不同诱导方法所得Treg样细胞的多方面比较
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摘要
目的
建立MSCV-FOXP3转导法和TGF-β诱导法获取调节性T淋巴细胞(regulatory T cells, Treg)样细胞的方法体系,比较两种方法获得Treg样细胞的产率及其免疫抑制功能的差异,并初步探讨两种Treg样细胞发挥免疫抑制作用相关机制的差异。
方法
1.构建重组逆转录病毒质粒pMSCV-FOXP3,转染PT67细胞,建立可稳定产毒单克隆细胞株；采用差速离心法对病毒进行浓缩。
2.免疫磁珠分选法获取CD4+CD25-T淋巴细胞；以CD4+CD25-T淋巴细胞为研究对象,采用优化后实验方法进行MSCV-FOXP3转导和TGF-β诱导获取Treg样细胞,以流式细胞术和Real time PCR技术对两种方法获得Treg样细胞的效率进行检测和比较。
3.应用活性染料CFSE染色和流式细胞术观察两种Treg样细胞和天然Treg细胞对自体CD4+T淋巴细胞增殖活性的影响,用ModFit软件分析CD4+T淋巴细胞增殖动力学模型。
4.应用ELISA技术,检测两种Treg样细胞培养上清IL-10和TGF-β的分泌水平；应用Real time PCR技术检测两种Treg样细胞颗粒酶A和颗粒酶B mRNA的转录水平差异。
结果
1.成功构建pMSCV-FOXP3重组质粒,转染PT67细胞,筛选建立了稳定产毒单克隆细胞株,最高病毒滴度为6×105cfu/ml。浓缩后病毒滴度可达1.2×107cfu／ml。
2.免疫磁珠分离获得CD4+CD25-T淋巴细胞,流式细胞术检测其纯度为92.46%,CD4+CD25+T淋巴细胞纯度为90.82%,台盼蓝染色检测细胞存活率高于90%。当病毒滴度为1×107cfu/ml时,诱导获得的Treg样细胞产率最高,可达49.12%；当TGF-β为5ng/ml,诱导时间为第6天时,诱导获得的Treg样细胞产率最高,可达41.08%；MSCV-FOXP3转导组CD4+T淋巴细胞的FOXP3 mRNA表达水平高于TGF-β组(P<0.05),MSCV-FOXP3转导组CD4+T淋巴细胞的FOXP3蛋白阳性率高于TGF-β组(P<0.05)。
3.流式细胞术检测结果显示,MSCV-FOXP3转导CD4+T淋巴细胞获得的Treg样细胞、TGF-β诱导获得的Treg样细胞以及天然型Treg细胞对自体CD4+T淋巴细胞的增殖均具有明显抑制作用,与对照组相比均具有显著性差异(P<0.05)。
4.MSCV-FOXP3转导CD4+T淋巴细胞获得的Treg样细胞与TGF-β诱导获得的Treg样细胞分泌IL-10水平明显上升,与对照组相比均具有显著性差异(P<0.05)。TGF-β诱导组和MSCV-FOXP3转导组TGF-β分泌水平较对照组无明显差异(P>0.05)。颗粒酶B在TGF-β诱导获得Treg样细胞表达水平较对照组明显升高(P<0.05)。颗粒酶B在TGF-β诱导获得Treg样细胞表达水平较显著高于MSCV-FOXP3转导组(P<0.05)。
结论
MSCV-FOXP3转导法和TGF-β诱导法均可在体外获得具有一定免疫抑制功能的Treg样细胞,但MSCV-FOXP3转导法的Treg样细胞产率要高于TGF-β诱导法,其获得的Treg样细胞免疫抑制活性可能高于TGF-β诱导法。在两种Treg样细胞发挥免疫抑制功能的过程中,IL-10可能均参与其中,而颗粒酶B可能只参与了TGF-β诱导的Treg样细胞发挥免疫抑制功能的过程。
Objective
To creat the methods of obtaining Treg-like cells via Retrovirus mediated gene transduction and TGF-βinduction. To compare the yield efficiency and activity of Treg-like cells of two methods and explore its relative mechanisms of immunosuppression.
Methods
1.FOXP3 gene was reconstructed in retrovirus vector and transferred into PT67 by lipofectamine mediated method. Stable monoclonal cell line which secreting high titer of MSCV-FOXP3 were selected. To further enhance the titer of retrovirus, the viral supernatant was concentrated by centrifugation.
