CD4~+CD25~+调节性T细胞体外扩增的研究
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摘要
目的：探讨体外分离及扩增CD4+CD25+T细胞的方法。
方法：采用免疫磁珠法(MACS)分选获得CD4+CD25+T细胞；采用IL-2 (200IU/m)+anti-CD3/CD28/beads对分离后的CD4+CD25+T细胞进行体外扩增。
结果：1、采用MACS分离的成人外周血CD4+CD25+T细胞,纯度高达81%-91%。2、IL-2(200IU/m)+anti-CD3/CD28/beads的扩增方法能达到一定程度的扩增效果。
结论：采用MACS分离成人外周血CD4+CD25+T细胞是可行的,分离纯度高。采用IL-2+anti-CD3/CD28/beads的方法扩增CD4+CD25+T细胞可达到显著的扩增效果。
Objective:To explore separation and amplification method of CD4+CD25+T cells in vitro.
Methods:By micro-magnetic beads method (MACS) isolating CD4+CD25+T cells; By using IL-2 (200IU/mL)+anti-CD3/CD28/beads to expansion in vitro after the separation of CD4+CD25+T cells.
Results:1. Using MACS separate adult peripheral CD4+CD25+T cells,the purity of the cells up to 81-91%.2. The expansion methods of using IL-2 (200IU/mL) +anti-CD3/CD28/beads can reach a certain degree of amplification effect.
Conclusion:Using MACS separation adult peripheral CD4+CD25+T cells is feasible, separation of high purity. Adopt IL-2+anti-CD3/CD28/beads method amplification CD4+CD25+T cells can reach significant amplification effect.
方法：采用免疫磁珠法(MACS)分选获得CD4+CD25+T细胞；采用IL-2 (200IU/m)+anti-CD3/CD28/beads对分离后的CD4+CD25+T细胞进行体外扩增。
结果：1、采用MACS分离的成人外周血CD4+CD25+T细胞,纯度高达81%-91%。2、IL-2(200IU/m)+anti-CD3/CD28/beads的扩增方法能达到一定程度的扩增效果。
结论：采用MACS分离成人外周血CD4+CD25+T细胞是可行的,分离纯度高。采用IL-2+anti-CD3/CD28/beads的方法扩增CD4+CD25+T细胞可达到显著的扩增效果。
Objective:To explore separation and amplification method of CD4+CD25+T cells in vitro.
Methods:By micro-magnetic beads method (MACS) isolating CD4+CD25+T cells; By using IL-2 (200IU/mL)+anti-CD3/CD28/beads to expansion in vitro after the separation of CD4+CD25+T cells.
Results:1. Using MACS separate adult peripheral CD4+CD25+T cells,the purity of the cells up to 81-91%.2. The expansion methods of using IL-2 (200IU/mL) +anti-CD3/CD28/beads can reach a certain degree of amplification effect.
Conclusion:Using MACS separation adult peripheral CD4+CD25+T cells is feasible, separation of high purity. Adopt IL-2+anti-CD3/CD28/beads method amplification CD4+CD25+T cells can reach significant amplification effect.
引文
[1]Sakaguchi S. Regulatory T cells:key controllers of Immunologic self-tolerance[J]. Cell,2000,101:455-458.
[2]Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases[J]. J Immunol,1995,155(3):1151-1164.
[3]Shevach EM. CD4+CD25+ suppressor T cells:More questions than answers [J]. Nat Rev Immunol,2002,2 (6):389-400.
[4]Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells:Thymus or periphery [J]. J Clin Invest,2003, 112(9):1310-1322.
[5]Earle K E, Tang Q, ZhonX et al. In vitro expanded human CD4+CD25+ Regulatory T cells suppress effector T cell prolileration[J]. Clin Inanunol,2005; 115(1):3-9.
[6]Salomon B,Lenschow DJ,Rhee L, et al.B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes[J]. Immunity.2000,12 (4):431-440.
[7]Shevach EM, McHugh RS, Piccirillo CA, et al. Control of T-cell activation by CD4+CD25+ suppressor T cells[J]. Immunol Rev. 2001,182:58-67.
