TGF-β1体外扩增/诱导的CD4~+CD25~+调节性T细胞逆转和预防慢性移植物抗宿主病鼠狼疮样综合征
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摘要
目的:明确转化生长因子-β1 (transforming growing factor-β1,TGF-β1)在CD4+CD25+调节性T细胞(regulatory T cell,Treg)发挥体内外免疫抑制功能中的作用,分析TGF-β1体外扩增/诱导的Treg细胞在逆转和预防慢性移植物抗宿主病(chronic graft-versus-host disease,cGVHD)鼠狼疮样综合征中的效应,明确Treg细胞体内外扩增和分化的信号通路,为系统性红斑狼疮(systemic lupus erythematosus,SLE)的免疫干预提供新的思路。
方法:采用磁活性标记的细胞分选方法(magnetic activated cell sorting,MACS)分选BALB/c小鼠的CD4+CD25-T和CD4+CD25+T细胞,应用流式细胞术(flow cytometry,FCM)检测分选细胞纯度。建立体外细胞培养体系,应用Trizol试剂提取培养后细胞总RNA ,采用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)方法检测叉状头/翅膀状螺旋转录因子(forkhead/ winged helix transcriptionfactor,Foxp3)的表达水平。同时,采用混合淋巴细胞反应方法,比较新鲜分离的CD4+CD25+Treg、TGF-β1体外扩增的Treg (eTreg)以及TGF-β1体外诱导CD4+CD25-T分化成的Treg (iTreg)细胞的体外抑制效应,应用β液闪烁仪测定CPM值。采用亲代淋巴细胞输注方法建立cGVHD鼠狼疮样综合征模型,以检测血常规(红细胞、白细胞和血小板)、ELISA法检测自身抗体(抗-dsDNA和ANA抗体)、尿蛋白试纸法检测尿蛋白、制备肾标本的石蜡切片、冰冻切片和电镜切片观察病理学改变等作为评价指标。在体外实验的基础上,将新鲜分离的CD4+CD25+Treg、eTreg和iTreg细胞分别于cGVHD鼠狼疮样综合征模型诱导后2周(逆转实验)或诱导前1天(预防实验)输入CB6F1小鼠,前瞻性观测输入不同类型Treg细胞后小鼠的临床表现和实验室指标的变化,评价Treg细胞在体内逆转和预防cGVHD鼠狼疮样综合征中的效应。应用SPSS 11.5软件,定量资料中两组均数比较采用t检验或Mann-Whitney秩和检验,三组及以上组间比较采用方差分析或重复测量数据的方差分析;定性资料的组间比较采用χ2检验或Kruskal-Wallis H检验。应用GraphPad Prism 4.0软件,采用Kaplan-Meier方法计算生存率、log rank方法进行统计学分析。
结果:体外实验结果显示,BALB/c小鼠脾脏单个核细胞经MACS分选后,CD4+CD25+T细胞纯度达98.5%。新鲜分离的CD4+CD25+T细胞在anti-CD3单克隆抗体(monoclonal antibody, mAb)和经过3 000 rad照射的同系基因型C57BL/6小鼠脾细胞的刺激下无反应;而当反应体系中加入IL-2时,细胞增殖为原来的4~5倍;若再加入TGF-β1,细胞增殖1~2倍。而新鲜分离的CD4+CD25-T细胞在anti-CD3 mAb和经照射后C57BL/6脾细胞的刺激下能够增殖;与此相比,当反应体系中加入IL-2,细胞增殖1～2倍;若再加入TGF-β1,细胞增殖效应降至原来的30%~40%。新鲜分离的CD4+CD25-T细胞表达极少量的Foxp3,相似于体外anti-CD3 mAb/ APC/ IL-2/ CD4+CD25-T细胞培养体系中Foxp3的表达水平;当培养体系中再加入TGF-β1,其诱导生成的iTreg细胞表达Foxp3的水平明显提高,近似于新鲜分离的CD4+CD25+T细胞的表达水平;此外,iTreg细胞体外也具有无反应性和免疫抑制性,同样相似于新鲜分离的CD4+CD25+Treg细胞。