T细胞疫苗抑制了Flt3-L介导的小鼠肝移植急性排斥反应
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摘要
目的:探讨供体抗原特异性T细胞疫苗诱导肝移植免疫耐受的作用及其机理,同时对肝移植"自动耐受"机制进行探讨。方法:以CBA小鼠作为受体,以B6小鼠作为供体,建立小鼠原位肝移植模型;制备T细胞疫苗:用B6小鼠的脾细胞免疫CBA小鼠,取CBA小鼠的脾淋巴细胞加以灭活,制备成T细胞疫苗(TCV)备用;实验分为3组:组1:单纯移植组,以CBA小鼠作为受体,以B6小鼠作为供体,做原位肝移植。组2:用Flt3-L治疗的B6小鼠作为供体,以CBA小鼠作为受体,做原位肝移植。组3:用Flt3-L治疗的B6小鼠作为供体,以TCV免疫过的CBA小鼠作为受体,进行原位肝移植。组间比较移植物存活时间(MST);测定肝移植小鼠血清中的IL-10、IL-4和IFN-γ水平;组内比较移植前后单向混合淋巴细胞反应(MLR);分离移植物浸润细胞(GICs)组间比较GICs凋亡率。结果:Flt3-L诱导了同种移植急性排斥反应,应用TCV于移植前和移植后可以显著抑制Flt3-L诱导的同种肝移植排斥反应。在没有Flt3-L刺激作用的情况下移植排斥反应非常轻微。肝移植小鼠血清中的IL-10、IL-4和IFN-γ水平检测发现,Flt3-L治疗组IFN-γ水平显著升高,IL-10、IL-4水平降低;TCV免疫组IL-10、IL-4水平显著升高,IFN-γ水平显著下降;单纯移植自动耐受组与TCV免疫组变化趋势一致。单向混合淋巴细胞反应(MLR)显示:在单纯移植组,淋巴细胞增殖效应不明显,CPM为3 318±1 190;在Flt3-L治疗组,效应细胞增殖显著增强,CPM为9 790±1 369,在TCV免疫组,TCV免疫加Flt3-L治疗,淋巴细胞增殖效应被显著抑制,CPM为4 017±880。分离肝脏移植物内浸润细胞凋亡,TCV免疫组引起TIL凋亡率为(54.20±3.12)%,有效抑制了TIL引起的免疫排斥反应,诱导了免疫耐受。而在Flt3-L治疗组,TIL凋亡率为(3.34±2.49)%。结论:小鼠同种肝移植存在"自动耐受"现象;这种"自动耐受"现象的免疫平衡可以被Flt3-L打破,从而诱发急性排斥反应。针对排斥效应T细胞的特异性T细胞疫苗可以显著抑制Flt3-L诱导的同种肝移植排斥反应。
Objective: To investigate the effect and mechanism of donor-Ag specific T cell vaccination on inducing specific immune tolerance of allogenic liver transplantation and the mechanism of immune privilege of liver transplantation. Methods: CBA mice were recipients,B6 mice were donors,T cell vaccination( TCV) were made from the attenuated spleen cells of CBA mice,which were stimulated by Con A and were challenged with the spleen cells of B6 mice. There are 3 groups in this experiment: Transplant control group: Orthotopic liver transplantation( OLT) were performed with the recipients of CBA mice and donors of B6 mice; Flt3-L treating group: OLT were performed with the recipients of CBA mice and donors of B6 mice treated with Flt3-L; TCV group: OLT were performed with the recipients of CBA mice inoculated with TCV and donors of B6 mice treated with Flt3-L. Median survival time( MST) of liver grafts was recorded,IL-4,IL-10 and IFN-γ in peripheral blood were tested after transplantation in each group. One-way mixed lymphocyte reaction( MLR) were carried out with effectors of spleen cells from CBA mice and stimulator of spleen cells from B6 mice at the5 th day after transplantation. The apoptosis of liver graft infiltrating cells( GICs) were analyzed by flow cytometric analysis at the 5th day after transplantation. Results: Flt3-L treating donor activated allogenic acute rejecting reaction,TCV vaccinating recipient before and after transplantation significantly depressed the acute immune rejecting reaction mediated by Flt3-L. The liver grafts were accepted by recipient without the presence of Flt3-L. The cytokines test show that the serum value of IL-4 and IL-10 were increased in Transplant control group and TCV group,but decreased in Flt3-L treating group. The value of IFN-γ was increased in Flt3-L treating group,but decreased in Transplant control group and TCV group. The result of one-way MLR show that the cpm values in Flt3-L treating group was9 790±1 369 which was higher than Transplant control group which was 3 318±1 190; the cpm values in TCV group was 3 318±1 190 which show that TCV inhibited Flt3-L mediated liver transplant acute immune rejection. The flow cytometric analysis showed that the percentage of apoptosis of TILs was markedly increased in TCV group( 54. 20±3. 12) in comparison with Flt3-L treating group( 3. 34±2. 49%). The apoptosis analysis certified that TCV inhibited the TIL-mediated immune rejecting reaction. Conclusion: Automatic tolerance can be observed at the situation of allogenic liver transplantation in mice,Flt3-L can break the balance of automatic tolerance and mediate acute rejection reaction. Donor-Ag specific T cell vaccination can successfully inhibit acute immune rejection mediated by Flt3-L.
