CD4~+CD25~+调节性T细胞与疾病相关性及其体外扩增的实验研究
|
摘要
正常人体内一般都有对自身抗原产生特异性反应的T细胞存在,但由于受到各种耐受机理的严格控制而不会导致免疫性疾病的发生。CD4+CD25+调节性T细胞(Regulatory T cells,Tregs)是自然产生并成熟于胸腺的独特细胞群,在正常人外周血和脾脏中占CD4+T细胞5%-10%。Tregs具有免疫无反应性和免疫抑制两大功能。随着对Tregs研究的深入,发现其在自身免疫病、器官移植、肿瘤免疫、慢性炎症反应、持续感染、母胎耐受等多方面发挥重要作用。本课题首先对CD4+CD25+、CD4+CD25highT细胞及其功能性转录因子Foxp3在正常人及疾病状态下(再生障碍性贫血、自身免疫病及恶性肿瘤)的表达特点进行了系统分析,结果表明:CD4+CD25+、CD4+CD25high T细胞及Foxp3在再生障碍性贫血及自身免疫病患者表达减低,而在肿瘤患者表达增高,表明Tregs与相关疾病的发生、发展密切相关。然而,其临床应用的主要限制之一为它们在人外周血和小鼠外周淋巴器官中比例低,没有足够数量用于疾病的治疗。因此我们进一步探讨了CD4+CD25+T细胞的体外分离和扩增方法,及分离和扩增后CD4+CD25+ T细胞的功能检测,结果表明:用免疫磁珠法分离正常人外周血CD4+CD25+ T细胞,可以得到很好的效果,分离的CD4+CD25+ T细胞纯度在80%以上;采用高剂量IL-2 +磁珠包被的anti-CD3mAb/anti-CD28mAb对CD4+CD25+ T细胞进行扩增,第9天增殖倍数达到68倍;分离和扩增后的CD4+CD25+ T细胞均具有免疫无反应及免疫抑制特性,并高表达Foxp3。为了解Treg细胞生长发育特点,我们采用流式细胞术及RT-PCR方法对新生儿脐血CD4+CD25+Treg细胞及Foxp3进行测定,并与正常人外周血进行比较,结果表明:脐血中Tregs的数量多,但占CD4+T细胞的百分比减低,考虑是由于脐血中CD4+T细胞比例较高的缘故,而脐血Foxp3表达减低,表明其功能还不成熟。进一步对脐血CD4+CD25+ T细胞的体外分离、功能鉴定及培养扩增的研究发现:脐血的分选及扩增结果同成人外周血相似,提示经外周抗原刺激后可以促使脐血Tregs成熟并发挥作用。
本课题研究的创新性在于在国内首次应用高剂量IL-2 +磁珠包被的anti-CD3mAb/anti-CD28mAb的方法对正常人外周血及新生儿脐血CD4+CD25+ T细胞进行体外培养和扩增,取得了较好的效果,并证实扩增后的CD4+CD25+ T细胞仍具有其免疫特性,我们上述实验的研究成果进一步证明了CD4+CD25+T细胞的临床应用前景。
There were T cells which produced specific reaction to autoantigen in normal adults, but one couldn’t get immune disease because the control of various kinds of tolerance mechanism. The immunologists had different ideas to the exist of regulatory T cells for the past many years because of the scared mark of cells and molecular basis of depressant effect. After the detection of CD4+CD25+ regulatory T cells(Tregs) by Sakaguchi in 1995,they are in the focus of intense research in immunology.Naturally occurring CD4+CD25+regulatory T cells are generated in thymus,and they represent 5%-10% of total CD4+T lymphocytes in periheral blood and spleen of normal adults .Tregs display immunoanergic and immunosuppressive activity. Activated Tregs can inhibit the proliferation and cytokines secretion of CD4+ and CD8+ T cells,and also inhibit NK,B cells and DC.Tregs is a kind of multi-cytokine secreting(interleukin 4,interleukin 10 and TGF-beta),affecting T cell suppressing cell. Tregs inhibit the proliferation of effector cells by cell-cell contact and/or secreting soluble cytokine such as TGF-βand IL-10. Its function in autoimmune disease occurring and developing was firstly studied. And gradually its effects in organ transplantion,tumor immunity,chronic inflammatory reaction and persistent infection were also explored. But there are several limitations to use them: one is the rare populations.So it is a quention which immunologists and clinicians search for constantly that how to culture and expand Tregs in vitro and then utilize in vivo.
Our projects are focusing on study the change and clinical significance of CD4+CD25+ T cells in normal adults and patients with morbid state,then in vitro isolation , purification and expansion of CD4+CD25+T cells and in vitro function assays, related mechanism studies,explore the express of CD4+CD25+ T cells in neonatal cord blood and in vitro isolation , purification and expansion of CD4+CD25+T cells in neonatal cord blood. Our study will contribute the experiment substructure to the clinical application of CD4+CD25+T cells in autoimmune disease,tumor and graft rejection.②The expression of Foxp3 between normal controls and patients:The percentage of Foxp3+ in CD4+CD25+, and CD4+CD25high T cells in patients with AA was lower than that in normal controls (P<0.05),and the percentage is also low in patients with autoallergic disease compare with normal controls(P<0.05). The percentage of Foxp3+ is higher than that in normal controls(P<0.01).
At first,the percentage and absolute numbers of CD4+ T cells、CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells in peripheral blood monouclear cell(PBMC) were examined with flow cytometry in normal controls,25 patients with AA, 37 patients with autoallergic disease,60 patients with cancer. The expression of Foxp3 in CD4+CD25+, CD4+CD25low and CD4+CD25high T cells was analyzed by flow cytometry.The levels of Foxp3mRNA in PBMC were determined by RT-PCR.①The comparesion of the percentage CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells between normal controls and patients:Compare with normal controls, the percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells are decreased in patients with AA(P<0.05), especially in patients with SAA(P<0.001), and they are also decreased in patients with autoallergic disease (P<0.05). The percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells are incresed in patients with cancers(Ⅰ+Ⅱ),but the difference is ont significance(P > 0.05),and they are obviously increased in patients with cancers(Ⅲ+Ⅳ) (P<0.01).③The expression of Foxp3 mRNA between normal controls and patients:it is lower in patients with AA and autoallergic disease than that in normal controls,and it is higher in patients with cancer. These findings suggeste that the expression of Tregs and Foxp3 is down regulated in patiens with AA and autoallergic disease,lead to Tregs’losing the ability of immunesuppression.It maybe provide a new idea of immunotherapy in patients with AA and immuneallergic disease through increasing the expression of Foxp3. It means that tumor cells could induce the proliferation,amplification and activation of CD4+CD25+ Treg by expressing tumor antigens or secreting immune inhibition factors,so it can impair effects of effector cells on tumor,and improve the tumor survival and development.It maybe provide an original way to immunotherapy in patients with tumor by removing Tregs of patients with tumor or block up the related cytokine.
