诱导性调节T细胞的体外扩增及其对小鼠角膜移植免疫排斥的抑制作用
유도성조절T세포적체외확증급기대소서각막이식면역배척적억제작용
In vitro-expansion of induced regulatory T cells and its inhibitory effects on corneal allograft rejection
  • 万方数据
    中华实验眼科杂志 중화실험안과잡지
    Chinese Journal of Experimental Ophthalmology
    2015年 10期 896-901 ,共6页

    魏彤心%李光玲%郭旭明

    위동심%리광령%곽욱명

    CD4+T淋巴细胞/细胞学%调节性T细胞/药物作用%角膜移植/治疗%免疫耐受%动物模型%小鼠 CD4+T淋巴細胞/細胞學%調節性T細胞/藥物作用%角膜移植/治療%免疫耐受%動物模型%小鼠
    CD4+T림파세포/세포학%조절성T세포/약물작용%각막이식/치료%면역내수%동물모형%소서
    CD4-positive T-lymphocytes/cytology%T-lymphocytes,regulatory/drug effects%Corneal transplantation/therapy%Immune tolerance%Disease model,animal%Mice
    背景 研究表明,CD4+CD25+自然调节性T细胞(nTregs)在维持外周免疫耐受及自身免疫平衡中起到重要作用,而体外诱导和扩增的诱导性调节性T细胞(iTregs)可通过过继转移的方式抑制器官移植的免疫排斥反应.目前iTregs的诱导方法仍在不断优化,且其对角膜移植的作用尚不明确.目的 研究iTregs的体外诱导和扩增方法,及其在体内对免疫排斥反应的抑制作用和在体外对效应T细胞(Teffs)增生的抑制作用.方法 从C57B L/6小鼠股骨骨髓组织中提取和培养骨髓来源树突状细胞(BMDCs);取BALB/c小鼠脾脏,磁珠分选CD4+ CD25+T细胞和CD4+ CD25-T细胞,将CD4+ CD25-T细胞分为阴性对照组(单纯CD4+CD25-T细胞)、CD3/28抗体珠组(CD4+ CD25-T细胞+抗鼠CD3/28抗体珠)、2.5 ng/ml转化生长因子-β1(TGF-β1)诱导组和10.0 ng/ml TGF-β1诱导组.在不同质量浓度TGF-β1和抗CD3/CD28抗体珠(细胞和抗体珠比例为1:1)条件下诱导iTregs的生成,并使用抗CD3/CD28抗体珠(细胞和抗体珠比例为1:2)、白细胞介素-2(IL-2)和TGF-β1体外扩增iTregs.采用流式细胞仪检测Tregs各表面因子的表达,采用混合淋巴细胞反应分析体外扩增iTregs对Teffs的抑制能力.建立同种异体小鼠角膜移植模型(C57BL/6→BALB/c),并将模型分为nTregs注射组、iTregs注射组和PBS组;根据分组经小鼠对侧眼球后静脉丛分别注射0.1 ml nTregs、体外扩增iTregs悬液和PBS,观察注射后3个组小鼠植片情况.结果 阴性对照组、CD3/28抗体珠组、2.5 ng/ml TGF-β1诱导组和10.0 ng/ml TGF-β1诱导组中CD4+CD25-T细胞表达CD4+ CD25+T细胞的比例分别为(6±3)％、(91±4)％、(91±3)％和(86±6)％,其中CD3/28抗体珠组、2.5 ng/ml TGF-β1诱导组和10.0 ng/mlTGF-β1诱导组CD4+CD25+T细胞比例明显高于阴性对照组,差异均有统计学意义(均P＜0.01).CD3/28抗体珠组、2.5 ng/ml TGF-β1和10.0 ng/ml TGF-β1诱导组Foxp3+T细胞比例分别为(1.18±0.20)％、(8.70±1.80)％和(21.80±3.36)％,其中2.5 ng/ml TGF-β1诱导组和10.0 ng/ml TGF-β1诱导组Foxp3+T细胞比例明显高于CD3/28抗体珠组,且10.0 ng/ml TGF-β1诱导组Foxp3+T细胞比例明显高于2.5 ng/ml TGF-β1诱导组,差异均有统计学意义(均P＜0.01).体外扩增iTregs中CD69+T细胞比例明显低于nTregs,PD-1+、Foxp3+和CD25+T细胞比例均明显高于nTregs,差异均有统计学意义(均P＜0.01).在1:1、1:2、1:4、1:8和1:16Tregs/Teffs比例条件下,iTregs与Teffs混合培养后Teffs的增生能力明显低于nTregs与Teffs混合培养组,差异均有统计学意义(均P＜0.01).iTregs注射组角膜植片存活时间为4周,永久耐受者占50％,而nTreg组小鼠角膜植片的存活时间为3周,永久耐受者占17％,2个组比较差异有统计学意义(P＜0.05).结论 TGF-β1可诱导CD4+ CD25-T细胞生成iTregs并高表达Foxp3,体外扩增iTregs较nTregs具有更强的抑制淋巴细胞增生能力,从而抑制角膜移植排斥反应的发生.