[8]Singh B, Read S, Asseman C, et al. Control of intestinal inflammation by regulatory T cells[J]. Immunol Rev.2001,182: 190-200.
[9]Sakaguchi S. Animal models of autoimmunity and their relevance to human diseases[J]. Curr Opin Immunol.2000,12(6):684-690.
[10]Aluvmare VR, Mand K, Betz AG. Regulatoty T cells mediate maternal tolerance to the fetus [J]. Nat Immunol.2004, 5(3):266-271.
[11]Viguier M, Lemait re F, Verola 0, et al. Foxp3 expressing CD4+CD25high regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit t he function of infilt rating T cells[J].J Immunol.2004,173(2):1444-1453.
[12]Wolf A, Wolf D, Steurer M, et al. Increase of regulatory T cells in the peripheral blood of cancer patients[J]. Clin Cancer Res.2003,9(2):606-612.
[13]Gregori S, Bacchetta R, Hauben E, et al.Regulatory T cells: prospective for clinical application in hematopoietic stem cell transplantation[J]. Curr Opin Hematol.2005,12(6):451-456.
[14]Curiel TJ,Coukos G,Zou L, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med.2004,10(9):942-949.
[15]Toda A, Piccirillo CA. Development and function of naturally occurring CD4+CD25+ regulatory T cells[J]. J Leukoc Biol.2006, 80(3):458-470.
[16]Nelson BH. IL-2, regulatory T cells, and tolerance [J]. J Immunol. 2004,172 (7):3983-3988.
[17]Bayer AL, Yu A, Adeegbe, D, et al. Essential role for interleukin-2 for CD4+CD25+T regulatory cell development during the neonatal period[J].J Exp Med.2005,201 (5):769-777.
[18]Malek TR. The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol[J].2003,74(6):961-965.
[19]Riley JL, June CH.The CD28 family:a T-cell rheostat for therapeutic control of T-cell activation[J]. Blood.2005, 105(1):13-21.
[20]Earle KE, Tang Q, Zhou X, et al. In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation[J]. Clin Immunol.2005,115(1):3-9.
[21]Hoffmann P, Eder R, Kunz-Schughart LA, et al. Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells[J]. Blood.2004,104(3):895-903.
[22]Horwitz DA, Zheng SG, Gray JD, et al. Regulatory T cells generated ex vivo as an approach for the therapy of autoimmune disease[J]. Semin Immunol.2004,16(2):135-143.
[23]Banerjee DK, Dhodapkar MV, Matayeva E, et al. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients[J]. Blood.2006,108(8):2655-2661.
[24]Yamazaki S, Iyoda T, Tarbell K, et al.Direct expansion of functional CD4+CD25+regulatory T cells by antigen-processing dendritic cells[J].J Exp Med.2003,198 (2):235-247.
[25]Battaglia M, S tabilini 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 (12):8338-8347.
[1]Bach JF. Regulatory T cells under scrutiny [J]. Nat Rev Immunol,2003,3 (3):189-198.
[2]Sakaguchi S. Regulatory T cells:key controllers of Immunologic self-tolerance[J]. Cell,2000,101:455-458.
[3]Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases [J]. J Immunol,1995,155(3):1151-1164.
[4]Shevach EM et al. Human CD4+CD25+ regulatory contact-dependent T cells induce [J]. Journal of Experimental Medicine,2002,196 (2):237-246
[5]Pacholczyk R,Kfaj P,Lgnatowicz LPeptide specificity of thymic selectin of CD4+CD25+T cells[J]. J Immunol,2002,168(2):613-620.
[6]Shevach EM. CD4+CD25+ suppressor T cells:More questions than answers [J]. Nat Rev Immunol,2002,2 (6):389-400.
[7]Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells: Thymus or periphery[J]. J Clin Invest,2003,112(9):1310-1322.
[8]Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production[J].Exp Med.1998,188(2):287-296.
