can’t be used to isolate viable Treg cells for functional study because of its intranuclear expression and the detection of it requires fixation and permeabilization. Latest research sugested that the coexpression of CD25 and CD27 could effectively differentiate Treg cells from activated CD25+ Tresp cells in synovia fluid of rheumatoid arthritis. Hower, this method has not been used in other researches on human autoimmune disease till now.
Although thymectomy (Tx) is a current treatment for MG and its effect has been recognized Nowdays, the exact mechnism of this method is still unclear. Then, futher researches on the changes of Treg and Tresp cells pre- and post-thymectomy might help us find out the influences of Tx on the peripheral autoimmune tolerance in MG.
Studies have showed that the peripheral blood levels of IL-6, IL-2 and TNF-a were higher in MG, IL-6 could enhance the activity of Tresp cells by blocking the supressive function of Tregs. Then, the study of the changes of their levels pre-TX and post-Tx might help us understand the functional state of MG Treg and Tresp cells.
Mthods 1. The detection of CD25 and Foxp3 expression in normal and MG thymus via immunohistological staining. 2. The detection of frequencies of CD4+ CD25+ , CD4+ CD25hi , CD4+ CD25+ CD27+ and CD4+ CD25+CD27- T cell and the expression of Foxp3, CTLA-4, GITR and CD62L in the herein before cell subgroups by FCM; 3. The detection of the frequencies CD4+CD25+, CD4+CD25hi , CD4+CD25+CD27+ and CD4+ CD25+CD27- T cell groups in the peripheral blood of normal controls , per-Tx and post-Tx MG patients via FCM; The detection of the expression of Foxp3, CTLA-4, GITR and CD62L in the herein before cell subgroups by FCM; The detection of IL-6, IL-2 and TNF-a in the peripheral blood of normal controls , pre-Tx and post-Tx MG patients via cytometric bead array system (CBA); The analysis of the relationships between QMG and the hereinbefore T cell subgroups, IL-6, IL-2 and TNF-a in pre-Tx and post-Tx MG patients.
Results 1. The CD25+ and Foxp3+ cells in MG and normal thymus were mainly located in the vascular septa area of medulla and a few were located in the cortex. In MG thymus, the CD25+ and Foxp3+ cells could also be found in the lymphoid follicles, surrounding the germinal centers and hassall corpuscles. 2. The expression of CD25 was significantly higher and the expression of Foxp3 was significantly lower in MG thymus than that in control tymus respectively(P<0.05). In addition, the expressions of CD25 and Foxp3 were both different between MGFA classes (P<0.05, P<0.01). 3. The percetages of CD4+CD25+ cells were signifacantly increased in MG thymus compared with control thymus (P<0.05), but there was no significant difference in the percentage of CD4+CD25hi cells between MG and control thymus (P>0.05). 4. The percentages of Foxp3+ and CTLA-4+ cells within the CD4+CD25+ subgroup of MG thymus were both significantly lower than that within the CD4+CD25+ subgroup of control thymus respctively (P<0.05,P<0.01); The percentages of Foxp3+ and CTLA-4+ cells with the CD4+CD25hi subgroup were also significantly decreased in MG thymus than controls thymus (P<0.05,P<0.01); the percentages of CD62L+ and GITR+ cells within the CD4+CD25+ /CD4+CD25hi subgroup have no significant difference between MG and control thymus (P>0.05). Both in control and MG thymus, the percentages of Foxp3+ and CTLA-4+ cells within the CD4+CD25hi subgroup were significantly higher than that within the CD4+CD25+ subgroup(P<0.01), the percentages of GITR+ cells within the CD4+CD25hi subgroup were significantly lower than that within the CD4+CD25+ subgroup(P<0.01)and there’s no significant difference in the percentage of CD62L+ cells between the hereinbefore cell subgroups was found(P>0.05). 