    배경 연구표명,CD4+CD25+자연조절성T세포(nTregs)재유지외주면역내수급자신면역평형중기도중요작용,이체외유도화확증적유도성조절성T세포(iTregs)가통과과계전이적방식억제기관이식적면역배척반응.목전iTregs적유도방법잉재불단우화,차기대각막이식적작용상불명학.목적 연구iTregs적체외유도화확증방법,급기재체내대면역배척반응적억제작용화재체외대효응T세포(Teffs)증생적억제작용.방법 종C57B L/6소서고골골수조직중제취화배양골수래원수돌상세포(BMDCs);취BALB/c소서비장,자주분선CD4+ CD25+T세포화CD4+ CD25-T세포,장CD4+ CD25-T세포분위음성대조조(단순CD4+CD25-T세포)、CD3/28항체주조(CD4+ CD25-T세포+항서CD3/28항체주)、2.5 ng/ml전화생장인자-β1(TGF-β1)유도조화10.0 ng/ml TGF-β1유도조.재불동질량농도TGF-β1화항CD3/CD28항체주(세포화항체주비례위1:1)조건하유도iTregs적생성,병사용항CD3/CD28항체주(세포화항체주비례위1:2)、백세포개소-2(IL-2)화TGF-β1체외확증iTregs.채용류식세포의검측Tregs각표면인자적표체,채용혼합림파세포반응분석체외확증iTregs대Teffs적억제능력.건립동충이체소서각막이식모형(C57BL/6→BALB/c),병장모형분위nTregs주사조、iTregs주사조화PBS조;근거분조경소서대측안구후정맥총분별주사0.1 ml nTregs、체외확증iTregs현액화PBS,관찰주사후3개조소서식편정황.결과 음성대조조、CD3/28항체주조、2.5 ng/ml TGF-β1유도조화10.0 ng/ml TGF-β1유도조중CD4+CD25-T세포표체CD4+ CD25+T세포적비례분별위(6±3)％、(91±4)％、(91±3)％화(86±6)％,기중CD3/28항체주조、2.5 ng/ml TGF-β1유도조화10.0 ng/mlTGF-β1유도조CD4+CD25+T세포비례명현고우음성대조조,차이균유통계학의의(균P＜0.01).CD3/28항체주조、2.5 ng/ml TGF-β1화10.0 ng/ml TGF-β1유도조Foxp3+T세포비례분별위(1.18±0.20)％、(8.70±1.80)％화(21.80±3.36)％,기중2.5 ng/ml TGF-β1유도조화10.0 ng/ml TGF-β1유도조Foxp3+T세포비례명현고우CD3/28항체주조,차10.0 ng/ml TGF-β1유도조Foxp3+T세포비례명현고우2.5 ng/ml TGF-β1유도조,차이균유통계학의의(균P＜0.01).체외확증iTregs중CD69+T세포비례명현저우nTregs,PD-1+、Foxp3+화CD25+T세포비례균명현고우nTregs,차이균유통계학의의(균P＜0.01).재1:1、1:2、1:4、1:8화1:16Tregs/Teffs비례조건하,iTregs여Teffs혼합배양후Teffs적증생능력명현저우nTregs여Teffs혼합배양조,차이균유통계학의의(균P＜0.01).iTregs주사조각막식편존활시간위4주,영구내수자점50％,이nTreg조소서각막식편적존활시간위3주,영구내수자점17％,2개조비교차이유통계학의의(P＜0.05).결론 TGF-β1가유도CD4+ CD25-T세포생성iTregs병고표체Foxp3,체외확증iTregs교nTregs구유경강적억제림파세포증생능력,종이억제각막이식배척반응적발생.
