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

魏彤心; 李光玲; 郭旭明

doi:10.3760/cma.j.issn.2095-0160.2015.10.007

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中华实验眼科杂志

• 实验研究 •

ENGLISH ABSTRACT

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

作者及单位信息

出版日期： 2015 -10 -10 ·

DOI： 10.3760/cma.j.issn.2095-0160.2015.10.007

In vitro-expansion of induced regulatory T cells and its inhibitory effects on corneal allograft rejection

Authors Info & Affiliations

Wei Tongxin

Shanxi Eye Hospital, Taiyuan 030002, China

Li Guangling

Guo Xuming

Published： 2015 -10 -10 ·

DOI： 10.3760/cma.j.issn.2095-0160.2015.10.007

322

APP内阅读

摘要

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C57BL/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-β ₁(TGF-β ₁)诱导组和10.0 ng/ml TGF-β ₁诱导组。在不同质量浓度TGF-β ₁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-β ₁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-β ₁诱导组和10.0 ng/ml TGF-β ₁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-β ₁诱导组和10.0 ng/ml TGF-β ₁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-β ₁和10.0 ng/ml TGF-β ₁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-β ₁诱导组和10.0 ng/ml TGF-β ₁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-β ₁诱导组Foxp3 ^＋ T细胞比例明显高于2.5 ng/ml TGF-β ₁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∶16 Tregs/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-β ₁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。

关键词： 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

ABSTRACT

BackgroundResearches 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.

ObjectiveThis study was to investigate the inducing and expansion method of iTregs and explore its inhibitory effects on corneal allograft rejection.

MethodsBone 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)-β ₁ induced group and 10.0 ng/ml TGF-β ₁ induced group.The iTregs was formed after induction of different concentrations of TGF-β ₁ and CD3/CD28 antibody bead (1∶1). CD3/CD28 antibody bead (1∶2), interleukin-2 (IL-2) and TGF-β ₁ 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.

ResultsThe 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-β ₁ induced group and 10.0 ng/ml TGF-β ₁ induced group, showing significant increases in the CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β ₁ induced group and 10.0 ng/ml TGF-β ₁ 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-β ₁ induced group and 10.0 ng/ml TGF-β ₁ 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-β ₁ induced group and 10.0 ng/ml TGF-β ₁ 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).

ConclusionsTGF-β ₁ 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.

KEYWORDS： CD4-positive T-lymphocytes/cytology;T-lymphocytes, regulatory/drug effects;Corneal transplantation/therapy;Immune tolerance;Disease model, animal;Mice

Corresponding author: Guo Xuming, Email: mocdef.3ab614211gnimuxoug

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引用本文

魏彤心,李光玲,郭旭明. 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[J]. 中华实验眼科杂志,2015,33(10)：896-901.

DOI:10.3760/cma.j.issn.2095-0160.2015.10.007

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角膜盲是眼科主要可治疗盲之一，穿透角膜移植术是目前主要的治疗方法。尽管角膜是器官移植的免疫赦免部位，但同种异体免疫排斥反应仍然是角膜移植术失败的主要原因，因此降低角膜移植术后免疫排斥反应，达到角膜植片长期存活尤为重要。调节性T细胞(regulatory T cells，Tregs)是一类具有免疫负向调节作用的细胞总称，表型为CD4 ^＋CD25 ^＋，其特异性标志物为Foxp3，其中自然调节性T细胞(natural Tregs，nTregs)在维持外周免疫耐受及自身免疫平衡方面发挥重要作用，而体外诱导和扩增的诱导性Tregs(induced Tregs，iTregs)则起到加强和过继nTregs的作用。效应T细胞(effector T cells，Teffs)是一类具有免疫正向调节作用的细胞总称，表型为CD4 ^＋CD25 ^－，Tregs与Teffs间的平衡对于预防和控制自身免疫性疾病的发生和发展至关重要。研究显示，将BALB/c小鼠来源的CD4 ^＋TCD25 ^－ T细胞注射到BALB/c无胸腺裸鼠体内可引起裸鼠发生自身免疫性疾病 ^{[
1
]}，表明外周血和组织中胸腺来源的nTregs在啮齿动物和人类免疫耐受中起重要作用。多项研究已证实，Tregs表面可表达肿瘤坏死因子(tumor necrosis factor，TNF)受体，并表现出免疫相关调节功能 ^{[
2
,

3
,

4
]}。治疗自身免疫性疾病的理想方法并不是去除自身Teffs，而是减少其细胞数量，尤其是病理性和记忆性T细胞，更重要的是诱导和扩增Tregs细胞数量，调节Tregs和Teffs之间的平衡。多种方式参与调节Tregs和Teffs之间的平衡，如CD3、CD4和CD40L单克隆抗体治疗可以减少抗原特异性T细胞的数量而不影响Tregs，从而达到Tregs占主导的免疫状态；Akt-mTOR通道抑制剂雷帕霉素可增加Tregs的Foxp3转录 ^{[
5
]}；组蛋白去乙酰基酶抑制剂曲古霉素A可促进Foxp3的乙酰化，从而促进Foxp3 ^＋ Tregs的扩增 ^{[
6
]}；目前有多种方法可以生成iTregs以治疗自身免疫性疾病、过敏、炎性疾病和移植耐受等疾病 ^{[
7
]}。本研究中探讨iTregs的诱导过程及其对鼠角膜移植排斥反应的治疗作用，为细胞疗法在临床上的应用提供实验依据。

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参考文献

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Sakaguchi S , Ono M , Setoquchi R ,et al. Foxp3 ^＋ CD25 ^＋ CD4 ^＋ natural regulatory T cells in dominant self-tolerance and autoimmune disease [J]．Immunol Rev, 2006,212:8-27. doi： 10.1111/j.0105-2896.2006.00427.x .