5. The percentages of CD4+CD25+CD27+ cells were significantly lower (P<0.05) and the percentages of CD4+CD25+CD27- cells were significantly higher (P<0.01) in MG thymus compared with their counterparts in control thymus respectively. Both in control and MG thymus, the percentages of CD27+ cells were greatly decreased(P<0.001) and the percentages of CD27- cells were greatly increased (P<0.001) within the CD4+CD25+ subgroup than that within the CD4+CD25hi subgroup. 6. There was no significant difference in the percentages of Foxp3+, CTLA-4+, CD62L+ and GITR+ cells had been found between control and MG thymus not only in the CD4+CD25+CD27+ subgroups but also in the CD4+CD25+CD27- subgroups (P>0.05). Both in control and MG thymus, the percentages of Foxp3+ and CTLA-4+ cells within the CD4+CD25+CD27+ subgroups were greatly higher than that within the CD4+CD25+CD27- subgroups (P<0.001) and there’s no difference in the percentages of CD62L+ and GITR+ cells within these two subgroups respectvely(P>0.05). 7. There’s no significant difference in the percentages of both peripheral CD4+CD25+ and CD4+CD25hi cells between pre-Tx MG patients and controls (P>0.05), and, there’s no significant difference had been found in the percentages between pre-Tx and post-Tx either (P>0.05). 8. In peripheral blood, the percentages of Foxp3+ cells within the CD4+CD25+ groups of pre-Tx MG patients were significantly lower than that of controls (P<0.05), and no significant difference had been found in the percentages of CTLA-4+, CD62L+ and GITR+ cells within the CD4+CD25+ groups between pre-Tx MG patients and controls (P>0.05); There was no difference in the percentages of Foxp3+, CTLA-4+, CD62L+ and GITR+ cells within the CD4+CD25hi subgroups between the pre-Tx MG patients and controls (P>0.05). 9. The expression of Foxp3, CTLA-4, CD62L and GITR in peripheral CD4+CD25+CD27+ subgroups of MG patients had no signifecant difference compared with controls (P>0.05). 10. There was no significant difference in the percentages of peripheral CD4+CD25+CD27+ cells between MG patients pre-Tx and controls (P>0.05), and no evident difference had been found in MG patients pre- and post-Tx either; The percentages of peripheral CD4+CD25+CD27- cells were greatly increased in MG patients pre-Tx compared with that in controls (P<0.01), while there was only a slight decrease in patients post-Tx compared with that in patients pre-Tx (P>0.05); The propotions of peripheral CD25+CD27+/CD25+CD27- (RCD27) in MG patients pre-Tx were significant lower than that in controls (P<0.01), and increased evidently after thymectomy in MG patients (P<0.05). 11. The levels of IL-2, IL-6 and TNF-a in the peripheral blood of MG patients pre-Tx were all higher than that of controls (P<0.05, P<0.01,P<0.05). The level of peripheral IL-6 decrased greatly in MG patients post-Tx compared with pre-Tx (P<0.05), but it was still higher than that in normal controls (P<0.05). There was no significant difference in the levels of IL-2 and TNF-a between the post-Tx and pre-Tx MG patients (P>0.05), but the difference of TNF-a level between the the post-Tx patients and the controls failed to achieve stastistical significance (P>0.05). 12. The dosage of pyridostigmin bromide decreased significantly after thymectomy (P<0.05), and the Quantative MG score (QMG) also significantly decreased (P<0.01). 13. The percentages of peripheral CD4+CD25+CD27- cells in patients pre-Tx were significantly correlated with the QMG scores (R= 0.723,P< 0.01), IL-6 level (R= 0.582,P< 0.05) and TNF-a level (R= - 0.675,P< 0.01); RCD27 in patients pre-Tx was also correlated significantly with the QMG score pre-Tx (R=-0.896, P<0.001), the difference value of QMG between pre-Tx and post-Tx (R=-0.786,P<0.001), and the peripheral IL-6 level of pre-Tx (R=-0.975,P<0.001). RCD27 in patients post-Tx was correlated significantly with the QMG score post-Tx (r= - 0.939,p<0.01), but was not significantly correlated with the difference value of QMG between pre-Tx and post-Tx (P>0.05). There’s no significant correlation between the percentages of peripheral CD4+CD25+CD27+ cells and the QMG scores (P>0.05).