2.The CD4+CD25-T cells were separated from PBMC by using MACS.The optimized methods were respectively applied in MSCV-FOXP3 transduction and TGF-βinduction to acquire Treg-like cells. Compare the higher yield efficiency of two methods and real-time quantitative PCR was used to detect the difference of FOXP3 mRNA expression.
3.CFSE staining and Flow cytometry assay was used to detect the influence of nature Treg and Treg-like cells on CD4+T proliferation. ModFit software was used to analysis the dynamic model of CD4+T proliferation.
4. The level of IL-10 and TGF-βsecreted from Treg-like cells was assayed by ELISA method. To compare the mRNA level difference of granzyme A and granzyme B which express from Treg-like cells, real-time quantitative PCR was used to observe them.
Result
1.The recombinant plasmid pMSCV-FOXP3 was constructed and transferred into PT67. The stable retrovirus-producing cell line which produces high titer retrovirus was selected. The titer of MSCV-FOXP3 can reach to 1.2×107cfu/ml by concentration.
2.The purity of CD4+CD25+ and CD4+CD25-T cells were 90.82% and 92.46% respectively identified by flow cytometry. The vitality of isolated T cells was above 90% by Trypan blue exclusion test. When the viral titer was 1×107cfu/ml, The Treg-like cells attained the highest yield efficiency of 49.12%.In the other method, while TGF-B was 5ng/ml and time at 6th days, the induction of the Treg-like cells gain the highest yield efficiency of 41.08%.The yield efficiency of the Treg-like cells in MSCV-FOXP3 transduction group was higher than it in TGF-βinduction group (P<0.05).The mRNA level of FOXP3 in MSCV-FOXP3 transduction group is high than it inTGF-βinduction group (P<0.05).
3.Compared with control group, the nTreg and Treg-like cells significantly inhibited the proliferation of autologous CD4+T cells (P<0.05).
4. The level of IL-10 was up-regulated significantly in the two groups of Treg-like cells compared with control group (P<0.05).There was no statistic difference of secrecting TGF-βin the two groups of Treg-like cells compared with control group (P>0.05).The mRNA level of Granzyme B in TGF-βinduction group of Treg-like cells was higher than its in control group (P<0.05).
Conclusion
The Treg-like cells having the ability of immunosupression can be obtained with the method of MSCV-FOXP3 transduction and the method of TGF-βinduction, While the higher yield efficiency was obtained with the method of MSCV-FOXP3 transduction and the ability of immunosupression of Treg-like cells with the method of MSCV-FOXP3 transduction was higher than the latter. IL-10 may join the proess of fuctioning immunosupression in two groups, and granzyme B maybe one factor of fuctioning immunosupression in Treg-like cells of TGF-βinduction group.
建立MSCV-FOXP3转导法和TGF-β诱导法获取调节性T淋巴细胞(regulatory T cells, Treg)样细胞的方法体系,比较两种方法获得Treg样细胞的产率及其免疫抑制功能的差异,并初步探讨两种Treg样细胞发挥免疫抑制作用相关机制的差异。
方法
1.构建重组逆转录病毒质粒pMSCV-FOXP3,转染PT67细胞,建立可稳定产毒单克隆细胞株；采用差速离心法对病毒进行浓缩。
2.免疫磁珠分选法获取CD4+CD25-T淋巴细胞；以CD4+CD25-T淋巴细胞为研究对象,采用优化后实验方法进行MSCV-FOXP3转导和TGF-β诱导获取Treg样细胞,以流式细胞术和Real time PCR技术对两种方法获得Treg样细胞的效率进行检测和比较。
3.应用活性染料CFSE染色和流式细胞术观察两种Treg样细胞和天然Treg细胞对自体CD4+T淋巴细胞增殖活性的影响,用ModFit软件分析CD4+T淋巴细胞增殖动力学模型。
4.应用ELISA技术,检测两种Treg样细胞培养上清IL-10和TGF-β的分泌水平；应用Real time PCR技术检测两种Treg样细胞颗粒酶A和颗粒酶B mRNA的转录水平差异。
结果
1.成功构建pMSCV-FOXP3重组质粒,转染PT67细胞,筛选建立了稳定产毒单克隆细胞株,最高病毒滴度为6×105cfu/ml。浓缩后病毒滴度可达1.2×107cfu／ml。
