γ-干扰素刺激培养imDC过继治疗抗-GBM肾炎的实验研究
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摘要
目的:经IFN-γ刺激培养的imDC,过继转入抗-GBM肾炎模型,观察经IFN-γ刺激后的imDC对抗-GBM肾炎肾脏病变的抑制效果。
方法:以合成多肽序列pCol (28-40)作为抗原,包入脂质体内制成抗原制剂,建立抗-GBM肾炎大鼠模型,具体为:第0天(d0)脂质体抗原制剂0. 25 ml,于脚掌和尾根部皮下注射初次致敏;第7天(d7)脂质体抗原制剂0. 1 ml,同部位加强致敏;采集wistar大鼠骨髓,采用经典的GM-CSF+IL-4方案制备imDC,经imDCIFN-γ刺激培养,以合成多肽pCol (28-40)多肽作为外源性抗原处理imDCIFN-γ,流式细胞仪检测表面MHC II类分子,及其共刺激分子CD80, CD86和DC表面相对特异性标记OX62的表达情况,在体外与脾脏分离的CD4+T共培养,采用MTT比色法检测CD4+T增殖情况,Annexin-V法检测CD4+T凋亡情况,酶联免疫吸附法检测脾细胞培养液中IL-4、IL-13、IFN-γ的含量,观察体外抗原特异性T细胞功能活性状态;并于首次致敏前7天(-d7)过继转入抗-GBM肾炎模型,间隔7天随机选取相关样本进行生化指标(24小时尿白蛋白,血清肌酐,血尿素氮)肾脏病理学、免疫学指标,病理学、免疫学检查,观察对抗-GBM肾炎肾小球病变抑制效果及抗原特异性免疫耐受的相关机制.
结果:(1)光镜下观察imDC IFN-γ细胞呈圆形或卵圆形,细胞膜略不规则,有少量的短小突起或伪足。流式细胞仪检测imDC和imDC IFN-γ表面低表达的MHC II类分子(14.01%)及共刺激分子CD80(4.78%)、CD86(19.79%),同样低表达DC表面相对特异性标记OX62( 15.31%)(p<0.05, n=4)的水平都显著低于mDC;
(2)抗-GBM肾炎大鼠模型组大鼠尿蛋白,血肌酐,血尿素氮短期内明显升高(P < 0.05,n=10)、HE染色肾小球系膜细胞数增多,炎细胞浸润,PAS染色均有细胞性新月体形成(占肾小囊>50%)、免疫荧光可见大量IgG线样沉积;电镜可见足突融合,基底膜增厚,内皮细胞增生,免疫复合物沉积。
(3)体外共培养的脾淋巴细胞,imDC组与对照组增殖无显著性差异;而imDCIFN-γ组与对照组增殖明显受抑制,对照组OD值(0.1950±0.0160.);imDC组OD值(0.1974±0.017);imDCIFN-γ组为OD值(0.836±0.012)(p<0.05, n=5);imDCIFN-γ组凋亡率为(12.98+0.02),与对照组和imDC组相比凋亡均明显增高(p<0.05, n=5);上调了Th2型细胞因子IL一4及IL-13的表达,而下调Th1型细胞因子IFN-γ;Th1型细胞因子明显向Th2型细胞因子偏移。进一步体内实验发现,过继转入imDC IFN-γ后均能明显降低尿蛋白,血肌酐,血尿素氮水平,同时期亦减少炎细胞浸润,细胞新月体形成比率和速度减少,免疫荧光检测发现IgG沉积减少,荧光强度减弱;过继转入imDCIFN-γ能够抑制细胞性新月体形成,能明显缓解抗-GBM肾炎病情的进展,对抗-GBM肾炎具有保护作用
结论:
1. imDC IFN-γ对外源性抗原多肽pCol(28-40)摄取及提呈能力明显下降;可抑制T细胞增殖,促进T细胞凋亡;实验还表明IFN-γ刺激培养的未成熟树突状细胞促使Th1型细胞因子向Th2型细胞因子偏移,诱导形成特异性抗原免疫耐受。
2.成功建立了抗-GBM肾炎大鼠模型
3. IFN-γ刺激培养的imDC过继转入模型大鼠,可诱导形成抗原特异性免疫耐受,能明显抑制抗-GBM肾炎病情的进展,对抗-GBM肾炎具有保护作用。
Objective: The imDC was stimulated by IFN-γand adoptive transferred into the anti-GBM model. The inhibition effect of the stimulated imDC to glomerular change of anti-GBM nephritis was observed.