Conclusions 1. CD4+CD25+ T cells within the thymus are not all Treg cells, some of them are autoreactive T cells in early active stage. The number of intrathymic CD4+CD25+ Treg cells is lower and the number of actvive autoreactive T cells is higher in MG patients , and there’s realy a disturbance of immune regulation in the MG thymus. 2. the usage of CD25hi as the marker molecule is the main reason for the inconsistent conclusions in the studies of human Tregs. 3. the coexpression of CD25 and CD27 can effectively differentiate Treg cells from early activated CD25+ Tresp cells and can be used to isolate viable Treg cell populations for functional studies. 4. There’s no quantitative deficiency of Treg cells in the peripheral blood of MG patients. The founctional defect is the main mechanism of the imbalance between Tregs and autoreactive T cells in the blood. 5. The peripheral level of IL-6 has a significant relationship with the percentage of CD25+ CD27- cells, RCD27, and QMG score. The increase of peripheral IL-6 is one of the crucial reasons for the functional down-regulation of Tregs in MG blood. 6. Thymectomy has no effect on the percentage of Tregs,but can reduce the percentage of active autoreactive cells and the IL-6 level in the peripheral blood , and recover the dynamic balance between the Tregs and active autoreactive T cells. 7. The RCD27 in the MG patients pre-Tx has a significant correlation with the decrease of QMG score after thymecomy, then, the detection of RCD27 pre-Tx might help us estimate the prognosis of thymectomy in MG patients.
最近的研究发现一些CD25呈中低表达(CD25low-int)的CD4+细胞也具有抑制功能,因此以CD25hi作为标记物就会遗漏许多CD4+CD25lo-int Treg细胞。在Treg细胞的研究中最重要的进展就是发现了Foxp3基因,它是目前最特异的nTreg细胞标记物。但由于Foxp3表达于细胞核内,其检测需要固定和破膜,因此不能用于鉴别和分选活性Treg细胞以进行体外功能检测。最近有研究发现CD25与CD27联合表达可以很好地区分风湿性关节炎滑液中的Treg细胞和活化的CD25+效应性T细胞,但目前尚未见用于其他人类自身免疫性疾病研究的报道。
胸腺切除是目前治疗MG的主要方法,虽然其疗效已经得到了公认,但确切机制尚不完全清楚。深入研究胸腺切除前后外周血Treg细胞和活化的Tresp细胞的变化,也有助于我们了解胸腺切除对MG外周免疫耐受的影响。
研究已发现IL-6可以通过阻断Treg细胞的抑制作用来促进Tresp细胞的活化和功能,并且MG外周血中IL-6、IL-2及TNF-a等细胞因子水平升高。因此分析它们在胸腺切除前后外周血中的变化,可能有助于了解手术前后MG Treg细胞和自身反应性T细胞的活动状态。
方法1、采用免疫组化染色方法同时检测MG胸腺组织中nTreg细胞标分子CD25和Foxp3的阳性表达及分布情况。2、通过流式细胞技术检测MG患者和正常人胸腺组织中CD4+ CD25+、CD4+ CD25hi、CD4+ CD25+ CD27+和CD4+ CD25+CD27- T细胞的数量及其功能表型Foxp3、CTLA-4、GITR和CD62L的表达。3、通过流式细胞技术检测正常对照和MG患者术前外周血中CD4+ CD25+、CD4+ CD25hi、CD4+ CD25+ CD27+ T细胞的表型特点和数量变化以及CD4+ CD25+CD27-T细胞的数量变化,同时分析上述细胞亚群数量变化与手术前后QMG评分的关系;通过CBA流式蛋白分析系统检测胸腺切除前后外周血IL-6、IL-2及TNF-a水平,并分析它们与各细胞亚群和QMG的关系。
