The Effect Of CD4~+CD25~+ And CD8~+CD103~+ Regulatory T Cell In Mice Corneal Transplantation

Corneal transplantation is the only effective therapy for the corneal blindness to improve the visual acuity at present.Though corneal graft survival is the highest in the clinical organ or tissue transplantation,the success rate of it is still less than is generally appreciated.Allograft rejection is the most common cause of corneal graft failure,which results in huge economic burden of the society and visual function lose of the patients.The development of immunosuppression and its clinical joint application reduced acute graft rejection obviously and improved graft survival significantly.The organ or issue transplantation becomes the effective method to save lives of the patients who have organ or issue failure in the terminal stage.However,it is the toxicity and adverse reaction,such as damaging the basilic organ and depressing the immunity of anti-tumor or anti-infection,and the low therapeutic effect to chronic graft rejection and so on that limits immunosuppression to its long time application in clinic.Thus,investigators are trying to avoid the long time application of immunosuppression and conquer the graft rejection by the way to induce immune tolerance in transplantation.Nowadays,many researches prove that inducing the anterior chamber associated immune deviation(ACAID) before corneal allograft transplantation could improve graft survival time notably.Recently,some studies show that CD4⁺CD25⁺ T lymphocytes and CD8⁺CD103⁺ T lymphocytes,which play a very important role in the immune privilege in the periphery and the course of ACAID induction,take a part in the inhibition of delayed-type hypersensitivity and cytotoxic T lymphocyte activity as the regulatory T cells.Currently, researches about the roles of regulatory T cell in the ACIAD induction focus on the aspects of uveitis.Therefore,this study,based on the model of corneal grafting in mice,is looking forward to the effect of CD4⁺CD25⁺ T lymphocytes and CD8⁺CD103⁺ T lymphocytes in the corneal transplantation and the immune tolerance. PARTâ… The effect of CD4⁺CD25⁺ and CD8⁺CD103⁺ regulatory T cell in mice isograft and allograft corneal transplantationPurpose:To study the changes of CD4⁺CD25⁺ and CD8⁺CD103⁺ regulatory T cells and their correlated cytokines in corneal isograft and allograft transplantation in mice.Methods:BABL/c(H-2^d) received corneal allografts from C3H/He(H-2^k) were the experimental group,and BABL/c(H-2^d) were the donor as well as the recipient in the control group.The corneal graft survival time was recorded. Pre-,3^rd days,7^th days,4^th weeks,8^th weeks post-operatively,the infiltration of inflammatory cells and the corneal neovascularization was evaluated by histopathology,the percentage of CD4⁺CD25⁺ and CD8⁺CD103⁺ T cell in peripheral blood and spleen was determined by flow cytometry,meanwhile, the expression of IL-10,IL-4,IFN-γand IL-1βin serum and aqueous humor was measured by ELISA.Result:The graft rejection in the experimental group occurred at from 7 days to 4 weeks,mean 14.79±1.02 days.But the grafts of the control group remained clear within the duration of observation which was 8 weeks and the survival time is much longer than that of the allografts(X²=46.934,P=0.000). Flow cytometry showed the percentage of CD4⁺CD25⁺ Treg in peripheral blood in the control group after surgery was(3.36±0.29)%,(4.09±0.44)%, (5.44±0.35)%,(5.73±0.53)%,which raised much higher than that in the experimental group as(2.50±0.39)%,(3.24±0.25)%,(4.20±0.45)%, (4.18±0.14)%(t=3.828,2.898,3.780,4.892,P＜0.05).On the other hand, CD8⁺CD103⁺ T cell in peripheral blood in the experimental group after surgery was(2.20±0.33)%,(2.79±0.57)%,(4.55±1.03)%,(4.31±0.07)%,which was much higher than that in