[9]Yaqi H, Nomura T,Nakamura K, et al. Crucial role of FOXP3 in the development and function of human CD4+CD25+ regulatory T cells[J]. Int Immunol.2004,16 (11),1643-1656.
[10]Ochs HD, Ziegler SF, Torgerson TR. FOXP3 acts as a rheostat of the immune response [J]. Immunol Rev.2005,203,156-164.
[11]Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells [J]. Nat Immunol.2003,4 (4), 330-336.
[12]Sakaguchi S. Regulatory T cells in the past and for the future [J]. Eur J Immunol.2008,38(4):901-937
[13]Walker MR,Kasprowicz DJ, Gersuk VH, et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25 T cells[J].J Clin Invest.2003,112 (9),1437-1443.
[14]Khattri R, Cox T, Yasayko SA, et al. An essential role for Scurfin in CD4+CD25+T regulatory cells[J].Nat Immunol.2003,4 (4),337-342.
[15]Gambineri E, Torgerson TR, Ochs HD. Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX),a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis[J]. Curr Opin Rheumatol. 2003,15 (4),430-435.
[16]Bach J F, Chatenoud L. Tolerance to islet autoantigens in type diabetes[J].Annu Rev Immunol.2001,19:131-161.
[17]Cureil TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduce survival[J].Nat Met,2004,10 (9):942-949
[18]Taylor KN, Shinde-Patil VR, Cohick E, et al. Induction of FoxP3+CD4+CD25+ regulatory T cells following hemopoietic stem cell transplantation:role of bone marrow-derived facilitating cells[J]. J Immunol.2007,179(4):2153-2162.
[19]Aluvmare VR, Mand K, Betz AG. Regulatoty T cells mediate maternal tolerance to the fetus [J]. Nat Immunol.2004,5(3):266-271.
[20]von Boehme H. Mechanisms of suppression by suppressor T cells [J]. Nat Immunol,2005,6(4):338-344
[21]Earle KE, Tang Q, Zhou X, et al. In vitro expanded human CD4+CD25+ regulatory T cells suppress effector Tcell proliferation[J]. Clin Immunol,2005,115 (1):3-9
[22]Horwitz DA, Zheng SG, Gray JD, etal Regulatory T cells Generated ex vivo as an approach for the therapy of autoimmune disease [J]. Scmin lmmunoI, 2004,16(2):135-143.
[23]曾冬竹,王自强,邦峻松,等.IL-2和IL-10对Treg细胞体外扩增影响的差异研究[J].第三军医大学学报,2007,29(4)；290-292,
[24]Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299(5609):1057-1061.
[25]Khattri R, Cox T, Yasayko SA, et al.An essential role for scurfy in CD+4 CD25+ regulatory T cells[J].Nat Immunol,2003,4(4):337-342.
[26]Fontenot JD Gavin MA Rudensky AY, et al. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[J]. Nat Immunol,2003(4): 330-336.
[27]张晓菊,田拥军,丁静民,等.TGF-β1上调CD4+CD25+谢节性T细胞FoxP3的表达[J].华中科技大学学报(医学版),2006.35(6)：709-711.
[28]Battaglia M, StabllTnl A}Ronca∞10 MG, Rapamycm selecnwjy expands CD4+CD25+FoxP3+Regulatory T cells [J]. B100d,2005,105(12):743-4748.
[29]刘华,何敏,刘建胜,等.雷帕霉素对TGF-β1诱导的Treg细胞的体外影响[J].中南药学,2010,8(6)：437-440
[30]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.
[31]卢凯华,李晓曦,郝莎,等.17 β-雌二醇对小鼠淋巴结、脾脏和胸腺调节性T细胞及其Foxp3表达的影响[J].东南大学学报,2008,2(27)
[2]Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases[J]. J Immunol,1995,155(3):1151-1164.
[3]Shevach EM. CD4+CD25+ suppressor T cells:More questions than answers [J]. Nat Rev Immunol,2002,2 (6):389-400.
[4]Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells:Thymus or periphery [J]. J Clin Invest,2003, 112(9):1310-1322.