即外源性TGF-β1可诱导CD4+CD25-T细胞分化为CD4+CD25+Treg细胞,进而发挥免疫抑制效应。剂量反应关系显示,在anti-CD3 mAb/ APC/ IL-2/ TGF-β1/ CD4+CD25-T细胞的培养体系中,CD25和Foxp3的表达水平随TGF-β1剂量的加大而增高,但升高幅度下降,提示1 ng/ml TGF-β1可能是适宜剂量。在混合淋巴细胞反应中,新鲜分离的Treg、eTreg和iTreg细胞均能够明显抑制CD4+CD25-T细胞的增殖;此外,iTreg细胞与应答细胞在1:1,1:2,1:5,1:10或1:20的比例下,均具有免疫抑制效应,且抑制作用与Treg细胞的剂量呈正相关;而当iTreg细胞与应答细胞的混合培养体系中加入anti-TGF-β1时,不但Foxp3的表达水平降低,且iTreg细胞的抑制效应被部分阻断。体内实验结果显示,当采用亲代淋巴细胞输注后,子代CB6F1小鼠可出现尿蛋白;体重降低,皮肤干涩;红细胞、白细胞和血小板计数降低;抗ds-DNA和ANA抗体的OD值上升;以及狼疮肾炎的病理学改变,如肾小球系膜细胞和内皮细胞增生、系膜增宽、基底膜增厚、免疫复合物沉积等;表明本次cGVHD鼠狼疮样综合征模型诱导成功。将体外新鲜分离的CD4+CD25+Treg、TGF-β1作用下的eTreg和iTreg细胞分别于cGVHD鼠狼疮样综合征模型诱导后2周输入CB6F1小鼠,结果显示,输入3组Treg细胞对已出现自身抗体的模型鼠均有明显的逆转效应,小鼠于输注后抗-dsDNA滴度即开始下降,并伴随其他临床症状和实验室指标的逐渐改善;其中以iTreg细胞的体内逆转效应较新鲜分离的CD4+CD25+Treg和eTreg细胞的作用更明显,提示TGF-β1可能作为CD4+CD25+Treg细胞发挥免疫抑制信号的中介,有助于维持体内Treg细胞的数量以及免疫抑制效应的循环。体内预防实验中,将不同剂量的iTreg细胞于cGVHD鼠狼疮样综合征模型诱导前1天输入CB6F1小鼠,结果显示,仅输入1×106 iTreg细胞即能延缓和减轻CB6F1小鼠的发病,但以输入25×106 iTreg细胞对自身免疫反应的抑制效应最明显,该组小鼠的表现与正常对照组的差异无统计学意义,提示iTreg细胞可延缓和预防cGVHD鼠狼疮样综合征的发生。
结论:TGF-β1与外周CD4+CD25+Treg细胞的活化、增殖,以及诱导CD4+CD25-T分化为CD4+CD25+Treg细胞密切相关,在维持Treg细胞体内外的免疫抑制功能方面发挥不可或缺的作用,尤其是TGF-β1诱导生成的iTreg细胞具有更明显的逆转和预防cGVHD鼠狼疮样综合征的效应,研究结果为SLE的免疫干预开辟了新的途径。
Objectives: To analyze and confirm the contribution of TGF-β1 on the function of CD4+CD25+ regulatory T cells (Tregs) in vitro. Furthermore,the important purpose of this study is to identify and compare in vivo suppressive effect of CD4+CD25+Tregs generated in vitro through TGF-β1 on reversing and preventing murine lupus-like syndrome of chronic graft-versus-host disease (cGVHD). Therefore, a better understanding of signals for the generation and development of CD4+CD25+Tregs will probably result in a new immunotherapy for systemic lupus erythematosus.
