The Role Of Foxp3～+CD4～+CD25～+ Regulatory T Cells In Naturally Tolerance Following Rat Allograft Liver Transplantation

Part 1: Establishment the Liver Transplantation Model ofRat and Detect the Proportion of CD4~+CD25~+ T Cells in livergraftAIM: To establish an acute rejection,tolerance and isogenic model of ratorthotopic liver transplantation (ROLT), and detect the proportion ofintrahepatic CD4~+CD25~+ T cells.METHODS: Forty two ROLT models were established by modifiedKamada two-cuff technique, with inbred male DA rats and male LEWrats served as donor and recipient respectively. The recipients wererandomly divided into 3 groups: rejection group (DA→LEW, n=12),tolerance group (LEW→DA, n=15), isogenic group (DA→DA, n=15).Every 2 recipients from each group sacrificed at 4, 7, 10dpost-transplantation to collect blood serum and liver tissues. 3 recipientsfrom the other two groups sacrificed at 30d post-transplantation to collectblood serum and liver tissues. The other 6 recipients were left in each group to observe the animal survival time. The proportion of intrahepaticCD4~+CD25~+T cells from three groups was determined by flow cytometry(FCM) in different times.RESULTS: The serum concentrations of AST and BILI in rejectiongroup were higher than the other two groups at 7 d and 10 dpost-transplantation(p＜0.05). The grafts Banff scales in rejection groupwere severer than in other groups (rejection group,Ⅲ; tolerance group,Ⅰ-Ⅱ; isogenic group, 0-Ⅰ). The median survival times in three groupswere 12d, 106d, 128d respectively. Beginning immediately aftertransplantation, the ratio of Treg cells increased over time in bothallogeneic groups but was significantly greater in the Rejection group.The quotient of Treg cells declined after day 4, a reduction that was moredramatic in the rejection group than in the tolerance group. Animals in theTolerance group showed a second increase in the quotient after day 30,and maintain in the high lever.CONCLUSION: DA and LEW rat strain combination can be applied toconstruct rejection and tolerance model of rat liver transplantation. Theliver function changes, graft histological manifestation, and survival timeare consistent with the features of rejection and tolerance model. Theproportion of CD4~+CD25~+T cells goes parallel with increasing amountsof activated memory T cells. This indicates that the recipient's immunesystem had initiated an alloresponse against antigens transferred with the MHC-incompatible liver allograft.Part 2: Isolation of Rat CD4~+CD25~+ Regulatory T Cell byMACS in Two positive Steps from Peripheral BloodAIM: To establish a stable and high efficiency method for ex vivoenrichment of CD4~+CD25~+ regulatory T cells in rats.METHODS: CD4~+CD25~+ regulatory T cells were isolated from theperipheral blood in two steps by magic cell sorting (MACS) system. Thefirst positive step was selected of CD4~+ T cells by anti-rat CD4-APC andAnti-APC MultiSort MicroBeads, the second positive was selected byanti-CD25 PE and anti-PE magic microbeads. The purity and viability ofenriched cells were measured by flow cytometry. The suppressive abilityof enriched cells on the proliferation of CD4~+CD25~- T cells was assessedby cell proliferation assay.RESULTS: The purity of two positive enriched CD4~+CD25~+ T cells was94.2%±1.1% (87%～98%) with the viability of 93.8%±3.4% (93%～95%).The enriched cells significantly suppressed the proliferation ofCD4~+CD25~-T cells in mixed lymphocyte culture(CD25~+/CD25~- vs CD25~-,P＜0.01).CONCLUSION: We established an effective procedure for enrichment of CD4~+CD25~+ regulatory T cells in two steps by MACS, with satisfiedcell purity, viability and function.Part 3: Expression of Transcription Factor Foxp3 in theOrthotopic Liver Transplantation Modle of Inbred Rats withSpontaneous ToleranceAIM: To investigate the expression of transcription factor Foxp3 in theorthotopic liver transplantation modle of inbred rats with spontaneoustolerance.METHODS: The real-time fluorescence quantitative PCR (RFQ-PCR)method was used to analyze the expression level of Foxp3 mRNA in theliver of tolerance group (TOL) and syngeneic group (SYN) after 4d,7d,10d,30d,60d following liver transplantation. The expression of Scurfinin hepatic tissue was assayed by Western blot and then analyzed withcomputer imaging system.RESULTS: The expression of Foxp3 mRNA was lower than normal liverin the early phase after liver transplantation. About one week later, thelever of Foxp3 mRNA ascensus to peak value. Latterly, the value ofFoxp3 mRNA decreased but still kept in a high level. The Western blotshows us the resemblance change about Scurfin. CONCLUSION Transcription factor Foxp3 may play an important rolein the spontaneous tolerance in the orthotopic liver transplantation ofinbred rat.