结果1、CD25+和Foxp3+细胞在正常胸腺中主要分布于胸腺的髓质和血管周围纤维间隔区,皮质中也有少量分布;在增生胸腺的淋巴滤泡内也有分布并围绕在生发中心的周围,髓质胸腺小体的周围也有CD25+和Foxp3+细胞环绕。2、MG胸腺CD25阳性表达明显高于正常胸腺(P<0.05),Foxp3表达明显低于正常胸腺(P<0.05),同时各MGFA分型中CD25和Foxp3的表达也明显不同(P<0.05,P<0.01)。3、与对照相比,MG胸腺中CD4+CD25+细胞的比例明显增加(P<0.05),CD4+CD25hi细胞的比例无明显变化(P>0.05)。4、MG胸腺CD4+CD25+、CD4+CD25hi细胞亚群中Foxp3+和CTLA-4+T细胞的比例与对照相比均明显降低(P<0.01,P<0.05),CD62L+和GITR+细胞的比例无明显差异(P>0.05) ;MG和正常胸腺CD4+CD25+T细胞中的Foxp3+和CTLA-4+ T细胞的比例都显著低于CD4+CD25hi T细胞中的比例(P<0.01),GITR+T细胞的比例高于CD4+CD25hi细胞(P<0.01),CD62L+T细胞的比例未见显著性差异。5、MG胸腺与正常胸腺相比,CD4+CD25+CD27+T细胞的比例显著下降(P<0.05),CD4+CD25+CD27-细胞的比例显著增加(P<0.01);在对照胸腺和MG胸腺中,CD4+CD25+亚群中CD27+的比例都明显低于CD4+ CD25hi亚群(P<0.001),CD27-细胞的比例都明显高于CD4+ CD25hi亚群(P<0.001)。6、MG胸腺CD4+ CD25+ CD27+、CD4+ CD25+ CD27- T细胞亚群中Foxp3+、CTLA-4+、GITR+和CD62L+ T细胞的比例与对照胸腺相比,均无明显差异(P>0.05);对照胸腺和MG胸腺CD4+CD25+ CD27+T细胞中Foxp3+、CTLA-4+T细胞的比例均明显高于CD4+ CD25+ CD27- T细胞中的比例(P<0.001),GITR+T细胞和CD62L+T细胞的比例在上述各组别之间未见显著性差异(P>0.05)。7、MG患者术前外周血CD4+CD25+和CD4+CD25hi T细胞的比例与健康对照相比均无明显差异(P>0.05),胸腺切除前后也无明显改变(P>0.05)。8、MG患者术前外周血CD4+CD25+细胞中Foxp3+的比例明显低于正常对照(P<0.05),CTLA-4+、GITR+及CD62L+的比例无明显差异(P>0.05); CD4+CD25hi细胞中Foxp3+、CTLA-4+、GITR+及CD62L+的比例与正常对照相比都无显著性差异(P>0.05)。9、MG患者术前外周血CD4+CD25+CD27+细胞中Foxp3+、CTLA-4+、GITR+、CD62L+的比例与正常对照相比无显著差异(P>0.05)。10、MG外周血CD4+CD25+CD27+细胞的比例与正常对照相比无明显差异(P>0.05),手术前后也无明显变化(P>0.05);CD4+ CD25+ CD27- T细胞的比例与对照相比明显升高(P<0.01),术后较术前稍有下降(P>0.05);CD25+ CD27+ /CD25+ CD27-比值(RCD27)与对照相比明显降低(P<0.01),术后较术前明显升高(P<0.05)。11、MG患者术前外周血IL-2、IL-6和TNF-a的水平与正常对照相比明显升高(P<0.05, P<0.01,P<0.05)。术后IL-6水平与术前相比下降明显(P<0.05),但与对照相比仍明显升高(P<0.05);术后IL-2和TNF-a与术前相比下降都不明显(P>0.05),IL-2仍明显高于对照(P<0.05),但TNF-a与对照相比无明显差异(P>0.05)。12、术后溴吡啶斯的明用量较术前明显减少(P<0.05), QMG评分明显下降(P<0.01)。13、术前CD25+CD27-细胞的比例与QMG评分呈正相关(R=0.723,P<0.01),与血中IL-6水平呈正相关(R=0.582,P<0.05)。术前RCD27值与QMG评分、手术前后QMG评分下降幅度及IL-6水平均呈负相关(R=-0.896, P<0.001; R=-0.786,P<0.001;R=-0.975,P<0.001),术后Rcd27值与QMG呈显著负相关(r=-0.939,p<0.01),但与手术前后QMG评分下降幅度无关(P>0.05)。外周血中CD4+CD25+CD27+细胞的比例与QMG评分无显著相关性(P>0.05)。
结论1、在胸腺中,并不是所有的CD4+CD25+T细胞都是调节性T细胞。MG胸腺内nTreg细胞的数量下降,活化的自身反应性T细胞大量增加,在MG胸腺内确实存在着免疫调节的失控。2、采用CD25hi作为Treg细胞标记物是导致目前MG患者外周血Treg细胞研究中结论相互矛盾的主要原因。3、CD27与CD25联合表达可以很好地区分CD4+CD25+Treg细胞和活化早期的CD25+自身反应性T细胞,可用于MG胸腺和外周血Treg细胞群整体抑制功能体外评价研究中的细胞分选。4、MG患者外周血Treg细胞的数量没有明显的改变,Treg细胞对自身反应性T细胞的抑制功能的下降是导致外周血中免疫调节失控的原因。5、外周血IL-6的水平与CD25+ CD27-的比例、RCD27值及QMG评分明显相关,IL-6水平的升高是外周血Treg细胞抑制作用下降的重要原因之一。6、胸腺切除对外周血Treg细胞的比例无影响,但可以降低外周血中活化的自身反应性T细胞的数量,降低IL-6水平,恢复Treg细胞与自身反应性T细胞的动态平衡。7、MG患者术前外周血RCD27值与手术前后QMG评分下降的幅度高度相关,术前检测外周血RCD27值对胸腺切除的预后判断有一定的意义。
Backgroud The concept of regulatory T cells(Tregs) was first proposed by Gershon in 1971. No attention was payed on it till 1995, when CD4+CD25+ T cells with anergy and immunoregulatory function were found in the spleen and lymphonodes of rats by Sakaguchi and his colleagues. Up to now, researchers have founded many kinds of Tregs, such as Tr1, Th3,CD8+ CD25+ T cell,γδT cell , NKT cell, etc. These Tregs together take part in the maintance of peripheral immune tolerance. More and more evidences shows that CD4+CD25+ Tregs are the key