Methods: CD4+CD25-T and CD4+CD25+T cells of BALB/c murine were separated with MACS and the purity was analyzed by FCM. First of all, we set up different cell culture systems. Total RNA was prepared from cultured cells with Trizol reagent and reverse transcribed into cDNA. And Foxp3 expression was detected by PCR. Also freshly isolated CD4+CD25+Tregs, in vitro-expanded CD4+CD25+Tregs (eTregs) and in vitro -induced CD4+CD25+Tregs (iTregs) conditioned with TGF-β1 were assessed by their abilities to inhibit the proliferation of CD4+CD25-T cell, CPM (cycles per minute) value was detected. Furthermore, we generated murine lupus-like syndrome of cGVHD by parental lymphocyte engraftment, and CB6F1 recipients were monitored for clinical signs by the following parameters: weight of body, red blood cell, white blood cell and platelet, serum anti-dsDNA and ANA autoantibodies measured by ELISA, proteinuria detected by albustix, pathologic changes of kidney tested through light microscope and electron microscope. To assess the reverse effect of different Tregs on murine lupus-like syndrome of cGVHD, freshly isolated CD4+CD25+Tregs, eTregs and iTregs through TGF-β1 were injected into CB6F1 mice after 2 wk of disease induction. And to analyze the preventive effect in vivo, different doses of iTregs were injected on 1 day before disease induction. Statistical analysis was performed by Mann-Whitney rank sum test or Student t test or ANOVA test depending on the dataset using SPSS 11.5 software. GraphPad Prism 4.0 software was used to analyze survival data, survival fractions were calculated by Kaplan-Meier method and compared by log rank test.
Results: The purity of BALB/c CD4+CD25-T and CD4+CD25+T cells were >98%. Freshly isolated CD4+CD25+T cells were unresponsive to anti-CD3 mAb and syngeneic antigen-presenting cells (irradiated C57BL/6 splenic cells), but it could expand up to 4- to 5-fold if IL-2 was added to the culture system. And adding IL-2 and TGF-β1 would lead to only 1- to 2-fold expansion on cells. Compared with the culture system including freshly isolated CD4+CD25-T cells, anti-CD3 mAb and syngeneic APCs, the expansion of cells was 1- to 2-fold in the culture system added with IL-2, but it was only 30% to 40%-fold in the culture system conditioned with IL-2 and TGF-β1. As to the level of Foxp3, freshly isolated CD4+CD25-T cells expressed little Foxp3, as well as CD4+CD25-T cells cultured with anti-CD3 mAb, syngeneic APCs and IL-2 in the absence of TGF-β1. When TGF-β1 was added to the culture protocol above, it led to a significant elevated Foxp3 expression. When these iTregs were re-stimulated with anti-CD3 mAb and APCs, this population did not proliferate in response to the stimulators, which was similar to freshly isolated CD4+CD25+Tregs. The dose-response analysis showed that CD25 and Foxp3 expression were rising with the increase of TGF-β1. And 1 ng/ml TGF-β1 was the most appropriated dose. Freshly isolated CD4+CD25+Tregs, eTregs and iTregs had visible effect on inhibiting the proliferation of CD4+CD25-T cells in vitro. iTregs and responder cells mixed with a different ratio (1:1, 1:2, 1:5, 1:10 or 1:20), could suppress the alloresponse markedly. Furthermore, the addition of graded numbers of iTregs resulted in a positive dose-dependent manner. When anti-TGF-β1 was added to the system of mixed lymphocyte reaction, it would partially block the Foxp3 expression and might impair the suppressive function of iTregs. After parental lymphocyte engraftment, proteinuria, weight loss, reduced RBC (red blood cell), WBC (white blood cell) and PLT (platelet), anti-dsDNA and ANA autoantibodies, glomerulonephritis occured on CB6F1 mice. Our results showed that in vitro-expanded and -induced Tregs by TGF-β1 could retroconverse the morbidity of cGVHD with lupus-like syndrome mice which have already developed autoantibody. And iTregs had more efficient inhibition than that of freshly isolated or eTregs in reversing the clinical symptom of the diseased mice. When comparing the protective effect among different groups which were injected with 25×106 or 5×106 or 1×106 iTregs respectively, the difference was distinguished. And the best inhibition on autoimmunity was mediated by 25 million iTregs, and there was no pathologic effect in this group.