And then,we make a study of isolation, purification and expansion of CD4+CD25+T cells in vitro.①We use the micro-magnetic beads to isolate the CD4+CD25+T cells. After the columns, the purity of the cells are usually about 80%-89%.It confirm the method of isolation is feasible.②The freshly purified CD4+CD25+T cells are anergic to the stimulators but they suppress the proliferation of CD4+ T cells and is dose-dependent.The inhibition ratio is 74% at a 1:2 ratio(CD4+:CD4+CD25+ T cells) by MTT.③The amplificated CD4+CD25+ T cells also keep their immunity characteristics,and the immunosuppressive action is enhanced.We adopted 2 kinds of concentration of IL-2(200u/mL and 500u/mL),and all plused anti-CD3/anti-CD28 beads. The data showed that it can be expanded by both of protocols but, in terms of yield, plate-bound IL-2 500u/mL is better than another one. Expansion gets its peak on day 9, then goes down.④The amplificated CD4+CD25- T cells can express the marker of CD25 but have no immunosuppression ability.
Many studies have made remarkable progress along with understand to Tregs by immunologists,but we have ont know the molecule mechanism of Tregs about its proceed,growth and mature.In order to explore the feature of Tregs ,we quantify the proportion of CD4+CD25highFoxp3+ regulatory T cells in neonatal cord blood and adult peripheral blood with flow cytometry , and explore the clinical significance of Treg in neonatal cord blood,and isolate、dentify、expand Tregs. It showde that the percentage and absolute numbers of CD4+ T cells were both increased in neonatal cord blood (P<0.01),the percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells were decreased in neonatal cord blood(P<0.05),but their absolute numbers were increased(P<0.01) compared with adult PBMC. The percentage of Foxp3 in CD4+CD25+ and CD4+CD25high T cells in neonatal cord blood were both lower than that in adult peripheral blood(P<0.01), the expression levels of Foxp3 mRNA were also lower than that in adult peripheral blood(P<0.05). It can’t distinguish CD4+CD25+ T and CD4+CD25-T cells in adult peripheral blood with flow cytometry ,but they can be distinguished well in neonatal cord blood.We observe the high express of Foxp3 in CD4+CD25highT cells.It confirm that CD4+CD25highT cells play a important role which educe the ability of immunosuppression in regulatory cells. We examine the express of Foxp3 in neonatal cord blood no matter with flow cytometry or RT-PCR,the expression levels of Foxp3 are both lower than that in adult PBMC.Tregs of neonatal cord blood maybe incipient T cells and seldomly be stimulated by antigen,so it can’t be activated.Tregs can obtain suppression function by reexcitation of antigen in periphery even though there are more Tregs in thymus. We isolate CD4+CD25+T cells from neonatal cord blood with micro-magnetic beads, the purity of the cells are usually about 83%-85%. The freshly purified and amplificated CD4+CD25+T cells are anergic to the stimulators but they suppress the proliferation of CD4+ T cells and is dose-dependent. Expansion gets its peak on day 12.
Our study proved the prospect of Tregs’clinical application.With the progress of medical science,we have used neonatal cord blood to treat many kinds of disease.It maybe provide theory basis of Tregs’clinical application and transplantation immunity. It’s significance that induce CD4+CD25highT cells in neonatal cord blood to mature and then treat various kinds of autollergic diease,allergic disorder,supress allograft rejection.
本课题研究的创新性在于在国内首次应用高剂量IL-2 +磁珠包被的anti-CD3mAb/anti-CD28mAb的方法对正常人外周血及新生儿脐血CD4+CD25+ T细胞进行体外培养和扩增,取得了较好的效果,并证实扩增后的CD4+CD25+ T细胞仍具有其免疫特性,我们上述实验的研究成果进一步证明了CD4+CD25+T细胞的临床应用前景。
There were T cells which produced specific reaction to autoantigen in normal adults, but one couldn’t get immune disease because the control of various kinds of tolerance mechanism. The immunologists had different ideas to the exist of regulatory T cells for the past many years because of the scared mark of cells and molecular basis of depressant effect. After the detection of CD4+CD25+ regulatory T cells(Tregs) by Sakaguchi in 1995,they are in the focus of intense research in immunology.Naturally occurring CD4+CD25+regulatory T cells are generated in thymus,and they represent 5%-10% of total CD4+T lymphocytes in periheral blood and spleen of normal adults .Tregs display immunoanergic and immunosuppressive activity. Activated Tregs can inhibit the proliferation and cytokines secretion of CD4+ and CD8+ T cells,and also inhibit NK,B cells and DC.Tregs is a kind of multi-cytokine secreting(interleukin 4,interleukin 10 and TGF-beta),affecting T cell suppressing cell. Tregs inhibit the proliferation of effector cells by cell-cell contact and/or secreting soluble cytokine such as TGF-βand IL-10. Its function in autoimmune disease occurring and developing was firstly studied. And gradually its effects in organ transplantion,tumor immunity,chronic inflammatory reaction and persistent infection were also explored. But there are several limitations to use them: one is the rare populations.So it is a quention which immunologists and clinicians search for constantly that how to culture and expand Tregs in vitro and then utilize in vivo.
Our projects are focusing on study the change and clinical significance of CD4+CD25+ T cells in normal adults and patients with morbid state,then in vitro isolation , purification and expansion of CD4+CD25+T cells and in vitro function assays, related mechanism studies,explore the express of CD4+CD25+ T cells in neonatal cord blood and in vitro isolation , purification and expansion of CD4+CD25+T cells in neonatal cord blood. Our study will contribute the experiment substructure to the clinical application of CD4+CD25+T cells in autoimmune disease,tumor and graft rejection.②The expression of Foxp3 between normal controls and patients:The percentage of Foxp3+ in CD4+CD25+, and CD4+CD25high T cells in patients with AA was lower than that in normal controls (P<0.05),and the percentage is also low in patients with autoallergic disease compare with normal controls(P<0.05). The percentage of Foxp3+ is higher than that in normal controls(P<0.01).
At first,the percentage and absolute numbers of CD4+ T cells、CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells in peripheral blood monouclear cell(PBMC) were examined with flow cytometry in normal controls,25 patients with AA, 37 patients with autoallergic disease,60 patients with cancer. The expression of Foxp3 in CD4+CD25+, CD4+CD25low and CD4+CD25high T cells was analyzed by flow cytometry.The levels of Foxp3mRNA in PBMC were determined by RT-PCR.①The comparesion of the percentage CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells between normal controls and patients:Compare with normal controls, the percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells are decreased in patients with AA(P<0.05), especially in patients with SAA(P<0.001), and they are also decreased in patients with autoallergic disease (P<0.05). The percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells are incresed in patients with cancers(Ⅰ+Ⅱ),but the difference is ont significance(P > 0.05),and they are obviously increased in patients with cancers(Ⅲ+Ⅳ) (P<0.01).③The expression of Foxp3 mRNA between normal controls and patients:it is lower in patients with AA and autoallergic disease than that in normal controls,and it is higher in patients with cancer. These findings suggeste that the expression of Tregs and Foxp3 is down regulated in patiens with AA and autoallergic disease,lead to Tregs’losing the ability of immunesuppression.It maybe provide a new idea of immunotherapy in patients with AA and immuneallergic disease through increasing the expression of Foxp3. It means that tumor cells could induce the proliferation,amplification and activation of CD4+CD25+ Treg by expressing tumor antigens or secreting immune inhibition factors,so it can impair effects of effector cells on tumor,and improve the tumor survival and development.It maybe provide an original way to immunotherapy in patients with tumor by removing Tregs of patients with tumor or block up the related cytokine.