[5]Earle K E, Tang Q, ZhonX et al. In vitro expanded human CD4+CD25+ Regulatory T cells suppress effector T cell prolileration[J]. Clin Inanunol,2005; 115(1):3-9.
[6]Salomon B,Lenschow DJ,Rhee L, et al.B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes[J]. Immunity.2000,12 (4):431-440.
[7]Shevach EM, McHugh RS, Piccirillo CA, et al. Control of T-cell activation by CD4+CD25+ suppressor T cells[J]. Immunol Rev. 2001,182:58-67.
[8]Singh B, Read S, Asseman C, et al. Control of intestinal inflammation by regulatory T cells[J]. Immunol Rev.2001,182: 190-200.
[9]Sakaguchi S. Animal models of autoimmunity and their relevance to human diseases[J]. Curr Opin Immunol.2000,12(6):684-690.
[10]Aluvmare VR, Mand K, Betz AG. Regulatoty T cells mediate maternal tolerance to the fetus [J]. Nat Immunol.2004, 5(3):266-271.
[11]Viguier M, Lemait re F, Verola 0, et al. Foxp3 expressing CD4+CD25high regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit t he function of infilt rating T cells[J].J Immunol.2004,173(2):1444-1453.
[12]Wolf A, Wolf D, Steurer M, et al. Increase of regulatory T cells in the peripheral blood of cancer patients[J]. Clin Cancer Res.2003,9(2):606-612.
[13]Gregori S, Bacchetta R, Hauben E, et al.Regulatory T cells: prospective for clinical application in hematopoietic stem cell transplantation[J]. Curr Opin Hematol.2005,12(6):451-456.
[14]Curiel TJ,Coukos G,Zou L, et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[J]. Nat Med.2004,10(9):942-949.
[15]Toda A, Piccirillo CA. Development and function of naturally occurring CD4+CD25+ regulatory T cells[J]. J Leukoc Biol.2006, 80(3):458-470.
[16]Nelson BH. IL-2, regulatory T cells, and tolerance [J]. J Immunol. 2004,172 (7):3983-3988.
[17]Bayer AL, Yu A, Adeegbe, D, et al. Essential role for interleukin-2 for CD4+CD25+T regulatory cell development during the neonatal period[J].J Exp Med.2005,201 (5):769-777.
[18]Malek TR. The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol[J].2003,74(6):961-965.
[19]Riley JL, June CH.The CD28 family:a T-cell rheostat for therapeutic control of T-cell activation[J]. Blood.2005, 105(1):13-21.
[20]Earle KE, Tang Q, Zhou X, et al. In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation[J]. Clin Immunol.2005,115(1):3-9.
[21]Hoffmann P, Eder R, Kunz-Schughart LA, et al. Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells[J]. Blood.2004,104(3):895-903.
[22]Horwitz DA, Zheng SG, Gray JD, et al. Regulatory T cells generated ex vivo as an approach for the therapy of autoimmune disease[J]. Semin Immunol.2004,16(2):135-143.
[23]Banerjee DK, Dhodapkar MV, Matayeva E, et al. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients[J]. Blood.2006,108(8):2655-2661.
[24]Yamazaki S, Iyoda T, Tarbell K, et al.Direct expansion of functional CD4+CD25+regulatory T cells by antigen-processing dendritic cells[J].J Exp Med.2003,198 (2):235-247.
[25]Battaglia M, S tabilini 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 (12):8338-8347.
[1]Bach JF. Regulatory T cells under scrutiny [J]. Nat Rev Immunol,2003,3 (3):189-198.
[2]Sakaguchi S. Regulatory T cells:key controllers of Immunologic self-tolerance[J]. Cell,2000,101:455-458.
[3]Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases [J]. J Immunol,1995,155(3):1151-1164.
[4]Shevach EM et al. Human CD4+CD25+ regulatory contact-dependent T cells induce [J]. Journal of Experimental Medicine,2002,196 (2):237-246
[5]Pacholczyk R,Kfaj P,Lgnatowicz LPeptide specificity of thymic selectin of CD4+CD25+T cells[J]. J Immunol,2002,168(2):613-620.