Conclusions: We established an essential correlation between TGF-β1 and CD4+CD25+Tregs’activation, expansion, as well as induction from precursor cells. It indicates that TGF-β1 signaling is required to maintain the suppressive effect of CD4+CD25+Tregs in vitro and in vivo. Importantly, iTregs have more inhibitive function on reversing an established disease and preventing the onset of lupus-like syndrome of cGVHD. In a word, the present study raises a possibility that CD4+CD25+Tregs induced by TGF-β1 in vitro have the potential that is used as an immunotherapy agent to control SLE.
方法:采用磁活性标记的细胞分选方法(magnetic activated cell sorting,MACS)分选BALB/c小鼠的CD4+CD25-T和CD4+CD25+T细胞,应用流式细胞术(flow cytometry,FCM)检测分选细胞纯度。建立体外细胞培养体系,应用Trizol试剂提取培养后细胞总RNA ,采用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)方法检测叉状头/翅膀状螺旋转录因子(forkhead/ winged helix transcriptionfactor,Foxp3)的表达水平。同时,采用混合淋巴细胞反应方法,比较新鲜分离的CD4+CD25+Treg、TGF-β1体外扩增的Treg (eTreg)以及TGF-β1体外诱导CD4+CD25-T分化成的Treg (iTreg)细胞的体外抑制效应,应用β液闪烁仪测定CPM值。采用亲代淋巴细胞输注方法建立cGVHD鼠狼疮样综合征模型,以检测血常规(红细胞、白细胞和血小板)、ELISA法检测自身抗体(抗-dsDNA和ANA抗体)、尿蛋白试纸法检测尿蛋白、制备肾标本的石蜡切片、冰冻切片和电镜切片观察病理学改变等作为评价指标。在体外实验的基础上,将新鲜分离的CD4+CD25+Treg、eTreg和iTreg细胞分别于cGVHD鼠狼疮样综合征模型诱导后2周(逆转实验)或诱导前1天(预防实验)输入CB6F1小鼠,前瞻性观测输入不同类型Treg细胞后小鼠的临床表现和实验室指标的变化,评价Treg细胞在体内逆转和预防cGVHD鼠狼疮样综合征中的效应。应用SPSS 11.5软件,定量资料中两组均数比较采用t检验或Mann-Whitney秩和检验,三组及以上组间比较采用方差分析或重复测量数据的方差分析;定性资料的组间比较采用χ2检验或Kruskal-Wallis H检验。应用GraphPad Prism 4.0软件,采用Kaplan-Meier方法计算生存率、log rank方法进行统计学分析。
结果:体外实验结果显示,BALB/c小鼠脾脏单个核细胞经MACS分选后,CD4+CD25+T细胞纯度达98.5%。新鲜分离的CD4+CD25+T细胞在anti-CD3单克隆抗体(monoclonal antibody, mAb)和经过3 000 rad照射的同系基因型C57BL/6小鼠脾细胞的刺激下无反应;而当反应体系中加入IL-2时,细胞增殖为原来的4~5倍;若再加入TGF-β1,细胞增殖1~2倍。而新鲜分离的CD4+CD25-T细胞在anti-CD3 mAb和经照射后C57BL/6脾细胞的刺激下能够增殖;与此相比,当反应体系中加入IL-2,细胞增殖1～2倍;若再加入TGF-β1,细胞增殖效应降至原来的30%~40%。