And then,we make a study of isolation, purification and expansion of CD4+CD25+T cells in vitro.①We use the micro-magnetic beads to isolate the CD4+CD25+T cells. After the columns, the purity of the cells are usually about 80%-89%.It confirm the method of isolation is feasible.②The freshly purified CD4+CD25+T cells are anergic to the stimulators but they suppress the proliferation of CD4+ T cells and is dose-dependent.The inhibition ratio is 74% at a 1:2 ratio(CD4+:CD4+CD25+ T cells) by MTT.③The amplificated CD4+CD25+ T cells also keep their immunity characteristics,and the immunosuppressive action is enhanced.We adopted 2 kinds of concentration of IL-2(200u/mL and 500u/mL),and all plused anti-CD3/anti-CD28 beads. The data showed that it can be expanded by both of protocols but, in terms of yield, plate-bound IL-2 500u/mL is better than another one. Expansion gets its peak on day 9, then goes down.④The amplificated CD4+CD25- T cells can express the marker of CD25 but have no immunosuppression ability.
Many studies have made remarkable progress along with understand to Tregs by immunologists,but we have ont know the molecule mechanism of Tregs about its proceed,growth and mature.In order to explore the feature of Tregs ,we quantify the proportion of CD4+CD25highFoxp3+ regulatory T cells in neonatal cord blood and adult peripheral blood with flow cytometry , and explore the clinical significance of Treg in neonatal cord blood,and isolate、dentify、expand Tregs. It showde that the percentage and absolute numbers of CD4+ T cells were both increased in neonatal cord blood (P<0.01),the percentage of CD4+CD25+/ CD4+ and CD4+CD25high / CD4+T cells were decreased in neonatal cord blood(P<0.05),but their absolute numbers were increased(P<0.01) compared with adult PBMC. The percentage of Foxp3 in CD4+CD25+ and CD4+CD25high T cells in neonatal cord blood were both lower than that in adult peripheral blood(P<0.01), the expression levels of Foxp3 mRNA were also lower than that in adult peripheral blood(P<0.05). It can’t distinguish CD4+CD25+ T and CD4+CD25-T cells in adult peripheral blood with flow cytometry ,but they can be distinguished well in neonatal cord blood.We observe the high express of Foxp3 in CD4+CD25highT cells.It confirm that CD4+CD25highT cells play a important role which educe the ability of immunosuppression in regulatory cells. We examine the express of Foxp3 in neonatal cord blood no matter with flow cytometry or RT-PCR,the expression levels of Foxp3 are both lower than that in adult PBMC.Tregs of neonatal cord blood maybe incipient T cells and seldomly be stimulated by antigen,so it can’t be activated.Tregs can obtain suppression function by reexcitation of antigen in periphery even though there are more Tregs in thymus. We isolate CD4+CD25+T cells from neonatal cord blood with micro-magnetic beads, the purity of the cells are usually about 83%-85%. The freshly purified and amplificated CD4+CD25+T cells are anergic to the stimulators but they suppress the proliferation of CD4+ T cells and is dose-dependent. Expansion gets its peak on day 12.
Our study proved the prospect of Tregs’clinical application.With the progress of medical science,we have used neonatal cord blood to treat many kinds of disease.It maybe provide theory basis of Tregs’clinical application and transplantation immunity. It’s significance that induce CD4+CD25highT cells in neonatal cord blood to mature and then treat various kinds of autollergic diease,allergic disorder,supress allograft rejection.
引文
[1]Chatenoud L, Salomon B, Bluesone JA. Suppressor T cell:They’re back and critical for regulation of autoimmunity [J]. Immunol Rev,2001,182:149-163.
[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]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.
[6]Jonuleit H,Schmitt E,Kakirman H,et al.Infectious tolerance:human CD25+Regulatory T cell s convey suppressor activity to conventional CD4+T helper cell s[J].J Exp Med,2002,196(2):255-260.
[7]Chen ML,Pittet MJ,Gorelik L,et al.Regulatory T clls suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo[J].Proc Natl Acad Sci USA,2005,102(2):419-424.
[8]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.
[9]Sakaguchi S. Regulatory T cells in the past and for the future[J].Eur J Immunol. 2008,38(4):901-937
[10]Ochs HD,Ziegler SF,Torgerson TR. FOXP3 acts as a rheostat of the immuneresponse[J].Immunol Rev. 2005,203,156–164.
[11]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.
[12]Hori S,Sakaguchi S.Foxp3:a critical regulator of the development and function of regulatory T cells[J].Microbes Infect. 2004,6(8),745–751.
[13]Hori S,Nomura T,Sakaguchi S.Control of regulatory T cell development by the transcription factor Foxp3[J].Science 2003,299(5609),1057–1061.
[14]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.
[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]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.
[17]Bach JF. Regulatory T cells under scrutiny[J]. Nat Rev Immunol. 2003,3(3):189-198.
[18]Fontenot JD, Dooley JL, Farr AG,et al.Developmental regulation of Foxp3 expression during ontogeny[J].J Exp Med. 2005 ,202(7):901-906.
[19]Fontenot JD, Rudensky AY.A well adapted regulator contrivance:regulatory T cell development and the forkhead family transcription factor Foxp3[J]. Nat Immunol, 2005, 6(4): 331-337.
[20]Sakaguchi S, Setoguchi R, Yagi H,et al.Naturally arising Foxp3-expressing CD4+CD25+ regulatory T cells in self- tolerance and autoimmune disease[J].Curr Top Microbiol Immunol. 2006,305:51-66.
[21]Roncarolo MG, Levings MK.The role of different subsets of T regulatory cells incontrolling autoimmunity[J]. Curr Opin Immunol, 2000,12(6):676-683.
[22]Fontenot JD, Rasmussen JP, Williams LM, et al.Regulatory T cell lineage specifica- tion by the forkhead transcription factor foxp3[J].Immunity. 2005,22(3):329-341.
[23]Jaeckel E,von Boehmer H,Manns MP.Antigen-specific FoxP3-transduced T- cells can control established type 1 diabetes[J].Diabetes,2005,54(2):306-310
[24]Fontenot JD, Rasmussen JP, Gavin MA,et al. A function for interleukin 2 in Foxp3-expressing regulatory T cells [J]. Nat Immunol.2005,6(11):1142-1151.
[25]Khatt ri R,Cox T,Yasayko SA,et al.An essential role for Scurfinin CD4+CD25+T regulatory cells[J].Nat Immunol,2003,4(4):337-342.
