The Changes Of Regulatory T Cells And T Helper Cells In The The Patients With Myasthenia Gravis And The Effects Of Immunosuppressants On Them

Myasthenia gravis （MG） is a typical organ specific autoimmune disease, and it isthe result of immune tolerance failure. CD4⁺CD25⁺regulatory T cells （Tregs） areinstrumental in the maintenance of immunologic self-tolerance, and thymus derivednatural Tregs （nTregs） playe the key roles in the number and function. Tregs andeffector T cells （Teffs） are in the interdependent relationship in the body and maintain acertain equilibrium state. Occurrence of autoimmune disease is associated with thisdamaged balance because of some congenital or acquired factors. The view has beenwidely accepted.Recently, the effect of Tregs in the pathogenesis of MG has been drawn attentionby domestic and international scholars, but the conclusions were not consistent.Relevant factors may include selecting Tregs associated molecule, different age bias.Therefore, we think it necessary to consider the interference of these confoundingfactors when we would like to comprehensively assess the changes of Tregs and Teffs inMG patients. Moreover, we want to understand how to the conventionalimmunosuppressants （IS） inhibitors such as GC and Ig, which are prescribed to MGpatients, work.This study was divided into three parts:1) detected Treg-association molecules inperipheral blood of MG patients and compared to those of the heathy donors, andobserved the effect of immunomodulators such as prednisone and gamma globulin onthem.2) investigated the expressions of Tregs、naǐve T cells and Th17cells in thethymus of the MG patients, and compare with those of the MG （-） thymoma patientsand the healthy subjects.3) explored the changes of different Th cells in peripheral blood of the MG patients, and investigated the effects of immunomodulators such asprednisone and gamma globulin on them.Changes of Treg-association Molecules in the Peripheral Blood of the MG Patientsand the Effects of Immunosuppresants such as Prednisone and Immunoglobulin onthemCD4⁺FOXP3⁺T cells、 CD4⁺CD25highT cells、 CD4⁺CD25⁺CD127-T cells andCD4⁺CD25⁺FOXP3⁺T cells respectively were regarded as human Tregs, we detected thechanges of them in the peripheral blood of the MG patients, and explored theircorrelation with the severity of clinical sympotom and the effects of immunosuppresantssuch as pred on them.We used the six colors fluorescent analysis technique of FACSCanto Ⅱflowcytometry by newly developed by BD, and take use of the characteristic of its simpleand quick operation and highly accurate test results.They were divided into two groups in order to understand the impact of Tregs onthe patient condition:57MG patients in active stage and8MG patients in remission.57MG patients in active stage were also divided into three groups according to thetreatment strategy in order to investigate the effect of GC, Ig and other immuneinhibitor on Tregs and Th subsets:1)17MG patients （untreated group）: who did notundergo any IS therapy before enrollment;2)29MG patients （GCs treated group）: whohad previously received oral prednisone （pred） alone or pred and azathioprine treatment;3)11MG patients （IVIG group）: who were suffering from progressive generalweakness and diagnosed clinically as myasthenia crisis. The healthy control group is90cases. The average age of the MG patients was39.4±13.5years old, and the averageage of the healthy donors was34.5±9.0years old. The sex ratio was11:8（male: female）in the patients, and it was3:2in the healthy donors.Our results showed that no significant difference was observed in the absolutenumber and frequency of CD4⁺CD25⁺CD127-T cells among different groups. The frequency of CD4⁺CD25highT cells in the patients in active stage obviouslydecreased when compared to that of the healthy donors, but no measurable differencewhen compared to that of the patents in remission.CD4⁺FOXP3⁺T cells are often taken as Tregs. Our results showed that nosignificant difference was observed in the absolute number and frequency ofCD4⁺FOXP3⁺T cells between different groups.Our results indicated that the numbers and frequencies of CD4⁺CD25⁺FOXP3⁺Tcells significantly decreased in the patients in active stage when compared to those ofthe patients in remission and the healthy donors, and no diffenrence was seen beweenthe latters. The dynamic analysis of Treg cell level in MG patients who accepted IStherapy suggested that IS such as GC and Ig had the capacity of increasing the size ofthe peripheral Tregs population. The analysis of relationship between the change rate ofQMGs and Treg cell levels suggested significant that there was