the control group as(0.73±0.12)%,(1.10±0.19)%, (1.43±0.14)%,(2.10±0.14)%(t=7.133,4.876,5.196,19.960,P＜0.05).The up-regulation of CD4⁺CD25⁺ and CD8⁺CD103⁺ T cell in spleen was earlier than that in peripheral blood.ELISA showed the expression of IL-10 and IL-4 in serum in the experimental group after the surgery was much lower than that in the control group(t=3.203,3.141,3.012,2.869 and 2.34,6.681,8.839,8.574, P=0.011,0.012,0.013,0.019 and 0.053,0.000,0.000,0.000).Otherwise,the level of IFN-γand IL-1βin serum in the experimental group grew higher than that in the control group(t=3.508,3.265,4.402,5.539 and 3.630,5.796,1.728, 0.660,P=0.006,0.011,0.002,0.000 and 0.005,0.000,0.115,0.524). Furthermore,the cytokine in aqueous humor behaved similarly with that in serum.Conclusions:The up-regulation of CD4⁺CD25⁺ Treg,IL-10 and IL-4 protected the corneal graft from rejection.And the raise of CD8⁺CD103⁺ Treg,IFN-γand IL-1βtook an important part in allograft rejection.PARTâ…¡The effect of CD4⁺CD25⁺ and CD8⁺CD103⁺ regulatory T cell in allograft corneal rejection in various inbred strains of micePurpose:To study the changes of CD4⁺CD25⁺ and CD8⁺CD103⁺ regulatory T cells and their correlated cytokines in corneal allograft transplantation in various inbred strains of mice.Methods:C57BL/6(H-2^b) received corneal allografts from BALB/c(H-2^d) were the group A,C3H/He(H-2^k) received allografts from C57BL/6(H-2^b) were the group B;and BABL/c(H-2^d) received corneal allografts from C3H/He(H-2^k) were the group C.The corneal graft survival time was recorded.Pre-,3^rd days, 7^th days,4^th weeks,8^th weeks post-operatively,the infiltration of inflammatory cells and the corneal neovascularization was evaluated by histopathology,the percentage of CD4⁺CD25⁺ and CD8⁺CD103⁺ T cell in peripheral blood and spleen was determined by flow cytometry,meanwhile,the expression of IL-10 and IFN-γin serum was measured by ELISA.Result:The graft rejection in the group A occurred at from 7 to 21 days,mean 12.38±0.81 days;in the group B from 7 to 24 days,mean 13.63±0.91 days; and in the group C from 7 to 28 days,mean 14.79±1.02 days(X²=3.688, P=0.055).Flow cytometry showed the percentage of CD4⁺CD25⁺ Treg in peripheral blood in the group A pre- and post operatively after surgery was (1.59±0.66)%,(1.22±0.33)%,(2.39±0.53)%,(1.37±0.28)%and(1.82±0.22)% respectively,which were lower than that in the group B as(3.03±0.82)%, (4.62±0.12)%,(2.70±0.16)%,(5.05±0.84)%and(4.64±0.51)%,and in the group C as(3.60±0.99)%,(2.50±0.39)%,(3.24±0.25)%,(4.20±0.45)%, (4.18±0.14)%(P=0.025,0.005,0.388,0.000,0.000).On the other hand, CD8⁺CD103⁺ T cell in peripheral blood in the group A and B after surgery increased earlier than that in the group C,but the levels that the three groups expressed had no statistical significance(P＞0.05).The up-regulation of CD4⁺CD25⁺ and CD8⁺CD103⁺ T cell in spleen was earlier than that in peripheral blood,but there was no difference in expression levels of the three groups.Furthermore,ELISA showed the expression of IL-10 in serum in the three groups after the surgery was much lower than that pre-operatively,but three groups had some difference in levels(P=0.025,0.001,0.009,0.484, 0.750).Otherwise,the level of IFN-γin serum in the three groups grew higher than that pre-operatively and there was some difference among the groups(P =0.097,0.029,0.063,0.539,0.012).Conclusions:The various inbred strains of mice had similar survival time of corneal allografts.Though there was difference in the expression levels of various strains,the tendency in the Tregs and cytokines expression of the various strains was basically with one accord.The low-level of CD4⁺CD25⁺Treg and IL-10 reduced the corneal graft survival.And the raise of CD8⁺CD103⁺Treg and IFN-γtook an important part in allograft rejection.