[26]Higgins J , Metcalf JA, Stevens RA ,et al. Effects of lymphocyte isolation and timing of processing on detection of CD127 expression on T cells in human immunodeficiency virus-infected patients[J]. Clin Diagn Lab Immunol, 2005, 12(1):228-230.
[27]Hartigan-O’Connor DJ, Poon C,Sinclair E,et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain(CD127) , allowing consistent identification and sorting of live cells[J].J Immunol Methods,2007,319(1-2):41-52.
[28]Vang KB, Yang J, Mahmud SA,etal. IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development[J].J Immunol. 2008,181(5):3285-3290.
[29]Wells AD, Walsh MC, Bluestone JA, et al.Signaling through CD28 and CTLA-4 controls two distinct forms of T cell anergy[J]. J Clin Invest,2001,108(6):895-903.
[30]Carreno BM, Bennett F, Chau TA, et al. CTLA-4(CD152) can inhibit T cell activation by two different mechanisms depending on its level of cell surface expression[J]. J Immunol,2000,165(3):1352-1356.
[31]Takahashi T, Tagami T, Yamazaki S, et al.Immunologic self-tolerance maintained by CD4+CD25+ regulatory T cells constitutively expressing cytotoxic T lymphocyte– associated antigen 4[J].J Exp Med. 2000,192(2):303-310.
[32]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.
[33]Read S,Malmstrom V,Powrie F.Cytotoxic T lymphocyte–associated antigen 4 plays an essential role in the function of CD4+CD25+ regulatory cells that control intestinal inflammation[J].J Exp Med. 2000,192(2):295-302.
[34]McHugh RS,Whitters MJ,Piccirillo CA,et a1. CD4+CD25+ immunoregulatory T cells:gene expression analysis reveals a functional role for the Glucocorticod 2 induced TNF receptor[J]. Immunity, 2002,16(2):311-323.
[35]Takahashi T, Kuniyasu Y, Toda M,et al. Immunologic self-tolerance maintained by CD4+CD25+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state[J]. Int Immunol, 1998,10(12):1969-1980.
[36]Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonalTcell activation in vitro by inhibit-ing interleukin 2 production[J]. J Exp Med, 1998, 20, 188(2):287-296.
[37]Chen ZM, O'Shaughnessy MJ, Gramaglia I, et al.IL-10 and TGF-beta induce alloreactive CD4+CD25- T cells to acquire regulatory cell function[J].Blood, 2003, 101(12): 5076-5083.
[38]Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+ regulatoryT cells is mediated by cell surface-bound transforming growth factor beta[J].J Exp Med, 2001 ,194(5):629-644.
[39] Schramm C, Huber S, Protschka M, et al.TGFbeta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo[J]. Int Immunol, 2004, 16(9): 1241-1249.
[40] Karube K, Ohshima K, Tsuchiya T, et al. Expression of FoxP3, a key molecule in CD4+CD25+ regulatory T cells, in adult T-cell leukaemia/lymphoma cells[J].Br J Haematol,2004,126(1):81-84.
[41]Parameswaran K, Liang H, Fanat A,et al. Role for cysteinyl leukotrienes in allergen-induced change in circulating dendritic cell number in asthma[J].J Allergy Clin Immunol, 2004,114(1):73-79.
[42]Malek TR,Yu A, Vincek V,et al.CD4 regulatory T cells prvent lethal autoimmunity in IL-2R beta deficient mice.Implication for the nonredundant function of IL-2[J]. Immunity. 2002,17(2):167-178.
[43]Setoguchi R, Hori S, Takahashi T, et al. Homeoststic maintenance of natural Foxp3+ CD4+CD25+ regulatory T cells by interleukin(IL)-2 and induction of autoimmune disease by IL-2 neutralization[J].J Exp Med.2005,201(5):723-735.
[44]Bensinger SJ, Walsh PT, Zhang J, et al. Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells[J].J Immunol.2004,172(9):5287-5296.
[45]Su L, Creusot RJ, Gallo EM, et al. Murine CD4+CD25+ regulatory T cells fail to undergo chromatin remodeling across the proximal promoter region of the IL-2 gene[J].J Immunol.2004,173(8):4994-5001.
[46]Barthlott T, Moncrieffe H, Veldhoen M, et al. CD4+CD25+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production[J].Int Immunol.2005,17(3):279-288.
[47]Furtado GC,Curotto de Lafaille MA,Kutchukhidze N, et al. Interleukin 2 signaling is required for CD4+ regulatory T cell function[J]. J Exp Med.2002;196(6):851-857.
[48]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.
[49]Thomton CA,Upham JW, Wikstrom ME,et al. Functional maturation of CD4+ CD25+ CTLA4+ CD45RA+ regulatory T cells in human neonatal T cell responses to environmental antigens/allergens[J]. J Immunol.2004,173(5):3084-3092.
[50]De-la-Rosa M.Rutz S Dominger H,et al.Interleukin-2 is essential for CD4+CD25+ regulatory T cell function[J]. Eur J Immunol.2004,34(9):2480-2488.
[51]Nelson BH. IL-2, regulatory T cells, and tolerance[J].J Immunol. 2004, 172(7): 3983-3988.
[52]Read S,Malmstrom V,Powrie F.Cytotoxic T lymphocyte– associated antigen 4 plays an essential role in the function of CD4+CD25+ regulatory cells that control intestinal inflammation[J].J Exp Med. 2000,192(2):295-302.
[53]McHugh RS,Whitters MJ,Piccirillo CA,et a1. CD4+CD25+ immunoregulatory T cells:gene expression analysis reveals a functional role for the Glucocorticod 2 induced TNF receptor[J]. Immunity.2002,16(2):311-323.
[54]Ko K, Yamazaki S, Nakamura K, et al,Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+ CD4+CD25+ regulatory T cells[J]. J Exp Med.2005,202(7):885-891.
[55]Sakaguchi S.Regulatory T cells:key controllers of immunologic selftolerance [J].Cell.2000,101(5):455-458.
[56]Shevach EM. Immunology. Regulating suppression. [J]. Science. 2008 , 322(5899): 202-203.
[57]Bach J F,Chatenoud L.Tolerance to islet autoantigens in type diabetes[J].Annu Rev Immunol.2001,19:131-161.
[58]Sakaguchi S, Yamaguchi T, Nomura T,et al. Regulatory T cells and immune tolerance.[J]. Cell. 2008,133(5):775-787.
[59]Shevach EM. Regulatory T cells. Introduction [J]. Semin Immunol. 2004 ,16(2):69-71
[60]Maloy K J,Powrie F.Regulatory T cells in the control of immune pathology[J]. Nat Immunol.2001,2(9):816-822.
[61]Bagley KC, Shata MT, Onyabe DY, et al. Immunogenicity of DNA vaccines that direct the coincident expression of the 120 KDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin[J]. Vaccine, 2003, 21(23): 3335-3341.
[62]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.
[63]Sakaguchi S,kaguchi N,Shimizu J et al.Immunologic tolerance maintained by CD4+CD25+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity,and transp lantation tolerance[J].Immunol Rev. 2001,182(8):18-32.