2.免疫磁珠分离获得CD4+CD25-T淋巴细胞,流式细胞术检测其纯度为92.46%,CD4+CD25+T淋巴细胞纯度为90.82%,台盼蓝染色检测细胞存活率高于90%。当病毒滴度为1×107cfu/ml时,诱导获得的Treg样细胞产率最高,可达49.12%；当TGF-β为5ng/ml,诱导时间为第6天时,诱导获得的Treg样细胞产率最高,可达41.08%；MSCV-FOXP3转导组CD4+T淋巴细胞的FOXP3 mRNA表达水平高于TGF-β组(P<0.05),MSCV-FOXP3转导组CD4+T淋巴细胞的FOXP3蛋白阳性率高于TGF-β组(P<0.05)。
3.流式细胞术检测结果显示,MSCV-FOXP3转导CD4+T淋巴细胞获得的Treg样细胞、TGF-β诱导获得的Treg样细胞以及天然型Treg细胞对自体CD4+T淋巴细胞的增殖均具有明显抑制作用,与对照组相比均具有显著性差异(P<0.05)。
4.MSCV-FOXP3转导CD4+T淋巴细胞获得的Treg样细胞与TGF-β诱导获得的Treg样细胞分泌IL-10水平明显上升,与对照组相比均具有显著性差异(P<0.05)。TGF-β诱导组和MSCV-FOXP3转导组TGF-β分泌水平较对照组无明显差异(P>0.05)。颗粒酶B在TGF-β诱导获得Treg样细胞表达水平较对照组明显升高(P<0.05)。颗粒酶B在TGF-β诱导获得Treg样细胞表达水平较显著高于MSCV-FOXP3转导组(P<0.05)。
结论
MSCV-FOXP3转导法和TGF-β诱导法均可在体外获得具有一定免疫抑制功能的Treg样细胞,但MSCV-FOXP3转导法的Treg样细胞产率要高于TGF-β诱导法,其获得的Treg样细胞免疫抑制活性可能高于TGF-β诱导法。在两种Treg样细胞发挥免疫抑制功能的过程中,IL-10可能均参与其中,而颗粒酶B可能只参与了TGF-β诱导的Treg样细胞发挥免疫抑制功能的过程。
Objective
To creat the methods of obtaining Treg-like cells via Retrovirus mediated gene transduction and TGF-βinduction. To compare the yield efficiency and activity of Treg-like cells of two methods and explore its relative mechanisms of immunosuppression.
Methods
1.FOXP3 gene was reconstructed in retrovirus vector and transferred into PT67 by lipofectamine mediated method. Stable monoclonal cell line which secreting high titer of MSCV-FOXP3 were selected. To further enhance the titer of retrovirus, the viral supernatant was concentrated by centrifugation.
2.The CD4+CD25-T cells were separated from PBMC by using MACS.The optimized methods were respectively applied in MSCV-FOXP3 transduction and TGF-βinduction to acquire Treg-like cells. Compare the higher yield efficiency of two methods and real-time quantitative PCR was used to detect the difference of FOXP3 mRNA expression.
3.CFSE staining and Flow cytometry assay was used to detect the influence of nature Treg and Treg-like cells on CD4+T proliferation. ModFit software was used to analysis the dynamic model of CD4+T proliferation.
4. The level of IL-10 and TGF-βsecreted from Treg-like cells was assayed by ELISA method. To compare the mRNA level difference of granzyme A and granzyme B which express from Treg-like cells, real-time quantitative PCR was used to observe them.
Result
1.The recombinant plasmid pMSCV-FOXP3 was constructed and transferred into PT67. The stable retrovirus-producing cell line which produces high titer retrovirus was selected. The titer of MSCV-FOXP3 can reach to 1.2×107cfu/ml by concentration.
2.The purity of CD4+CD25+ and CD4+CD25-T cells were 90.82% and 92.46% respectively identified by flow cytometry. The vitality of isolated T cells was above 90% by Trypan blue exclusion test. When the viral titer was 1×107cfu/ml, The Treg-like cells attained the highest yield efficiency of 49.12%.In the other method, while TGF-B was 5ng/ml and time at 6th days, the induction of the Treg-like cells gain the highest yield efficiency of 41.08%.The yield efficiency of the Treg-like cells in MSCV-FOXP3 transduction group was higher than it in TGF-βinduction group (P<0.05).The mRNA level of FOXP3 in MSCV-FOXP3 transduction group is high than it inTGF-βinduction group (P<0.05).
3.Compared with control group, the nTreg and Treg-like cells significantly inhibited the proliferation of autologous CD4+T cells (P<0.05).
4. The level of IL-10 was up-regulated significantly in the two groups of Treg-like cells compared with control group (P<0.05).There was no statistic difference of secrecting TGF-βin the two groups of Treg-like cells compared with control group (P>0.05).The mRNA level of Granzyme B in TGF-βinduction group of Treg-like cells was higher than its in control group (P<0.05).