    Background Researches showed that CD4+CD25+ natural regulatory T cells (nTregs) play an important role in maintaining peripheral immune tolerance, while immunotherapy using in vitro-expanded induced regulatory T cells (iTregs) suppresses allograft rejection in multiple organ transplantation.The inducing method of iTregs still needs to be optimized.Furthermore,the effect of iTregs on grafts of keratoplasty is unclear.Objective This study was to investigate the inducing and expansion method of iTregs and explore its inhibitory effects on corneal allograft rejection.Methods Bone marrow-derived dendritic cells (BMDCs) were isolated from C57BL/6 mice femora and cultured.CD4+ CD25+ T cells and CD4+ CD25-T cells were isolated from mouse spleen and separated using flow cytometry.The CD4+CD25-T cells were divided into negative control group (CD4+CD25-T cells), CD3/ 28 antibody bead group (CD4+CD25-T cells+CD3/28 antibody bead) ,2.5 ng/ml transforming growth factor (TGF)-β1 induced group and 10.0 ng/ml TGF-β1 induced group.The iTregs was formed after induction of different concentrations of TGF-β1 and CD3/CD28 antibody bead (1 : 1).CD3/CD28 antibody bead (1 : 2) , interleukin-2 (IL-2) and TGF-β1 were used to expand iTregs.The phenotype and proliferation of iTregs were assayed by flow cytometry,and the inhibitory effect of iTregs on effector T cells (Teffs) was analyzed by mixed lymphocyte reaction.Allogenic keratoplasty model (C57BL/6→BALB/c) was build,and 0.1 ml iTregs or nTregs suspension or PBS was injected via posterior venous plexus of fellow eyes to assess the graft survival time.The use and care of the mice followed the ARVO statement.Results The CD4+CD25+ T cell proportions were (6±3)％ ,(91±4)％ ,(91±3)％ and (86± 6) ％ in the negative control group,CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1induced group, showing significant increases in the CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group compared with the negative control group (all at P＜0.01).The Foxp3+ T cell proportions of the CD3/CD28 antibody bead group,2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group were (1.18 ±0.20) ％ , (8.70± 1.80) ％ and (21.80±3.36) ％ , showing significant increases in the 2.5 ng/ml TGF-β1 induced group and 10.0 ng/ml TGF-β1 induced group compared with the CD3/CD28 antibody bead group (both at P＜0.01).Compared with the nTregs, the expression of CD69 was lower, and the expressions of PD-1 and Foxp3 were raised in the iTregs (all at P＜0.01).The proliferation of Teffs were decreased when cocultured with iTregs in comparison with nTregs at 1 : 1,1 : 2,1 : 4,1 : 8,1 : 16 Tregs/Teffs rations (all at P＜ 0.01).The survival time of mouse corneal grafts was 4 weeks with the permanent tolerance of 50％ in the iTregs injected group,which was superior to the 3 weeks survival time and 17％ permanent tolerance in the nTregs injected group(P＜0.05).Conclusions TGF-β1 can induce CD4+ CD25-T cells to form iTregs, which highly express Foxp3.iTregs show a stronger inhibitory effect on the growth of lymphocytes than nTregs, and therefore suppress the graft rejection after keratoplasty.
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