[64]Zelenika D,Adams E,Humm S et al.The role of CD4+T cell subsets in determining transp lantation rejection or tolerance[J].Immunol Rev.2001,182(8):164-179.
[65]Zou WP.Immunosuppressive networks in the tumor environment and their therapeutic relevance[J].Nat Rev Cancer, 2005, 5(4):263-274.
[66]Viguier M,Lemait re F,Verola O,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.
[67]Woo EY,Yeh H,Chu CS,et al.Cutting edge:Regulatory T cells from lung cancer patient s directly inhibit autologous T cell proliferation[J].J Immunol.2002,168(9):4272-4276.
[68]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.
[69]Yanade U K,Moore TT,Joo HG,et al.Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patient s with pancreas or breast adenocarcinoma[J].J Immunol.2002, 169(5):2756-2761.
[70]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.
[71]Apostolou I,Von Boehmer H.In vivo inst ruction of suppressor commitment in nave T cells[J].J Exp Med. 2004,199(10):1401-1408.
[72]Sakaguchi S. Naturally arising Foxp3 expressing CD4+CD25+ regulatory T cells in immunological tolerance to self and nonself[J].Nat Immunol.2005, 6(4):345-352.
[73]Lim HW,Hillsamer P,Banham AH, et al. Cutting edge: Direct suppression of B cells by CD4+CD25+ regulatory T cells [J]. J Immunol.2005,175(7):4180-4183.
[74]Khazaie K, Von Boehmei H. The impact of CD4+CD25+Treg on tumor specific CD8+ T cell cytotoxicity and cancer[J].Semin Cancer Biol.2006,12(2):124-136.
[75]Zou WP. Immunosuppressive networks in the tumor environment and their therapeutic relevance[J]. Nat Rev Cancer.2005,5(4):263-274.
[76]Sasaki Y,SakaiM,Miyazaki S,et al.Decidual and peripheral blood CD4+CD25+ regulatory T cells in early pregnancy subjects and spontaneous abortion cases[J]. Mol Hum Rep rod.2004,10(5):347-353.
[77]Aluvmare VR, Mand K, Betz AG. Regulatoty T cells mediate maternal tolerance to the fetus [J]. Nat Immunol. 2004, 5(3):266-271.
[78]Somerset DA,zheng Y,KilbyM D et al.Normai human pregnancy is associated with an elevation in the immune suppressive CD4+CD25+ regulatory T-cell subet[J]. Immunology. 2004,112(1):38-43.
[79]Saito S, Shima T, Nakashima A et al. What is the role of regulatory T cells in the success of implantation and early pregnancy?[J].J Assist Reprod Genet. 2007,24(9):379-386.
[80]Shevach EM. Certified professionals: CD4+CD25+ suppressor T cells[J]. J Exp Med, 2001, 193(11): F41-46.
[81]Schreiber TH. The use of FoxP3 as a biomarker and prognostic factor for malignant human tumors[J]. Cancer Epidemiol Biomarkers Prev. 2007,16(10):1931-1934.
[82]Young NS, calado RT, scheinberg P et al. Current concepts in the pathophysiology and treatment of aplastic anemia [J]. Blood,2006,108(8):2509-2519.
[83]Young NS. Autoimmunity and its treatment in aplastic anemia [J].Ann Intern Med,1997, 126(2):166-168.
[84]Solomou EE, Rezvani K, Mielke S, et al. Deficient CD4+CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia [J]. Blood.2007,110(5):1603-1606.
[85]Crispin JC,Martines A,Alcocer Varela J,et al.Quantification of regulatory T cells in patients with systemic lupus erythematosus[J].J Autoimmun.2003,21 (3): 273-276.
[86]Liu MF,Wang CR,Fung LL,Wu CR.Decreased CD4+CD25+T cells in peripheral blood of patients with systemic lupus erythematosus[J].Scand J Immunol.2004,59(2):198-202.
[87]Murphy TJ, Ni Choileain N, Zang Y,et al.CD4+CD25+ regulatory T cells control innate immune reactivity after injury[J].J Immunol. 2005,174(5):2957-2963.
[88]HoriS, NornuraT, SakaguchiSetal. Contro of regulatory T cell development by the transcription factor Foxp3 [J]. Science. 2003,299(5609): 1057-1061.
[89]Terabe M, Berzofsky J A. Immuno regulatory T cells in tumor immunity[J]. Curr Opin Immunol, 2004, 16(2): 157-162.
[90]Wolf AM, 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.
[91]Sasada T, Kimunre M, Yoshida Y, et al. CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies possible involvement of regulatory T cells in disease progression[J]. Cancer, 2003, 98(5): 1089-1099.
[92]Rosenberg SA. Progress in human tumor immunology and immunotherapy[J]. Nature, 2001, 411(6835): 380-384.
[93]Baecher-Allan C, Viglietta V, Hafler DA. Human CD4+CD25+ regulatory T cells[J]. Semin Immunol, 2004, 16(2): 89-97 .
[94]Gregg R, Smith CM, Clark FJ, et al.The number of human peripheral blood CD4+CD25+ regulatory T cells increases with age[J]. Clin Exp Immunol, 2005, 140(3): 540-546.
[95]Sutmuller RP, van Duivenvoorde LM, van Elsas A, et al. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitum or the rapy reveals altemative pathways for suppression of autoreactive cytotoxic T lymphocyte responses[J]. J Exp Med, 2001, 194(6): 823-832.
[96]Tanaka H, Tanaka I, Kjaergaard I, et al. Depletion of CD4+CD25+ regulatory T cells augments the generation of specific immune T cells in tumor-draining lymph nodes[J]. J Immunother, 2002, 25(3):207-217.
[97]Sakaguch S., Naturally arising CD4+ regulatory T cells for immunologic self- tolerance and negative control of immune responses[J]. Annu Rev Immunol. 2004.,22: 531-562.
[98]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.
[99]Singh B, Read S, Asseman C, et al.Control of intestinal inflammation by regulatory T cells[J]. Immunol Rev.2001,182: 190-200.
[100]Sakaguchi S.Animal models of autoimmunity and their relevance to human diseases[J]. Curr Opin Immunol.2000,12(6): 684-690.
[101]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.
[102]Shevach EM.Regulatory T cells in autoimmmunity[J]. Annu Rev Immunol. 2000,18:423-449.
[103]Barthlott T,Moncrieffe H,Veldhoen M,et al. CD4+CD25+ T cells compete with naive CD4+T cells for IL-2 and exploit it for the induction of IL-10 production[J]. Int Immunol. 2005,17(3):279-288.
[104]Bruniquel D,Schwartz RH.Selective,stable demethylation of the interleukin-2 gene enhances transcription by an active process[J].Nat Immunol. 2003,4(3):235-240.
[105]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.
[106]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.
[107]Fehervari Z,Sakaguchi S.Control of CD4+CD25+ regulatory cell activation and function by dendritic cells[J].Int Immunol.2004,16(12):1769-1780.