Conclusion
The Treg-like cells having the ability of immunosupression can be obtained with the method of MSCV-FOXP3 transduction and the method of TGF-βinduction, While the higher yield efficiency was obtained with the method of MSCV-FOXP3 transduction and the ability of immunosupression of Treg-like cells with the method of MSCV-FOXP3 transduction was higher than the latter. IL-10 may join the proess of fuctioning immunosupression in two groups, and granzyme B maybe one factor of fuctioning immunosupression in Treg-like cells of TGF-βinduction group.
引文
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[5]Wang J, Ioan-Facsinay Avander Voort E, Huizinga TW, Toes RE.Transient expression of FOXP3 in human activated nonregulatory CD4(+)T cells [J].Eur J Immunol,2007,37(1):129-138.
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[9]Prieto GA, Rosenstein Y. Oestradiol potentiates the suppressive function of human CD4 CD25 regulatory T cells by promoting their proliferation [J]. Immunology,2006,118(1):58-65.
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[1]Horwitz DA, Zheng SG, Wang J et al. Critical role of IL-2 and TGF-beta in generation, function and stabilization of Foxp3+CD4+ Treg [J].Eur J Immunol, 2008,38:912-915.
[2]Gupta S,Shang W, Sun Z.Mechanisms regulating the development and function of natural regulatory T cells [J].ArchImmunol Ther Exp,2008,56:85-102
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[4]Lio CW, Hsieh CS.A two-step process for thymic regulatory T cell development [J].Immunity,2008,28:100-111.
[5]Wang J, Ioan-Facsinay Avander Voort E, Huizinga TW, Toes RE.Transient expression of FOXP3 in human activated nonregulatory CD4(+)T cells [J].Eur J Immunol,2007,37(1):129-138.
[6]Tang Q, Henriksen KJ, Bi M et al. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes [J].J Exp Med,2004,199:1455-1465.
[7]Tarbell KV, Yamazaki S,Olson K et al. CD25+CD4+T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes [J].J ExpMed,2004,199:1467-1477.
[8]Battaglia M, Stabilini A, Migliavacca B et al. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+regulatory T cells of both healthy subjects and type 1 diabetic patients [J].J Immunol,2006,177:8338-8347.
[9]Prieto GA, Rosenstein Y. Oestradiol potentiates the suppressive function of human CD4 CD25 regulatory T cells by promoting their proliferation [J]. Immunology,2006,118(1):58-65.
[10]Zheng SG, Wang JH, Gray JD et al. Natural and induced CD4+CD25+ cells educate CD4+CD25-cells to develop suppressive activity:the role of IL-2, TGF-beta, and IL-10.[J]JImmunol,2004,172:5213-5221.
[11]Wang Z, Hong J, Sun W et al.Role of IFN-gamma in induction of Foxp3 and conversion of CD4+CD25-T cells to CD4+ Tregs [J].J Clin Invest,2006, 116:2434-2441.
[12]Hong J, Li N, Zhang X et al. Induction of CD4+CD25+regulatory T cells by copolymer through activation of transcription factor Foxp3 [J].Proc Natl Acad Sci,2005,102:6449-6454.
[13]Luo X, Tarbell KV, Yang H et al.Dendritic cells with TGF-betal differentiate naive CD4+CD25-T cells into isletprotective Foxp3+ regulatory T cells [J]. Proc Natl Acad Sci,2007,104:2821-2826.
[14]Coombes JL, Siddiqui KR, rancibia-Carcamo CV et al. A functionally specialized population of mucosal CD103+DCs induces Foxp3+ regulatory T cells via a TGF-beta andretinoic acid-dependent mechanism [J].J Exp Med, 2007,204:1757-1764.
[15]Selvaraj RK, Geiger TL.A kinetic and dynamic analysisof Foxp3 induced in T cells by TGF-beta [J].J Immunol,2007,178:7667-7677.
[16]Brandenburg S,Takahashi T, de la RM et al.IL-2 induces in vivo suppression by CD4(+) CD25(+)Foxp3(+)regulatory T cells [J].Eur J Immunol,2008, 38:1643-1653.
[17]Wang Z, Hong J, Sun W et al.Role of IFN-gamma ininduction of Foxp3 and conversion of CD4+CD25-T cells to CD4+Tregs [J].J Clin Invest,2006, 116:2434-2441.
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[21]Liang S,Alard P, Zhao Y et al.Conversion of CD4+ CD25-T cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus [J].J Exp Med,2005,201:127-137.
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