controllers in the peripheral immunological self-tolerance, and their quantitative deficiency or the functional down-regulation had significant correlations with the onset and persistance of autoimmune diseases in animal models. Now the CD4+CD25+ Tregs are called natural regulatory T cells (nTregs) so as to be differentiated from other Tregs. Because the human responsive T cells could also express CD25 in the early stage of activation ,CD25hi is used as the marker molecule in most human Treg cell Researches now. Unfortunately, while some researches got the conclusion that the deficiency of peripheral CD4+CD25hi Treg cells had close relationships with the onsets of many kinds of human autoimmune diseases , such as systemic lupus erythematous , rehumatoid arthritis and multiple sclerosis etc, there are still some other researches with contradictory conclusions exist. Although myasthenia gravis (MG) is a typical autoimmune disease, few studies have been down on the roles of CD4+CD25hi Treg cells in the onset and progression of MG so far and the conclusions of them are not in accordance either. As the CD4+ T cells with low-intermediate expression of CD25 (CD25low-int) also have immuno-suppressive function in recent researches, a lot of CD4+CD25low-int Treg cells might be lost in the studies using CD25hi as the marker of Treg cells. Then, the usage of CD25hi as the marker molecule might be the main reason of the inconsistent conclusions in the studies of human Tregs. The most important progress in the research of Tregs is the discovery of Foxp3, which is the most specific marker molecule for Tregs up to date. Hower, Foxp3 can’t be used to isolate viable Treg cells for functional study because of its intranuclear expression and the detection of it requires fixation and permeabilization. Latest research sugested that the coexpression of CD25 and CD27 could effectively differentiate Treg cells from activated CD25+ Tresp cells in synovia fluid of rheumatoid arthritis. Hower, this method has not been used in other researches on human autoimmune disease till now.
Although thymectomy (Tx) is a current treatment for MG and its effect has been recognized Nowdays, the exact mechnism of this method is still unclear. Then, futher researches on the changes of Treg and Tresp cells pre- and post-thymectomy might help us find out the influences of Tx on the peripheral autoimmune tolerance in MG.
Studies have showed that the peripheral blood levels of IL-6, IL-2 and TNF-a were higher in MG, IL-6 could enhance the activity of Tresp cells by blocking the supressive function of Tregs. Then, the study of the changes of their levels pre-TX and post-Tx might help us understand the functional state of MG Treg and Tresp cells.