[108]Szanya V, Ermann J, Taylor C, et al. The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7[J]. J Immunol. 2002,169(5):2461-2465.
[109]Liu H, Hu B, Xu D,et al. CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4[J]. J Immunol. 2003,171(10):5012-7.
[110]Ermann J, Hoffmann P, Edinger M, Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD[J]. Blood. 2005,105(5):2220-6.
[111]Cederbom L, Hall H, Ivars F. CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells[J]. Eur J Immunol. 2000, 30(6): 1538-1543.
[112]Toda A, Piccirillo CA. Development and function of naturally occurring CD4+CD25+ regulatory T cells[J]. J Leukoc Biol.2006,80(3):458-470.
[113]Nelson BH.IL-2,regulatory T cells,and tolerance[J]. J Immunol. 2004,172(7):3983- 3988.
[114]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.
[115]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.
[116]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.
[117]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.
[118]Hoffmann P, Eder R, Kunz-Schughart LA,et al. Large-scale in vitro expansion ofpolyclonal human CD4+CD25high regulatory T cells[J]. Blood. 2004,104(3):895-903.
[119]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.
[120]Mihu CM, Mihu D, Costin N, et al.Isolation and charact- erization of stem cells from the placenta and the umbilical cord[J].Rom J Morphol Embryol. 2008,49(4): 441- 446.
[121]Bensinger SJ,Bandeira A,Jordan MS,et al. Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells[J].J Exp Med.2001,194(4):427-438.
[122]van Santen HM,Benoist C,Mathis D. Number of T reg cells that differentiate does not increase upon encounter of agonist ligand on thymic epithelial cells[J]. J Exp Med,2004,200(10):1221-1230.
[123]Wing K, Ekmark A, Karlsson H,et al. Characterization of human CD4+CD25+ T cells in thymus, cord and adultblood [J]. Immunology.2002,106(2):190-199.
[124]Ziegler SF. FOXP3: ofmice andmen[J].AnnuRev Immunol. 2006,24 (1): 209-226.
[125]Godfrey WR, Spoden DJ, Ge YG,et al. Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function[J].Blood.2005, 105(2): 750-758.
[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]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.
[6]Jonuleit H,Schmitt E,Kakirman H,et al.Infectious tolerance:human CD25+Regulatory T cell s convey suppressor activity to conventional CD4+T helper cell s[J].J Exp Med,2002,196(2):255-260.
[7]Chen ML,Pittet MJ,Gorelik L,et al.Regulatory T clls suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo[J].Proc Natl Acad Sci USA,2005,102(2):419-424.
[8]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.
[9]Sakaguchi S. Regulatory T cells in the past and for the future[J].Eur J Immunol. 2008,38(4):901-937
[10]Ochs HD,Ziegler SF,Torgerson TR. FOXP3 acts as a rheostat of the immuneresponse[J].Immunol Rev. 2005,203,156–164.
[11]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.
[12]Hori S,Sakaguchi S.Foxp3:a critical regulator of the development and function of regulatory T cells[J].Microbes Infect. 2004,6(8),745–751.
[13]Hori S,Nomura T,Sakaguchi S.Control of regulatory T cell development by the transcription factor Foxp3[J].Science 2003,299(5609),1057–1061.
[14]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.
[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]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.
[17]Bach JF. Regulatory T cells under scrutiny[J]. Nat Rev Immunol. 2003,3(3):189-198.
[18]Fontenot JD, Dooley JL, Farr AG,et al.Developmental regulation of Foxp3 expression during ontogeny[J].J Exp Med. 2005 ,202(7):901-906.
[19]Fontenot JD, Rudensky AY.A well adapted regulator contrivance:regulatory T cell development and the forkhead family transcription factor Foxp3[J]. Nat Immunol, 2005, 6(4): 331-337.
[20]Sakaguchi S, Setoguchi R, Yagi H,et al.Naturally arising Foxp3-expressing CD4+CD25+ regulatory T cells in self- tolerance and autoimmune disease[J].Curr Top Microbiol Immunol. 2006,305:51-66.
[21]Roncarolo MG, Levings MK.The role of different subsets of T regulatory cells incontrolling autoimmunity[J]. Curr Opin Immunol, 2000,12(6):676-683.
[22]Fontenot JD, Rasmussen JP, Williams LM, et al.Regulatory T cell lineage specifica- tion by the forkhead transcription factor foxp3[J].Immunity. 2005,22(3):329-341.
[23]Jaeckel E,von Boehmer H,Manns MP.Antigen-specific FoxP3-transduced T- cells can control established type 1 diabetes[J].Diabetes,2005,54(2):306-310
[24]Fontenot JD, Rasmussen JP, Gavin MA,et al. A function for interleukin 2 in Foxp3-expressing regulatory T cells [J]. Nat Immunol.2005,6(11):1142-1151.
[25]Khatt ri R,Cox T,Yasayko SA,et al.An essential role for Scurfinin CD4+CD25+T regulatory cells[J].Nat Immunol,2003,4(4):337-342.
[26]Higgins J , Metcalf JA, Stevens RA ,et al. Effects of lymphocyte isolation and timing of processing on detection of CD127 expression on T cells in human immunodeficiency virus-infected patients[J]. Clin Diagn Lab Immunol, 2005, 12(1):228-230.
[27]Hartigan-O’Connor DJ, Poon C,Sinclair E,et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain(CD127) , allowing consistent identification and sorting of live cells[J].J Immunol Methods,2007,319(1-2):41-52.
[28]Vang KB, Yang J, Mahmud SA,etal. IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development[J].J Immunol. 2008,181(5):3285-3290.
[29]Wells AD, Walsh MC, Bluestone JA, et al.Signaling through CD28 and CTLA-4 controls two distinct forms of T cell anergy[J]. J Clin Invest,2001,108(6):895-903.
[30]Carreno BM, Bennett F, Chau TA, et al. CTLA-4(CD152) can inhibit T cell activation by two different mechanisms depending on its level of cell surface expression[J]. J Immunol,2000,165(3):1352-1356.
[31]Takahashi T, Tagami T, Yamazaki S, et al.Immunologic self-tolerance maintained by CD4+CD25+ regulatory T cells constitutively expressing cytotoxic T lymphocyte– associated antigen 4[J].J Exp Med. 2000,192(2):303-310.
[32]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.
[33]Read S,Malmstrom V,Powrie F.Cytotoxic T lymphocyte–associated antigen 4 plays an essential role in the function of CD4+CD25+ regulatory cells that control intestinal inflammation[J].J Exp Med. 2000,192(2):295-302.
[34]McHugh RS,Whitters MJ,Piccirillo CA,et a1. CD4+CD25+ immunoregulatory T cells:gene expression analysis reveals a functional role for the Glucocorticod 2 induced TNF receptor[J]. Immunity, 2002,16(2):311-323.
[35]Takahashi T, Kuniyasu Y, Toda M,et al. Immunologic self-tolerance maintained by CD4+CD25+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state[J]. Int Immunol, 1998,10(12):1969-1980.
