Protective Effects Of Intravenous Adoptive Transfusion Of In Vitro Expanded Regulatory T Cells In Rats With Cerebral Infarction

Objectives1.To establish rat CD4+CD25+regulatory T cells (regulatory T cells, Tregs)separation and in vitro expansion method, and to identify the cell characteristicsof separated and expanded CD4+CD25+regulatory T cells (Tregs).2.To replicate the permanent middle cerebral artery occlusion (MCAO)model in rats, and to investigate the protective effects of intravenous adoptivetransfusion of in vitro expanded CD4+CD25+Tregs in rats with cerebralinfarction.Methods1. Spleen and lymph nodes from healthy adult male Sprague-Dawley (SD)rats were used and single cell suspension was prepared. Tregs were isolated andpurified with magnetic cell sorting (MACS) method (also calledimmunomagnetic beads), i.e. positive and negative selection of separation andpurification of CD4+CD25+Tregs. The isolated and purified Tregs werestimulated with monoclonal antibodies of anti CD3and anti CD28, IL-2andrapamycin and other stimuli in vitro. In this way Tregs were expanded. Flowcytometry was used to analyze the purity of the cells.Trypan blue staining wasused to detect cell survival. The inhibitory effects of CD4+CD25+Tregs toCD4+CD25-T cells was determined by proliferation inhibition test (MTT).2.The amplified CD4+CD25+Tregs were prophylactic intravenous infused tohealthy adult male SD rats. One day after the infusion, the rats were subjected topermanent middle cerebral artery occlusion (MCAO).The animal were randomlydivided into normal group, MCAO group, HBSS group (MCAO+HBSS infusion), group Treg (MCAO+Tregs infusion). Three days,1week,2weeks,3weeks,4weeks after MCAO, The rats were underwent neurological assessment. Threedays,1week,4week after MCAO, the rats were killed and brain tissue wasstained by TTC, and a computer image analysis system was utilized to measurethe cerebral infarction volume.Three days,1week,4weeks after MCAO, thebrains were removed for preparation of brain slices. Nissl staining was ued toexame the nurons in hippocampus CA1region. the TUNEL staining with theImage-ProPlus image analysis system was applied to detect the cell apoptosisin peripheral zone of ischemic lesions.Result1.The purity of CD4+CD25+Treg cells obtained by MACS separation was88.31%, the cell survival rate was96.00%. In vitro proliferation assay showedthat CD4+CD25+Treg could significantly inhibit the proliferation ofCD4+CD25-T cells (P<0.01). After3weeks of culture of CD4+CD25+Treg cellsin vitro amplification the purity was77.20%, and activity was96.00%, theinhibitory function was slightly stronger than the freshly isolatedCD4+CD25+Treg cells, the difference was statistically significant (P<0.01).2. Bederson score in Treg group was significantly lower than that of MCAOgroup and HBSS group (P<0.05). Beam balance test scores in Rts of Treg groupwere significantly lower than that of MCAO group and HBSS group (P<0.01).Morris water maze experiment showed that the time platform-seeking latency ofrats in Treg group significantly reduced compared with the that of MCAO group,HBSS group (P<0.01). A significant increase in the number of plateformpassing(P<0.01) was found in Treg group than that t of MCAO and HBSSgroups. Cerebral infarction volume and cerebral infarction volume to thecontralateral hemisphere percentage in Treg group were significantly decreasedcompared with that in MCAO group and HBSS group (P<0.01). The normalpyramidal cells in hippocampus CA1area in Treg group increased higher thanthat in MCAO group and HBSS group (P<0.01). The number of apoptotic cellsin Treg group around focal ischemia area decreased significantly, as comparedwith that in MCAO group and HBSS group (P<0.01).Conclusion1. CD4+CD25+Treg cells of high purity, high activity were obtained byMACS, and CD4+CD25+Treg cells have the normal immune suppression function. Use of rapamycin plus anti CD3/CD28monoclonal antibody+IL-2invitro expanded CD4+CD25+Treg cells with a high purity, and does not affect theinhibitory function of CD4+CD25+Treg cells.2. intravenous adoptive transfusion of in vitro expanded CD4+CD25+Tregsin rats significantly decrease infarct volume of rats with cerebral infarction,reduce the defect of neurological function after ischemia, relieve cell injury ofhippocampal neurons after cerebral infarction, reduce the number of apoptosiscells around ischemic lesions.