The Role Of CD₄⁺CD₂₅⁺Foxp₃⁺ Regulatory T Cells And Th17 Cells In Portal Vein Induced Tolerance

Partâ… The roles of different subsets of T lymphocytes in portal veininjection induced tolerance.Objective: To analyze the role of different subsets of T lymphocytes in transferring toleranceinduced by portal vein injection.Methods: C57BL/6 (B6) mice were used as donors. 7 days after portal vein injection ofmitomycin-treated B6 splenocytes into BALB/c mice, the splenocytes of the recipient wereisolate. Mitomycin-treated B6 splenocytes were used as stimulator, PVI splenocytes or normalBALB/c splenocytes were used as reactor. And lymphocytes proliferations were conducted byAlamar Blue. CD4⁺, CD8⁺T lymphocytes were then sorted from PVI mice by FACSAria. Thesorted CD4⁺ (CD8⁺) T lymphocytes were then transfused into another BALB/c mice followedby heterotopic implantation of a B6 heart allograft. A C3H heart allograft and normal BALB/cwere used as controls. The rejection and survival of the allograft were observed. And 7 daysafter transplantation, the grafts were collected for HE stain.Results: Portal vein injection of alloantigen can prolong the subsequently transplantedallograft survival, when transfusion with CD4⁺T lymphocytes isolated from PVI mice, thesubsequent transplanted B6 allograft was survival longer than C3H allograft. Similar results were not observed from BALB/c with transfusion of CD8⁺T lymphocytes from PVI mice orCD₄⁺ T cells from normal Balb/c mice.Conclusion: This finding indicates that portal vein injection of alloantigen can induceantigen-specific tolerance. CD₄⁺T lymphocytes, rather than CD₈⁺T lymphocytes, play animportant role in transferring tolerance induced by portal vein injection and this tolerance isantigen-specific.Partâ…¡Increased CD₄⁺CD₂₅⁺Foxp₃⁺ regulatory T cells in toleranceinduced by portal vein injectionObjective: To assess the proportion and function CD₄⁺CD₂₅⁺Foxp₃⁺ regulatory T cells inportal vein injection induced tolerance.Methods: Mitomycin-treated C57BL/6 (B6) splenocytes were injected into Balb/c mice viathe portal vein. The proportions of CD₄⁺CD₂₅⁺Foxp₃⁺ Treg cells were determined in the blood,liver, and spleen in the 7, 14 or 21 days after injection. CD₄⁺CD₂₅⁺ Treg cells were isolatedfrom PVI mice and added into B6 stimulated Balb/c mixed lymphocytes culture at differentconcentrations. The proliferations of Balb/c lymphocytes were tested by Alamar Blue.Anti-CD₂₅ mAb were used to delete CD₄⁺CD₂₅⁺Foxp₃⁺ Treg in mice. B6 or C3H heartallografts were implanted into anti-CD25 mAb-treated PVI and isotype antibody PVI mice,and graft survivals were compared. The percentages of CD₄⁺CD₂₅⁺Foxp₃⁺ Treg, cytokineprofiles, and ratios of apoptosis were determined in anti-CD25 mAb-treated PVI anduntreated PVI mice.Results: Portal vein injection of allogeneic cells induced antigen-specific tolerance andincreased the percentage of CD₄⁺CD₂₅⁺Foxp₃⁺ Treg. The CD₄⁺CD₂₅⁺ Treg isolated from PVImice can inhibit the lymphocytes proliferation in a dose-dependent manner. Depletion ofCD₄⁺CD₂₅⁺ T cells prevented the induction of tolerance and decreased the percentage ofCD₄⁺CD₂₅⁺Foxp₃⁺ Treg in the CD₃⁺ T cell pool, and thus was associated with decreasedproduction of IL-4 and apoptosis of T cells. Conclusion: Increased CD₄⁺CD₂₅⁺Foxp₃⁺ Treg play an important role in portal vein toleranceinduction, at least partly via increasing the production of IL-4 and promoting apoptosis of Tcells.Partâ…¢Th17 cells were involved in acute allograft rejectionObjective: To analyze the role of Th17 cells in acute allograft rejection.Methods: B6 skin allografts were implanted into Balb/c mice. Seven days postoperation, skinallografts were collected to assess the expression of IL-17 by immunohistochemistry. CD₄⁺Tcells were isolated from normal Balb/c, and were induced by IL-6 and TGF-β. Then IL-1β,TNF-αand IL-23 were added to the culture to analysis their roles in differentiation of Th17.In order to test Th17 cells in rejection induction, B6 skin allografts were transplanted toBalb/c nude mice, and in vitro induced Th17 cells were injection into recipients at differentconcentrations. Skin allograft survival was conducted.Results: There were a lot of Th17 cells infiltrated into rejected skin allograft, whereas nearlyno Th17 cell was infiltrated into sygeneous grafts. IL-6 and TGF-βcan induce CD₄⁺Tdifferentiation into Th17 cells and IL-1βand TNF-αcan enhanced the differentiation. IL-23can augment the differentiation in second stimulation. Balb/c nude mouse is thymus deficient.It can not induce acute rejection of allograft. When Th17 cells were adoptively transfused intoBalb/c nude, the C57 allografts were rejected immediately.Conclusions: IL-6 and TGF-βare responsible for differentiation of Th17 cells, while IL-11β,TNF-αand IL-23 can enhanced the differentiation. Th17 cells are, at least partly, involved inacute rejection. Partâ…£The interaction of CD₄⁺CD₂₅⁺Foxp₃⁺ regulatory T cellsand Th17 cells in portal vein toleranceObjective: To explore the interaction of CD₄⁺CD₂₅⁺Foxp₃⁺T and Th17 cells in portal veininjection induced tolerance.Methods: Seven days after portal vein injection of B6 splenocytes, Balb/c mice received B6skin allograft transplantation. The allografts were collected for detection of IL-17 and Foxp3expression 7 days after transplantation, and sygeneous portal vein injection was used ascontrol. IL-2 and TGF-βwere added to culture of CD₄⁺CD₂₅^- T cells, and induced CD₄⁺CD₂₅⁺T cells were isolated from the culture. Then IL-6 was added into na(i|¨)ve CD₄⁺CD₂₅⁺T orinduced CD₄⁺CD₂₅⁺T culture to evaluate the differentiation bias of CD₄⁺CD₂₅⁺T cells. IL-2,with or without TGF-β, was added into Th17 culture to evaluate the differentiation bias ofTh17 cells. IL-6 and in vitro induced Th17 cells were transfused into PVI mice. And the skinallografts survival was conduced.Results: Compared to sygeneous antigen, portal vein injection of alloantigen induced highexpression of Foxp3 and low level of IL-17 in subsequently transplanted skin allografts. IL-6can cause trans-differentiation of Treg into Th17 cells. And IL-2 together with TGF-βcancause trans-differentiation of Th17 into Treg cells. The injection of IL-6, Th17 can cause therejection of B6 heart allograft transplanted into PVI mice.Conclusions: Portal vein injection of alloantigen can increase the accumulation of Treg cellsand reduced the infiltration of Th17 cells. In vitro, Treg and Th17 cells can differentiated intoeach other depending on the cytokine in the microenviroment. Adoptive transfused of IL-6and Th17 can reverse the tolerance induced by portal vein injection of alloantigen.