[36]Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonalTcell activation in vitro by inhibit-ing interleukin 2 production[J]. J Exp Med, 1998, 20, 188(2):287-296.
[37]Chen ZM, O'Shaughnessy MJ, Gramaglia I, et al.IL-10 and TGF-beta induce alloreactive CD4+CD25- T cells to acquire regulatory cell function[J].Blood, 2003, 101(12): 5076-5083.
[38]Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+ regulatoryT cells is mediated by cell surface-bound transforming growth factor beta[J].J Exp Med, 2001 ,194(5):629-644.
[39] Schramm C, Huber S, Protschka M, et al.TGFbeta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo[J]. Int Immunol, 2004, 16(9): 1241-1249.
[40] Karube K, Ohshima K, Tsuchiya T, et al. Expression of FoxP3, a key molecule in CD4+CD25+ regulatory T cells, in adult T-cell leukaemia/lymphoma cells[J].Br J Haematol,2004,126(1):81-84.
[41]Parameswaran K, Liang H, Fanat A,et al. Role for cysteinyl leukotrienes in allergen-induced change in circulating dendritic cell number in asthma[J].J Allergy Clin Immunol, 2004,114(1):73-79.
[42]Malek TR,Yu A, Vincek V,et al.CD4 regulatory T cells prvent lethal autoimmunity in IL-2R beta deficient mice.Implication for the nonredundant function of IL-2[J]. Immunity. 2002,17(2):167-178.
[43]Setoguchi R, Hori S, Takahashi T, et al. Homeoststic maintenance of natural Foxp3+ CD4+CD25+ regulatory T cells by interleukin(IL)-2 and induction of autoimmune disease by IL-2 neutralization[J].J Exp Med.2005,201(5):723-735.
[44]Bensinger SJ, Walsh PT, Zhang J, et al. Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells[J].J Immunol.2004,172(9):5287-5296.
[45]Su L, Creusot RJ, Gallo EM, et al. Murine CD4+CD25+ regulatory T cells fail to undergo chromatin remodeling across the proximal promoter region of the IL-2 gene[J].J Immunol.2004,173(8):4994-5001.
[46]Barthlott T, Moncrieffe H, Veldhoen M, et al. CD4+CD25+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production[J].Int Immunol.2005,17(3):279-288.
[47]Furtado GC,Curotto de Lafaille MA,Kutchukhidze N, et al. Interleukin 2 signaling is required for CD4+ regulatory T cell function[J]. J Exp Med.2002;196(6):851-857.
[48]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.
[49]Thomton CA,Upham JW, Wikstrom ME,et al. Functional maturation of CD4+ CD25+ CTLA4+ CD45RA+ regulatory T cells in human neonatal T cell responses to environmental antigens/allergens[J]. J Immunol.2004,173(5):3084-3092.
[50]De-la-Rosa M.Rutz S Dominger H,et al.Interleukin-2 is essential for CD4+CD25+ regulatory T cell function[J]. Eur J Immunol.2004,34(9):2480-2488.
[51]Nelson BH. IL-2, regulatory T cells, and tolerance[J].J Immunol. 2004, 172(7): 3983-3988.
[52]Read S,Malmstrom V,Powrie F.Cytotoxic T lymphocyte– associated antigen 4 plays an essential role in the function of CD4+CD25+ regulatory cells that control intestinal inflammation[J].J Exp Med. 2000,192(2):295-302.
[53]McHugh RS,Whitters MJ,Piccirillo CA,et a1. CD4+CD25+ immunoregulatory T cells:gene expression analysis reveals a functional role for the Glucocorticod 2 induced TNF receptor[J]. Immunity.2002,16(2):311-323.
[54]Ko K, Yamazaki S, Nakamura K, et al,Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+ CD4+CD25+ regulatory T cells[J]. J Exp Med.2005,202(7):885-891.
[55]Sakaguchi S.Regulatory T cells:key controllers of immunologic selftolerance [J].Cell.2000,101(5):455-458.
[56]Shevach EM. Immunology. Regulating suppression. [J]. Science. 2008 , 322(5899): 202-203.
[57]Bach J F,Chatenoud L.Tolerance to islet autoantigens in type diabetes[J].Annu Rev Immunol.2001,19:131-161.
[58]Sakaguchi S, Yamaguchi T, Nomura T,et al. Regulatory T cells and immune tolerance.[J]. Cell. 2008,133(5):775-787.
[59]Shevach EM. Regulatory T cells. Introduction [J]. Semin Immunol. 2004 ,16(2):69-71
[60]Maloy K J,Powrie F.Regulatory T cells in the control of immune pathology[J]. Nat Immunol.2001,2(9):816-822.
[61]Bagley KC, Shata MT, Onyabe DY, et al. Immunogenicity of DNA vaccines that direct the coincident expression of the 120 KDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin[J]. Vaccine, 2003, 21(23): 3335-3341.
[62]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.
[63]Sakaguchi S,kaguchi N,Shimizu J et al.Immunologic tolerance maintained by CD4+CD25+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity,and transp lantation tolerance[J].Immunol Rev. 2001,182(8):18-32.
[64]Zelenika D,Adams E,Humm S et al.The role of CD4+T cell subsets in determining transp lantation rejection or tolerance[J].Immunol Rev.2001,182(8):164-179.
[65]Zou WP.Immunosuppressive networks in the tumor environment and their therapeutic relevance[J].Nat Rev Cancer, 2005, 5(4):263-274.
[66]Viguier M,Lemait re F,Verola O,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.
[67]Woo EY,Yeh H,Chu CS,et al.Cutting edge:Regulatory T cells from lung cancer patient s directly inhibit autologous T cell proliferation[J].J Immunol.2002,168(9):4272-4276.
[68]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.
[69]Yanade U K,Moore TT,Joo HG,et al.Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patient s with pancreas or breast adenocarcinoma[J].J Immunol.2002, 169(5):2756-2761.
[70]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.
[71]Apostolou I,Von Boehmer H.In vivo inst ruction of suppressor commitment in nave T cells[J].J Exp Med. 2004,199(10):1401-1408.
[72]Sakaguchi S. Naturally arising Foxp3 expressing CD4+CD25+ regulatory T cells in immunological tolerance to self and nonself[J].Nat Immunol.2005, 6(4):345-352.
[73]Lim HW,Hillsamer P,Banham AH, et al. Cutting edge: Direct suppression of B cells by CD4+CD25+ regulatory T cells [J]. J Immunol.2005,175(7):4180-4183.
[74]Khazaie K, Von Boehmei H. The impact of CD4+CD25+Treg on tumor specific CD8+ T cell cytotoxicity and cancer[J].Semin Cancer Biol.2006,12(2):124-136.
[75]Zou WP. Immunosuppressive networks in the tumor environment and their therapeutic relevance[J]. Nat Rev Cancer.2005,5(4):263-274.
[76]Sasaki Y,SakaiM,Miyazaki S,et al.Decidual and peripheral blood CD4+CD25+ regulatory T cells in early pregnancy subjects and spontaneous abortion cases[J]. Mol Hum Rep rod.2004,10(5):347-353.