新鲜分离的CD4+CD25-T细胞表达极少量的Foxp3,相似于体外anti-CD3 mAb/ APC/ IL-2/ CD4+CD25-T细胞培养体系中Foxp3的表达水平;当培养体系中再加入TGF-β1,其诱导生成的iTreg细胞表达Foxp3的水平明显提高,近似于新鲜分离的CD4+CD25+T细胞的表达水平;此外,iTreg细胞体外也具有无反应性和免疫抑制性,同样相似于新鲜分离的CD4+CD25+Treg细胞。即外源性TGF-β1可诱导CD4+CD25-T细胞分化为CD4+CD25+Treg细胞,进而发挥免疫抑制效应。剂量反应关系显示,在anti-CD3 mAb/ APC/ IL-2/ TGF-β1/ CD4+CD25-T细胞的培养体系中,CD25和Foxp3的表达水平随TGF-β1剂量的加大而增高,但升高幅度下降,提示1 ng/ml TGF-β1可能是适宜剂量。在混合淋巴细胞反应中,新鲜分离的Treg、eTreg和iTreg细胞均能够明显抑制CD4+CD25-T细胞的增殖;此外,iTreg细胞与应答细胞在1:1,1:2,1:5,1:10或1:20的比例下,均具有免疫抑制效应,且抑制作用与Treg细胞的剂量呈正相关;而当iTreg细胞与应答细胞的混合培养体系中加入anti-TGF-β1时,不但Foxp3的表达水平降低,且iTreg细胞的抑制效应被部分阻断。体内实验结果显示,当采用亲代淋巴细胞输注后,子代CB6F1小鼠可出现尿蛋白;体重降低,皮肤干涩;红细胞、白细胞和血小板计数降低;抗ds-DNA和ANA抗体的OD值上升;以及狼疮肾炎的病理学改变,如肾小球系膜细胞和内皮细胞增生、系膜增宽、基底膜增厚、免疫复合物沉积等;表明本次cGVHD鼠狼疮样综合征模型诱导成功。将体外新鲜分离的CD4+CD25+Treg、TGF-β1作用下的eTreg和iTreg细胞分别于cGVHD鼠狼疮样综合征模型诱导后2周输入CB6F1小鼠,结果显示,输入3组Treg细胞对已出现自身抗体的模型鼠均有明显的逆转效应,小鼠于输注后抗-dsDNA滴度即开始下降,并伴随其他临床症状和实验室指标的逐渐改善;其中以iTreg细胞的体内逆转效应较新鲜分离的CD4+CD25+Treg和eTreg细胞的作用更明显,提示TGF-β1可能作为CD4+CD25+Treg细胞发挥免疫抑制信号的中介,有助于维持体内Treg细胞的数量以及免疫抑制效应的循环。体内预防实验中,将不同剂量的iTreg细胞于cGVHD鼠狼疮样综合征模型诱导前1天输入CB6F1小鼠,结果显示,仅输入1×106 iTreg细胞即能延缓和减轻CB6F1小鼠的发病,但以输入25×106 iTreg细胞对自身免疫反应的抑制效应最明显,该组小鼠的表现与正常对照组的差异无统计学意义,提示iTreg细胞可延缓和预防cGVHD鼠狼疮样综合征的发生。
结论:TGF-β1与外周CD4+CD25+Treg细胞的活化、增殖,以及诱导CD4+CD25-T分化为CD4+CD25+Treg细胞密切相关,在维持Treg细胞体内外的免疫抑制功能方面发挥不可或缺的作用,尤其是TGF-β1诱导生成的iTreg细胞具有更明显的逆转和预防cGVHD鼠狼疮样综合征的效应,研究结果为SLE的免疫干预开辟了新的途径。
Objectives: To analyze and confirm the contribution of TGF-β1 on the function of CD4+CD25+ regulatory T cells (Tregs) in vitro. Furthermore,the important purpose of this study is to identify and compare in vivo suppressive effect of CD4+CD25+Tregs generated in vitro through TGF-β1 on reversing and preventing murine lupus-like syndrome of chronic graft-versus-host disease (cGVHD). Therefore, a better understanding of signals for the generation and development of CD4+CD25+Tregs will probably result in a new immunotherapy for systemic lupus erythematosus.