Mthods 1. The detection of CD25 and Foxp3 expression in normal and MG thymus via immunohistological staining. 2. The detection of frequencies of CD4+ CD25+ , CD4+ CD25hi , CD4+ CD25+ CD27+ and CD4+ CD25+CD27- T cell and the expression of Foxp3, CTLA-4, GITR and CD62L in the herein before cell subgroups by FCM; 3. The detection of the frequencies CD4+CD25+, CD4+CD25hi , CD4+CD25+CD27+ and CD4+ CD25+CD27- T cell groups in the peripheral blood of normal controls , per-Tx and post-Tx MG patients via FCM; The detection of the expression of Foxp3, CTLA-4, GITR and CD62L in the herein before cell subgroups by FCM; The detection of IL-6, IL-2 and TNF-a in the peripheral blood of normal controls , pre-Tx and post-Tx MG patients via cytometric bead array system (CBA); The analysis of the relationships between QMG and the hereinbefore T cell subgroups, IL-6, IL-2 and TNF-a in pre-Tx and post-Tx MG patients.
Results 1. The CD25+ and Foxp3+ cells in MG and normal thymus were mainly located in the vascular septa area of medulla and a few were located in the cortex. In MG thymus, the CD25+ and Foxp3+ cells could also be found in the lymphoid follicles, surrounding the germinal centers and hassall corpuscles. 2. The expression of CD25 was significantly higher and the expression of Foxp3 was significantly lower in MG thymus than that in control tymus respectively(P<0.05). In addition, the expressions of CD25 and Foxp3 were both different between MGFA classes (P<0.05, P<0.01). 3. The percetages of CD4+CD25+ cells were signifacantly increased in MG thymus compared with control thymus (P<0.05), but there was no significant difference in the percentage of CD4+CD25hi cells between MG and control thymus (P>0.05). 4. The percentages of Foxp3+ and CTLA-4+ cells within the CD4+CD25+ subgroup of MG thymus were both significantly lower than that within the CD4+CD25+ subgroup of control thymus respctively (P<0.05,P<0.01); The percentages of Foxp3+ and CTLA-4+ cells with the CD4+CD25hi subgroup were also significantly decreased in MG thymus than controls thymus (P<0.05,P<0.01); the percentages of CD62L+ and GITR+ cells within the CD4+CD25+ /CD4+CD25hi subgroup have no significant difference between MG and control thymus (P>0.05). Both in control and MG thymus, the percentages of Foxp3+ and CTLA-4+ cells within the CD4+CD25hi subgroup were significantly higher than that within the CD4+CD25+ subgroup(P<0.01), the percentages of GITR+ cells within the CD4+CD25hi subgroup were significantly lower than that within the CD4+CD25+ subgroup(P<0.01)and there’s no significant difference in the percentage of CD62L+ cells between the hereinbefore cell subgroups was found(P>0.05). 5. The percentages of CD4+CD25+CD27+ cells were significantly lower (P<0.05) and the percentages of CD4+CD25+CD27- cells were significantly higher (P<0.01) in MG thymus compared with their counterparts in control thymus respectively. Both in control and MG thymus, the percentages of CD27+ cells were greatly decreased(P<0.001) and the percentages of CD27- cells were greatly increased (P<0.001) within the CD4+CD25+ subgroup than that within the CD4+CD25hi subgroup. 6. There was no significant difference in the percentages of Foxp3+, CTLA-4+, CD62L+ and GITR+ cells had been found between control and MG thymus not only in the CD4+CD25+CD27+ subgroups but also in the CD4+CD25+CD27- subgroups (P>0.05). Both in control and MG thymus, the percentages of Foxp3+ and CTLA-4+ cells within the CD4+CD25+CD27+ subgroups were greatly higher than that within the CD4+CD25+CD27- subgroups (P<0.001) and there’s no difference in the percentages of CD62L+ and GITR+ cells within these two subgroups respectvely(P>0.05). 7. There’s no significant difference in the percentages of both peripheral CD4+CD25+ and CD4+CD25hi cells between pre-Tx MG patients and controls (P>0.05), and, there’s no significant difference had been found in the percentages between pre-Tx and post-Tx either (P>0.05). 8. In peripheral blood, the percentages of Foxp3+ cells within the CD4+CD25+ groups of pre-Tx MG patients were significantly lower than that of controls (P<0.05), and no significant difference had been found in the percentages of CTLA-4+, CD62L+ and GITR+ cells within the CD4+CD25+ groups between pre-Tx MG patients and controls (P>0.05); There was no difference in the percentages of Foxp3+, CTLA-4+, CD62L+ and GITR+ cells within the CD4+CD25hi subgroups between the pre-Tx MG patients and controls (P>0.05). 