Adoptive Transfusion Of The In Vitro Expanded Regulatory T Cells Protects Against Ischemic Stroke And Its Anti-neuroinflammation Mechanism

Experiment purpose:1.To establish a method for isolating and expanding rat CD4+CD25+regulatory Tcells (Tregs) in vitro, and examined the purity, activity and the immunosuppressivefunction of fresh and expanded Treg cells.2.To explore the protective effects of introvenous transplantation of In vitroexpanded CD4+CD25+regulatory T cells (Tregs) after cerebral ischemia-reperfusion inRats.3.To research the effect of Tregs transfer in rodent models of ischemic stroke onactivating of resident and invading inflammatory cells（including microglia，astrocyle andneutrophils）respectively, and further investigated the mechanism underlying Treg-affordedneuroprotection.Research Methods:1.Two-steps method of Magnetic cell sorting (MACS) was used to extractCD4+CD25+regulatory T cells from healthy adult Sprague-Dawley (SD) rats spleen andlymph node. The Tregs were expanded in vitro in response to anti-CD3-anti-CD28-coatedmicrobeads、rapamycin and IL-2. Purity and activities of the fresh and expanded cellswere detected by FCM (flow cytometry) and trypan blue stainting respectively, and the invitro cell proliferation inhibition assay were used to exam the function of Tregs.2.Cerebral focal ischemia was induced in Sprague-Dawley rats by intraluminalocclusion of the right middle cerebral artery (MCA) for120minutes. Immediately aftersurgery, animals were randomly assigned to sham, phosphate-buffered saline(PBS),splenocyte, or Tregs treatment groups. Neuronogical behavior was evaluated by Longa’s scoring, forelimb placing test, foot-fault test and the cylinder test at3d、7d、14d afteroperation. At2w after operation, the water maze was used to detect long-term deficits inspatial information processing. Infarct size was determined by CV and TTC staining.Brain edema was assessed using the wet-dry method; Immunofluorescence was used toobserve the expression of caspase-3for detection of apoptosis, Fluro-JadeB staining wasused to detect degenerating neurons following temporary focal cerebral ischemia.3.The model processing and grouping as before. Immunofluorescence andImmunohistochemistry was used to detect the expression of MPO, Iba1and GFAP inischemic hemisphere, observe the infiltration of neutrophi, and the activation of microglialand astrocyle at3d,14d, and used westernblot to further validate.Result:1.The purity of CD4+CD25+Treg cells obtained by MACS separation was84.50%, thecell survival rate was95.00%. After3weeks of culture, the purity of Tregs was76.00%,the cell survival rate was94.30%. In vitro cell proliferation inhibition showed Tregssignificantly suppressed the proliferation of CD4+CD25-T cells.2.Neurological deficits after modeling were peaked at3d (P＜0.05, compared with7d), postischemic sensorimotor dysfunction significantly improved in Treg-treated Ratscompared to PBS-and SP-treated animals until14days (P＜0.05, respectively). MorrisWater Maze test showed that the spatial learning and memory ability of Tregs treatmentimproved significantly compared with PBS and SP treatment. TTC and Cresyl violetstaining revealed profoundly reduced infarct volumes at any detecting time after MCAO.The degree of brain edema of Tregs treatment group reduced significantly compared withthe PBS treatment group and SP treatment group. In the sham groups, there were scatteredcaspase-3and flruo-JadeB positive cells, while PBS and SP groups appeared numerouspositive cells at1d after reperfusion in the peri-ischemia area, then they reduced gradually,until two weeks there remain have little positive cells. caspase-3and flruo-JadeB positivecells of Tregs treatment decreased significantly compared with PBS and SP treatmentgroups (P＜0.01).3. Immunofluorescence staining revealed MPO+cells infiltration was prominent at1to3days after MCAO in PBS-and SP-treated rat, but remarkably attenuated inTreg-treated rat. In the sham groups, there were scattered Iba1positive cells, while PBSand SP groups appeared numerous Iba1positive cells at3d after reperfusion in theperi-ischemia area, Iba1positive cells of Tregs treatment decreased significantly compared with PBS and SP treatment groups at14d (P＜0.05). We observe the similiar changingtrends about GFAP. The results of western blot for Iba1and GFAP were similar to theimmunohistochemial data.Conclusion:1.The method established in the study for isolation and expansion of rat CD4+CD25+Treg cells is effective obtain satisfactory cells, which have high purity and activity, and thesuppressive function is influenced.2.The In vitro expanded CD4+CD25+regulatory T cells transplantation after transientischemia markedly reduce the cerebral infarct volume, improve the neurological functionand inhibit neuronal degeneration and apoptosis.3.The In vitro expanded CD4+CD25+regulatory T cells transplantation Attenuatepostischemic activation of resident and invading inflammatory cells. Our findingssuggested that neutrophil, microglial and astrocytes are targets for CD4+CD25+regulatoryT cells after cerebral ischemia. To inhibite neutrophil invade and modulate glial responsemay be a mechanism of Tregs-mediated neuroprotection in ischemia-reperfusion..