Objective: To investigate the effect and mechanism of donor-Ag specific T cell vaccination on inducing specific immune tolerance of allogenic liver transplantation and the mechanism of immune privilege of liver transplantation. Methods: CBA mice were recipients,B6 mice were donors,T cell vaccination( TCV) were made from the attenuated spleen cells of CBA mice,which were stimulated by Con A and were challenged with the spleen cells of B6 mice. There are 3 groups in this experiment: Transplant control group: Orthotopic liver transplantation( OLT) were performed with the recipients of CBA mice and donors of B6 mice; Flt3-L treating group: OLT were performed with the recipients of CBA mice and donors of B6 mice treated with Flt3-L; TCV group: OLT were performed with the recipients of CBA mice inoculated with TCV and donors of B6 mice treated with Flt3-L. Median survival time( MST) of liver grafts was recorded,IL-4,IL-10 and IFN-γ in peripheral blood were tested after transplantation in each group. One-way mixed lymphocyte reaction( MLR) were carried out with effectors of spleen cells from CBA mice and stimulator of spleen cells from B6 mice at the5 th day after transplantation. The apoptosis of liver graft infiltrating cells( GICs) were analyzed by flow cytometric analysis at the 5th day after transplantation. Results: Flt3-L treating donor activated allogenic acute rejecting reaction,TCV vaccinating recipient before and after transplantation significantly depressed the acute immune rejecting reaction mediated by Flt3-L. The liver grafts were accepted by recipient without the presence of Flt3-L. The cytokines test show that the serum value of IL-4 and IL-10 were increased in Transplant control group and TCV group,but decreased in Flt3-L treating group. The value of IFN-γ was increased in Flt3-L treating group,but decreased in Transplant control group and TCV group. The result of one-way MLR show that the cpm values in Flt3-L treating group was9 790±1 369 which was higher than Transplant control group which was 3 318±1 190; the cpm values in TCV group was 3 318±1 190 which show that TCV inhibited Flt3-L mediated liver transplant acute immune rejection. The flow cytometric analysis showed that the percentage of apoptosis of TILs was markedly increased in TCV group( 54. 20±3. 12) in comparison with Flt3-L treating group( 3. 34±2. 49%). The apoptosis analysis certified that TCV inhibited the TIL-mediated immune rejecting reaction. Conclusion: Automatic tolerance can be observed at the situation of allogenic liver transplantation in mice,Flt3-L can break the balance of automatic tolerance and mediate acute rejection reaction. Donor-Ag specific T cell vaccination can successfully inhibit acute immune rejection mediated by Flt3-L.
引文
[1]赵振林,郭永章,李立.T细胞疫苗诱导异种特异性免疫耐受的作用探讨[J].中国免疫学杂志,2003,19(6):388-391.
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[7]Morelli AE,Antonysamy MA,Takayama T,et al.Microchimerism,donor dendritic cells,and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells:modulation by tacrolimus[J].J Immunol,2000,165:(1):226-237.