9. The expression of Foxp3, CTLA-4, CD62L and GITR in peripheral CD4+CD25+CD27+ subgroups of MG patients had no signifecant difference compared with controls (P>0.05). 10. There was no significant difference in the percentages of peripheral CD4+CD25+CD27+ cells between MG patients pre-Tx and controls (P>0.05), and no evident difference had been found in MG patients pre- and post-Tx either; The percentages of peripheral CD4+CD25+CD27- cells were greatly increased in MG patients pre-Tx compared with that in controls (P<0.01), while there was only a slight decrease in patients post-Tx compared with that in patients pre-Tx (P>0.05); The propotions of peripheral CD25+CD27+/CD25+CD27- (RCD27) in MG patients pre-Tx were significant lower than that in controls (P<0.01), and increased evidently after thymectomy in MG patients (P<0.05). 11. The levels of IL-2, IL-6 and TNF-a in the peripheral blood of MG patients pre-Tx were all higher than that of controls (P<0.05, P<0.01,P<0.05). The level of peripheral IL-6 decrased greatly in MG patients post-Tx compared with pre-Tx (P<0.05), but it was still higher than that in normal controls (P<0.05). There was no significant difference in the levels of IL-2 and TNF-a between the post-Tx and pre-Tx MG patients (P>0.05), but the difference of TNF-a level between the the post-Tx patients and the controls failed to achieve stastistical significance (P>0.05). 12. The dosage of pyridostigmin bromide decreased significantly after thymectomy (P<0.05), and the Quantative MG score (QMG) also significantly decreased (P<0.01). 13. The percentages of peripheral CD4+CD25+CD27- cells in patients pre-Tx were significantly correlated with the QMG scores (R= 0.723,P< 0.01), IL-6 level (R= 0.582,P< 0.05) and TNF-a level (R= - 0.675,P< 0.01); RCD27 in patients pre-Tx was also correlated significantly with the QMG score pre-Tx (R=-0.896, P<0.001), the difference value of QMG between pre-Tx and post-Tx (R=-0.786,P<0.001), and the peripheral IL-6 level of pre-Tx (R=-0.975,P<0.001). RCD27 in patients post-Tx was correlated significantly with the QMG score post-Tx (r= - 0.939,p<0.01), but was not significantly correlated with the difference value of QMG between pre-Tx and post-Tx (P>0.05). There’s no significant correlation between the percentages of peripheral CD4+CD25+CD27+ cells and the QMG scores (P>0.05).
Conclusions 1. CD4+CD25+ T cells within the thymus are not all Treg cells, some of them are autoreactive T cells in early active stage. The number of intrathymic CD4+CD25+ Treg cells is lower and the number of actvive autoreactive T cells is higher in MG patients , and there’s realy a disturbance of immune regulation in the MG thymus. 2. the usage of CD25hi as the marker molecule is the main reason for the inconsistent conclusions in the studies of human Tregs. 3. the coexpression of CD25 and CD27 can effectively differentiate Treg cells from early activated CD25+ Tresp cells and can be used to isolate viable Treg cell populations for functional studies. 4. There’s no quantitative deficiency of Treg cells in the peripheral blood of MG patients. The founctional defect is the main mechanism of the imbalance between Tregs and autoreactive T cells in the blood. 5. The peripheral level of IL-6 has a significant relationship with the percentage of CD25+ CD27- cells, RCD27, and QMG score. The increase of peripheral IL-6 is one of the crucial reasons for the functional down-regulation of Tregs in MG blood. 6. Thymectomy has no effect on the percentage of Tregs,but can reduce the percentage of active autoreactive cells and the IL-6 level in the peripheral blood , and recover the dynamic balance between the Tregs and active autoreactive T cells. 7. The RCD27 in the MG patients pre-Tx has a significant correlation with the decrease of QMG score after thymecomy, then, the detection of RCD27 pre-Tx might help us estimate the prognosis of thymectomy in MG patients.
引文
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