Methods: CD4+CD25-T and CD4+CD25+T cells of BALB/c murine were separated with MACS and the purity was analyzed by FCM. First of all, we set up different cell culture systems. Total RNA was prepared from cultured cells with Trizol reagent and reverse transcribed into cDNA. And Foxp3 expression was detected by PCR. Also freshly isolated CD4+CD25+Tregs, in vitro-expanded CD4+CD25+Tregs (eTregs) and in vitro -induced CD4+CD25+Tregs (iTregs) conditioned with TGF-β1 were assessed by their abilities to inhibit the proliferation of CD4+CD25-T cell, CPM (cycles per minute) value was detected. Furthermore, we generated murine lupus-like syndrome of cGVHD by parental lymphocyte engraftment, and CB6F1 recipients were monitored for clinical signs by the following parameters: weight of body, red blood cell, white blood cell and platelet, serum anti-dsDNA and ANA autoantibodies measured by ELISA, proteinuria detected by albustix, pathologic changes of kidney tested through light microscope and electron microscope. To assess the reverse effect of different Tregs on murine lupus-like syndrome of cGVHD, freshly isolated CD4+CD25+Tregs, eTregs and iTregs through TGF-β1 were injected into CB6F1 mice after 2 wk of disease induction. And to analyze the preventive effect in vivo, different doses of iTregs were injected on 1 day before disease induction. Statistical analysis was performed by Mann-Whitney rank sum test or Student t test or ANOVA test depending on the dataset using SPSS 11.5 software. GraphPad Prism 4.0 software was used to analyze survival data, survival fractions were calculated by Kaplan-Meier method and compared by log rank test.
Results: The purity of BALB/c CD4+CD25-T and CD4+CD25+T cells were >98%. Freshly isolated CD4+CD25+T cells were unresponsive to anti-CD3 mAb and syngeneic antigen-presenting cells (irradiated C57BL/6 splenic cells), but it could expand up to 4- to 5-fold if IL-2 was added to the culture system. And adding IL-2 and TGF-β1 would lead to only 1- to 2-fold expansion on cells. Compared with the culture system including freshly isolated CD4+CD25-T cells, anti-CD3 mAb and syngeneic APCs, the expansion of cells was 1- to 2-fold in the culture system added with IL-2, but it was only 30% to 40%-fold in the culture system conditioned with IL-2 and TGF-β1. As to the level of Foxp3, freshly isolated CD4+CD25-T cells expressed little Foxp3, as well as CD4+CD25-T cells cultured with anti-CD3 mAb, syngeneic APCs and IL-2 in the absence of TGF-β1. When TGF-β1 was added to the culture protocol above, it led to a significant elevated Foxp3 expression. When these iTregs were re-stimulated with anti-CD3 mAb and APCs, this population did not proliferate in response to the stimulators, which was similar to freshly isolated CD4+CD25+Tregs. The dose-response analysis showed that CD25 and Foxp3 expression were rising with the increase of TGF-β1. And 1 ng/ml TGF-β1 was the most appropriated dose. Freshly isolated CD4+CD25+Tregs, eTregs and iTregs had visible effect on inhibiting the proliferation of CD4+CD25-T cells in vitro. iTregs and responder cells mixed with a different ratio (1:1, 1:2, 1:5, 1:10 or 1:20), could suppress the alloresponse markedly. Furthermore, the addition of graded numbers of iTregs resulted in a positive dose-dependent manner. When anti-TGF-β1 was added to the system of mixed lymphocyte reaction, it would partially block the Foxp3 expression and might impair the suppressive function of iTregs. After parental lymphocyte engraftment, proteinuria, weight loss, reduced RBC (red blood cell), WBC (white blood cell) and PLT (platelet), anti-dsDNA and ANA autoantibodies, glomerulonephritis occured on CB6F1 mice. Our results showed that in vitro-expanded and -induced Tregs by TGF-β1 could retroconverse the morbidity of cGVHD with lupus-like syndrome mice which have already developed autoantibody. And iTregs had more efficient inhibition than that of freshly isolated or eTregs in reversing the clinical symptom of the diseased mice. When comparing the protective effect among different groups which were injected with 25×106 or 5×106 or 1×106 iTregs respectively, the difference was distinguished. And the best inhibition on autoimmunity was mediated by 25 million iTregs, and there was no pathologic effect in this group.
Conclusions: We established an essential correlation between TGF-β1 and CD4+CD25+Tregs’activation, expansion, as well as induction from precursor cells. It indicates that TGF-β1 signaling is required to maintain the suppressive effect of CD4+CD25+Tregs in vitro and in vivo. Importantly, iTregs have more inhibitive function on reversing an established disease and preventing the onset of lupus-like syndrome of cGVHD. In a word, the present study raises a possibility that CD4+CD25+Tregs induced by TGF-β1 in vitro have the potential that is used as an immunotherapy agent to control SLE.
引文
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