[6]Shevach EM. CD4+CD25+ suppressor T cells:More questions than answers [J]. Nat Rev Immunol,2002,2 (6):389-400.
[7]Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells: Thymus or periphery[J]. J Clin Invest,2003,112(9):1310-1322.
[8]Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production[J].Exp Med.1998,188(2):287-296.
[9]Yaqi H, Nomura T,Nakamura K, et al. Crucial role of FOXP3 in the development and function of human CD4+CD25+ regulatory T cells[J]. Int Immunol.2004,16 (11),1643-1656.
[10]Ochs HD, Ziegler SF, Torgerson TR. FOXP3 acts as a rheostat of the immune response [J]. Immunol Rev.2005,203,156-164.
[11]Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells [J]. Nat Immunol.2003,4 (4), 330-336.
[12]Sakaguchi S. Regulatory T cells in the past and for the future [J]. Eur J Immunol.2008,38(4):901-937
[13]Walker MR,Kasprowicz DJ, Gersuk VH, et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25 T cells[J].J Clin Invest.2003,112 (9),1437-1443.
[14]Khattri R, Cox T, Yasayko SA, et al. An essential role for Scurfin in CD4+CD25+T regulatory cells[J].Nat Immunol.2003,4 (4),337-342.
[15]Gambineri E, Torgerson TR, Ochs HD. Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX),a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis[J]. Curr Opin Rheumatol. 2003,15 (4),430-435.
[16]Bach J F, Chatenoud L. Tolerance to islet autoantigens in type diabetes[J].Annu Rev Immunol.2001,19:131-161.
[17]Cureil TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduce survival[J].Nat Met,2004,10 (9):942-949
[18]Taylor KN, Shinde-Patil VR, Cohick E, et al. Induction of FoxP3+CD4+CD25+ regulatory T cells following hemopoietic stem cell transplantation:role of bone marrow-derived facilitating cells[J]. J Immunol.2007,179(4):2153-2162.
[19]Aluvmare VR, Mand K, Betz AG. Regulatoty T cells mediate maternal tolerance to the fetus [J]. Nat Immunol.2004,5(3):266-271.
[20]von Boehme H. Mechanisms of suppression by suppressor T cells [J]. Nat Immunol,2005,6(4):338-344
[21]Earle KE, Tang Q, Zhou X, et al. In vitro expanded human CD4+CD25+ regulatory T cells suppress effector Tcell proliferation[J]. Clin Immunol,2005,115 (1):3-9
[22]Horwitz DA, Zheng SG, Gray JD, etal Regulatory T cells Generated ex vivo as an approach for the therapy of autoimmune disease [J]. Scmin lmmunoI, 2004,16(2):135-143.
[23]曾冬竹,王自强,邦峻松,等.IL-2和IL-10对Treg细胞体外扩增影响的差异研究[J].第三军医大学学报,2007,29(4)；290-292,
[24]Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299(5609):1057-1061.
[25]Khattri R, Cox T, Yasayko SA, et al.An essential role for scurfy in CD+4 CD25+ regulatory T cells[J].Nat Immunol,2003,4(4):337-342.
[26]Fontenot JD Gavin MA Rudensky AY, et al. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[J]. Nat Immunol,2003(4): 330-336.
[27]张晓菊,田拥军,丁静民,等.TGF-β1上调CD4+CD25+谢节性T细胞FoxP3的表达[J].华中科技大学学报(医学版),2006.35(6)：709-711.
[28]Battaglia M, StabllTnl A}Ronca∞10 MG, Rapamycm selecnwjy expands CD4+CD25+FoxP3+Regulatory T cells [J]. B100d,2005,105(12):743-4748.
[29]刘华,何敏,刘建胜,等.雷帕霉素对TGF-β1诱导的Treg细胞的体外影响[J].中南药学,2010,8(6)：437-440
[30]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.
[31]卢凯华,李晓曦,郝莎,等.17 β-雌二醇对小鼠淋巴结、脾脏和胸腺调节性T细胞及其Foxp3表达的影响[J].东南大学学报,2008,2(27)