[8]Wei Li,Lina Lu,Zhiliang Wang,et al.Constimulation blocade promotes the apoptotic death of graft-infiltrating T cells and prolongs survival of hepatic allografts from Flt3L-treated donors[J].Transplantation,2001,72(8):1423-1432.
[9]Maraskovsky E,Brasel K,Teepe M,et al.In vivo ad ministration of Ftl3-Ligand results in generation of large numbers of dentritic cells in the lymphoid tissue of mice[J].J Exp Med,1996,184(5):1953-1962.
[10]Steptoe RJ,Fu F,Li W,et al.Augmentayion of dentritic cells in murine organ donor by Flt3-Ligand alters the balance between transplant tolerance and immunity[J].J Imuunol,1997,159(11):5483-5491.
[11]Shapira OM,Mor E,Reshef T,et al.Prolongation of survival of rat cardiac allografts by T cell vaccination[J].J Clin Invest,1993,91(2):388-390.
[12]Kalscheuer H,Onoe T,Dahmani A,et al.Xenograft tolerance and immune function of human T cells developing in pig thymus xenografts[J].J Immunol,2014,192(7):3442-3450.
[13]Hermans G,Denzer U,Lohse A,et al.Cellular and humoral immune response against autoreactive T cells in multiple sclerosis patients after T cell vaccination[J].J Autoimmu,1999,13(2):233-246.
[14]Jiang H,Braunstein NS,Yu B,et al.CD8+T cells control the Thphenotype of MBP-reactive CD4+T cells in EAE mice[J].Proc Natl Acad Sci USA,2001,98(11):6301-6306.
[2]Antonella Sistigu,Sophie Viaud,Nathalie Chaput,et al.Immunomodulatory effects of cyclophosphamide and implementations for vaccine design[J].Seminars Immunopathol,2011,33(4):369-383.
[3]Cohen IR,Weiner HL.T-cell vaccination[J].Immunol Today,1988,9(11):332-335.
[4]吴剑英,王孟龙,吕随峰,等.三袖套法大鼠原位肝移植模型的建立[J].第二军医大学学报,1996,17(2):186-187.
[5]杨扬,邓宜南.抗体免疫诱导治疗在肝移植中的应用[J].临床肝胆病杂志,2015,31(12):2031-2034.
[6]Thomson AW,Lu L,Murase N.Microchimerism,dendritic cell progenitors and transplantation tolerance[J].Stem Cells,1995,13(6):622-639.
[7]Morelli AE,Antonysamy MA,Takayama T,et al.Microchimerism,donor dendritic cells,and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells:modulation by tacrolimus[J].J Immunol,2000,165:(1):226-237.
[8]Wei Li,Lina Lu,Zhiliang Wang,et al.Constimulation blocade promotes the apoptotic death of graft-infiltrating T cells and prolongs survival of hepatic allografts from Flt3L-treated donors[J].Transplantation,2001,72(8):1423-1432.
[9]Maraskovsky E,Brasel K,Teepe M,et al.In vivo ad ministration of Ftl3-Ligand results in generation of large numbers of dentritic cells in the lymphoid tissue of mice[J].J Exp Med,1996,184(5):1953-1962.
[10]Steptoe RJ,Fu F,Li W,et al.Augmentayion of dentritic cells in murine organ donor by Flt3-Ligand alters the balance between transplant tolerance and immunity[J].J Imuunol,1997,159(11):5483-5491.
[11]Shapira OM,Mor E,Reshef T,et al.Prolongation of survival of rat cardiac allografts by T cell vaccination[J].J Clin Invest,1993,91(2):388-390.
[12]Kalscheuer H,Onoe T,Dahmani A,et al.Xenograft tolerance and immune function of human T cells developing in pig thymus xenografts[J].J Immunol,2014,192(7):3442-3450.
[13]Hermans G,Denzer U,Lohse A,et al.Cellular and humoral immune response against autoreactive T cells in multiple sclerosis patients after T cell vaccination[J].J Autoimmu,1999,13(2):233-246.
[14]Jiang H,Braunstein NS,Yu B,et al.CD8+T cells control the Thphenotype of MBP-reactive CD4+T cells in EAE mice[J].Proc Natl Acad Sci USA,2001,98(11):6301-6306.