significant negativerelationship between them.The changes of Tregs and naǐve T cells in the Thymus Tissue of the MG PatientsConsidering some confounding factors such as age, so we used multiple groupanalysis to investigate the expression of Tregs in the thymus of the MG patients.Control thymus tissues were obtained from the young adults suffering from bluntthoracic trauma and accepting thoracic surgery and the adolescents accepting correctivetherapy because of suffering from congenital cardiovascular malformation. They allsigned informed consent. They had not suffered from autoimmune disease and took anydrugs. The MG patient group included39patients, their mean age was40.92±14.4years, and the male to female ratio was10:29. The healthy donor group included14,their mean age was14.43±6.28years, and the male to female ratio was3:11. Thethymoma patients without myasthenia gravis were29, and their mean age was45.24±12.70years, and the male to female ratio was8:21. Different from the peripheralcirculation, CD25expression in thymus strongly is suggestive of Tregs, and FOXP3 expression in thymus also decided whether thymus produced Tregs are mature and playa key role in the inhibition function. Therefore, we examined the expression of CD25and FOXP3in the thymus.Our results showed that the numbers and expression intensities of CD25⁺thymocytes and FOXP3⁺thymocytes, significantly decreased when compared to those ofthe healthy donors. However, there was age bias between them, and the discrepancymay be ascribed to age bias. Therefore, we compared the age-mathched MG （-） patientswith thymoma to the MG patients, and found no measurably difference in the numbersand expression intensities of CD25⁺thymocytes and FOXP3⁺thymocytes between them.Teffs precursor naǐve T cells, corresponding to Tregs, develop and output in thethymus.Our immunohistochemical results showed that the numbers of CD4⁺Tlymphocytes and CD8+T lymphocytes in the thymus of the MG patients with thymomasignificantly were higher than those of the MG （-） patients with thymoma and thehealthy donors. The similar result was seen when compared to the non-neoplastic MGpatients. Moreover, we found the phenomenon that the frequencies of CD45RA+CD4⁺Tcells in peripheral blood of some MG patients were higher than that of healthy donors.We detected the express of Th17cell in the fresh thymus specimens of a few casesby the method of flow cytometry. Th17cells were not found in the thymi of the MGpatients with thymoma and the MG （-） patients, and the similar result was found in thehealthy donors.Changes of Th₁/Th₂/Th₁₇in the Peripheral Blood of MG Patients and the Effects ofImmunosuppresants on themPeripheral blood mononuclear cells （PBMNC） freshly extracted were added intoappropriate stimulating agents，placed in a CO2incubator and incubated for4⁶hours.Then they were labeled by the flow type antibodies. Considering that CD4moleculemay be coated in CD4⁺T cells and difficult to detect, we labeled CD4⁺T cells by usingCD3and CD8antibodies. The cytokines representative of Th cell subsets are intracellularly expressed and fixed. Their cell membranes were ruptured. The resultsdetected by flow cytometry showed that no significant difference was seen in thefrequencies and absolute numbers of Th cell substs among the patients and the healthyhealthy donors. No obvious effect of IS such as GC and Ig on Th cell subsets wasobserved. Similarly, no significant difference was seen in the levels of the plasmainflammatory cytokines representive of different Th subsets among the MG patients andthe healthy donors.In conclusion: according to our results, several conclusions can be drawn:1) Our results indicated that significant reduction in the numbers and frequencies ofCD4⁺CD25⁺FOXP3⁺T cells, the strongest regulation function in immune homeostasisand the most stable subset of nTregs, could not be attributed to Tregs deficiency inthymic development. Moreover, no significant difference in the number and frequenciesof CD4⁺FOXP3⁺T cells was seen between the patients and the healthy donors.2) No measureable difference was observed in the absolute numbers and frequencies ofTh₁/Th₂/Th₁₇between the patients and the healthy donors.3) Myasthenic symptoms can improve in the most patients with thymoma or thymichyperplasia, who accepted Tx therapy. The possible reason lies in the active patientswith thymic hyperplasia, generation and output of naǐve T cells.4) IS such as GC and Ig has been the most commonly prescribed drug for MG. Ourresults confirmed that their therapeutic effect is related to increasing the number and thefequency of CD4⁺CD25⁺FOXP3⁺T cells in peripheral blood of the MG patients.