The Changes Of Frequencies And Phenotypes Of Intrathymicand Peripheral CD4～+CD25～+Tregs In MG Patients

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.