[77]Aluvmare VR, Mand K, Betz AG. Regulatoty T cells mediate maternal tolerance to the fetus [J]. Nat Immunol. 2004, 5(3):266-271.
[78]Somerset DA,zheng Y,KilbyM D et al.Normai human pregnancy is associated with an elevation in the immune suppressive CD4+CD25+ regulatory T-cell subet[J]. Immunology. 2004,112(1):38-43.
[79]Saito S, Shima T, Nakashima A et al. What is the role of regulatory T cells in the success of implantation and early pregnancy?[J].J Assist Reprod Genet. 2007,24(9):379-386.
[80]Shevach EM. Certified professionals: CD4+CD25+ suppressor T cells[J]. J Exp Med, 2001, 193(11): F41-46.
[81]Schreiber TH. The use of FoxP3 as a biomarker and prognostic factor for malignant human tumors[J]. Cancer Epidemiol Biomarkers Prev. 2007,16(10):1931-1934.
[82]Young NS, calado RT, scheinberg P et al. Current concepts in the pathophysiology and treatment of aplastic anemia [J]. Blood,2006,108(8):2509-2519.
[83]Young NS. Autoimmunity and its treatment in aplastic anemia [J].Ann Intern Med,1997, 126(2):166-168.
[84]Solomou EE, Rezvani K, Mielke S, et al. Deficient CD4+CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia [J]. Blood.2007,110(5):1603-1606.
[85]Crispin JC,Martines A,Alcocer Varela J,et al.Quantification of regulatory T cells in patients with systemic lupus erythematosus[J].J Autoimmun.2003,21 (3): 273-276.
[86]Liu MF,Wang CR,Fung LL,Wu CR.Decreased CD4+CD25+T cells in peripheral blood of patients with systemic lupus erythematosus[J].Scand J Immunol.2004,59(2):198-202.
[87]Murphy TJ, Ni Choileain N, Zang Y,et al.CD4+CD25+ regulatory T cells control innate immune reactivity after injury[J].J Immunol. 2005,174(5):2957-2963.
[88]HoriS, NornuraT, SakaguchiSetal. Contro of regulatory T cell development by the transcription factor Foxp3 [J]. Science. 2003,299(5609): 1057-1061.
[89]Terabe M, Berzofsky J A. Immuno regulatory T cells in tumor immunity[J]. Curr Opin Immunol, 2004, 16(2): 157-162.
[90]Wolf AM, 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.
[91]Sasada T, Kimunre M, Yoshida Y, et al. CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies possible involvement of regulatory T cells in disease progression[J]. Cancer, 2003, 98(5): 1089-1099.
[92]Rosenberg SA. Progress in human tumor immunology and immunotherapy[J]. Nature, 2001, 411(6835): 380-384.
[93]Baecher-Allan C, Viglietta V, Hafler DA. Human CD4+CD25+ regulatory T cells[J]. Semin Immunol, 2004, 16(2): 89-97 .
[94]Gregg R, Smith CM, Clark FJ, et al.The number of human peripheral blood CD4+CD25+ regulatory T cells increases with age[J]. Clin Exp Immunol, 2005, 140(3): 540-546.
[95]Sutmuller RP, van Duivenvoorde LM, van Elsas A, et al. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitum or the rapy reveals altemative pathways for suppression of autoreactive cytotoxic T lymphocyte responses[J]. J Exp Med, 2001, 194(6): 823-832.
[96]Tanaka H, Tanaka I, Kjaergaard I, et al. Depletion of CD4+CD25+ regulatory T cells augments the generation of specific immune T cells in tumor-draining lymph nodes[J]. J Immunother, 2002, 25(3):207-217.
[97]Sakaguch S., Naturally arising CD4+ regulatory T cells for immunologic self- tolerance and negative control of immune responses[J]. Annu Rev Immunol. 2004.,22: 531-562.
[98]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.
[99]Singh B, Read S, Asseman C, et al.Control of intestinal inflammation by regulatory T cells[J]. Immunol Rev.2001,182: 190-200.
[100]Sakaguchi S.Animal models of autoimmunity and their relevance to human diseases[J]. Curr Opin Immunol.2000,12(6): 684-690.
[101]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.
[102]Shevach EM.Regulatory T cells in autoimmmunity[J]. Annu Rev Immunol. 2000,18:423-449.
[103]Barthlott T,Moncrieffe H,Veldhoen M,et al. CD4+CD25+ T cells compete with naive CD4+T cells for IL-2 and exploit it for the induction of IL-10 production[J]. Int Immunol. 2005,17(3):279-288.
[104]Bruniquel D,Schwartz RH.Selective,stable demethylation of the interleukin-2 gene enhances transcription by an active process[J].Nat Immunol. 2003,4(3):235-240.
[105]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.
[106]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.
[107]Fehervari Z,Sakaguchi S.Control of CD4+CD25+ regulatory cell activation and function by dendritic cells[J].Int Immunol.2004,16(12):1769-1780.
[108]Szanya V, Ermann J, Taylor C, et al. The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7[J]. J Immunol. 2002,169(5):2461-2465.
[109]Liu H, Hu B, Xu D,et al. CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4[J]. J Immunol. 2003,171(10):5012-7.
[110]Ermann J, Hoffmann P, Edinger M, Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD[J]. Blood. 2005,105(5):2220-6.
[111]Cederbom L, Hall H, Ivars F. CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells[J]. Eur J Immunol. 2000, 30(6): 1538-1543.
[112]Toda A, Piccirillo CA. Development and function of naturally occurring CD4+CD25+ regulatory T cells[J]. J Leukoc Biol.2006,80(3):458-470.
[113]Nelson BH.IL-2,regulatory T cells,and tolerance[J]. J Immunol. 2004,172(7):3983- 3988.
[114]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.
[115]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.
[116]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.
[117]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.
[118]Hoffmann P, Eder R, Kunz-Schughart LA,et al. Large-scale in vitro expansion ofpolyclonal human CD4+CD25high regulatory T cells[J]. Blood. 2004,104(3):895-903.
[119]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.
[120]Mihu CM, Mihu D, Costin N, et al.Isolation and charact- erization of stem cells from the placenta and the umbilical cord[J].Rom J Morphol Embryol. 2008,49(4): 441- 446.
[121]Bensinger SJ,Bandeira A,Jordan MS,et al. Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells[J].J Exp Med.2001,194(4):427-438.
[122]van Santen HM,Benoist C,Mathis D. Number of T reg cells that differentiate does not increase upon encounter of agonist ligand on thymic epithelial cells[J]. J Exp Med,2004,200(10):1221-1230.
[123]Wing K, Ekmark A, Karlsson H,et al. Characterization of human CD4+CD25+ T cells in thymus, cord and adultblood [J]. Immunology.2002,106(2):190-199.
[124]Ziegler SF. FOXP3: ofmice andmen[J].AnnuRev Immunol. 2006,24 (1): 209-226.
[125]Godfrey WR, Spoden DJ, Ge YG,et al. Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function[J].Blood.2005, 105(2): 750-758.