Therapeutic Effect Of CD4+CD25+regulatory T Cells Amplified In Vitro On Experimental Autoimmune Neuritis In Rats

Background:In 1955,Waksman and Adams first proposed that experimental autoimmune neuritis（EAN）and Guillain-Barrésyndrome（GBS）were identical in clinical features,morphology,electrophysiology,pathology,immunology and other aspects.Therefore,EAN as an animal model of GBS has been internationally recognized and widely used in the study of pathogenesis and immunotherapy of GBS.The infiltration of inflammatory cells and demyelination of the peripheral nerve are the gold standards for the diagnosis of EAN and GBS.Previous studies have shown that P2_53-78polypeptide can successfully induce EAN in sensitive rats.Regulatory T cells（CD4⁺CD25⁺regulatory T cells,Tregs）are a kind of cells with immunomodulatory function in vivo and in vitro.By inhibiting the activation and proliferation of effectual T cells to regulate the level of immune response to autoimmune and foreign antigens and maintain the state of autoimmune tolerance,which is closely related to the occurrence of autoimmune diseases.At present,there are few studies on the relationship between Tregs cells and GBS at home and abroad,and we know about the immunological mechanism of Tregs cells in GBS and the therapeutic effect after transfusion littlely.Therefore,the purpose of this study was to explore whether the adoptive transfusion of autologous CD4+CD25+Tregs has a therapeutic effect on EAN model rats and to provide new experimental and theoretical bases for the immunotherapy of GBS.Objective:To explore whether the adoptive transfusion of autologous CD4+CD25+Tregs has a therapeutic effect on EAN model rats,and it provides new experimental and theoretical bases for the immunotherapy of GBS.Method:1.EAN was successfully induced by sensitizer and was randomly divided into four groups（n=8）:healthy control group,non-intervention group,low-dose treatment group and high-dose treatment group.The rats were anesthetized with 10%chloral hydrate.2.Firstly,CD4+T cells were selected from normal rat spleen by immunomagnetic bead technique,and then Tregs were separated from CD4+T cells by flow cytometry.Then,cells were cultured in an incubator containing 50 ml/L of CO2 at 37°C,and were continuously amplified for 15days.3.The activity and purity and in vitro inhibitory function of amplified CD4⁺CD25⁺Tregs were determined after isolation in vitro.4.Treatment of EAN rat model with CD4⁺CD25⁺Tregs.5.The pathological changes of sciatic nerve,the morphologic changes of electron microscope and the level of Tregs in each group were compared before and after transfusion of EAN respectively.It is helpful to analyze and evaluate the therapeutic effect of Tregs in EAN.Results:1.The EAN model was successfully made.In the model group,a large number of inflammatory cells could be seen in the sciatic nerve,and primary myelin sheath damage and even axonal damage could be observed by Fleming staining and electron microscopy.2.After sorting using immunomagnetic beads separation techniques combined with flow cytometry,the purity of CD4+CD25+Tregs was>95%,with an average of 98.23±0.42;and activity was>95%.3.After four rounds of amplification for 15 days,CD4+CD25+Tregs had proliferated by 40-100 times and were able to maintain a stable phenotype during the amplification process.After in vitro amplification,cell purity was>85%,with an average of 86.47±0.78（t=23.327,P=0.002<0.05）,compared with cells immediately after sorting;and the difference was statistically significant.4.This suggests that CD4+CD25+Tregs maintained its inhibitory function after amplification.5.Compared with rats in the untreated group,rats in the treatment groups had significantly reduced infiltration of inflammatory cells in the sciatic nerve and myelin and axonal damage;and CD4+CD25+Treg levels in peripheral blood were significantly higher than those in the untreated group（P<0.05）.Moreover,the therapeutic effect became more significant with the increase in dose of adoptive transfusion.Conclusions:Adoptive transfusion of CD4+CD25+Tregs to EAN model rats has significant therapeutic effect on EAN model rats.These research results will provide some experimental theoretical basis for the clinical treatment of GBS.