Methods: The synthesize Peptide sequence pCol(28-40) was enwrapped into lipid for antigen preparation, the anti-GBM nephritis model was established. Steps are as below: day 0 (d0) 0.25ml liposome antigen was prepared, feet and the root of the tail were initially subcutaneous injected the allergens. The 7th day, 0.1ml liposome antigen were injected in the same parts for enhancement. Based on the ability of imDC to induce immune tolerance, we collected the bone marrow of wistar rat, and prepared imDC with the classic GM-CSF + IL-4 plan. And the imDCIFN-γwas cultured with synthetic peptide pCol (28-40) peptide as an antigen stimulus. We detected the expression of surface MHC II molecules, other costimulatory molecules CD80, CD86 and DC-specific marker OX62 by flow cytometry. And we co-cultured the isolated spleen CD4 + T in vitro. The CD4 + T proliferation were detected by the MTT ratio color assay, the CD4 + T apoptosis were detected by Annexin-V assay, the spleen cell culture medium level of IL-4,IL-13 and IFN-γwere detected by ELISA. We observed the function active state of in vitro antigen-specific T-cell. 7 days before sensitization (-d7), adoptive transfer into anti-GBM nephritis model. Every other 7 days we randomly selected sample for related biochemical indicators (24-hour urinary albumin, serum creatinine, blood urea nitrogen) renal pathology, immunology parameters, serum IgG antibodies and pathology and immunohistochemistry testing to observe the inhibitory effect for glomerular change of anti-GBM nephritis and study the mechanisms of antigen-specific immune tolerance.
Results:
(1) optical microscope showed that the imDC IFN-γcells were round or oval, the cell membrane were slightly irregular with a small number of short protrusions or pseudopodia. The result of flow cytometry show low level of MHC II molecules (14.01%), costimulatory molecules CD80 (4.78%), CD86 (19.79%) on the surface of imDC and imDC IFN-γ. The level of DC specific markers OX62 (15.31%) (p <0.05, n = 4) were significantly lower than imDC;
(2) The urine protein, serum creatinine, blood urea nitrogen of anti-GBM nephritis rats were significantly increased during a short term (P <0.05, n = 10). HE staining showed the increase of mesangial, stromal cells and the infiltration of inflammatory cells. PAS staining showed the formation of crescent (accounting for more than 50% in renal cysts). Immune fluorescence showed that IgG linear deposited. Electron microscopy showed fusion of foot processes, basement membrane thickening, endothelial cell proliferation and immune complex deposition.
(3) The in vitro co-culture of spleen lymphocytes of imDC group and the control group had no significant difference in proliferation, however the proliferation of imDCIFN-γgroup and control group were inhibited significantly. OD values of the control group was 0.1950±0.0160, imDC group was 0.1974±0.017, imDCIFN-γgroup was 0.836±0.012 (p <0.05, n = 5). The apoptosis rate of imDCIFN-γgroup is 12.98 +0.02 which was significantly increased compared with the control group and imDC group (p <0.05, n = 5); The spleen cell supernatants level of IL-4, IL-13, IFN-γand the Thl/Th2 cytokines ratio were detected by ELISA. The Th1 type cytokines IFN-γwere down-regulated while the Th2 type cytokines IL-4 and IL-13 were up-regulated. Th1 type cytokines clearly shifted to Th2 type cytokines. Further in vivo experiments found that adoptive transfer of imDC IFN-γcould significantly reduce proteinuria, serum creatinine, BUN level and crescent formation rate and decrease inflammatory cell infiltration at the same time. Immune fluorescence showed the IgG deposition was reduced. High-dose and multiple-turn in of imDCIFN-γcould inhibit cellular crescent formation and significantly alleviate the development of anti-GBM nephritis.
Conclusion:
1.The uptake and presentation capability of the imDCIFN-γto exogenous antigen pCol (28-40) peptide decreased significantly, the T cell proliferation were inhibited and the T cell apoptosis were promoted. Experiments also showed that the IFN-γstimulated immature dendritic cells promote shift of Th1 type cytokines to Th2 type cytokine and induced the specific antigen immune tolerance.
2. The anti-GBM nephritis rat model was successfully established.
3. IFN-γstimulated imDC adoptive transferred into the rat model could induce immune tolerance and inhibit the development of anti-GBM nephritis which shows its protection to anti-GBM nephritis.
方法:以合成多肽序列pCol (28-40)作为抗原,包入脂质体内制成抗原制剂,建立抗-GBM肾炎大鼠模型,具体为:第0天(d0)脂质体抗原制剂0. 25 ml,于脚掌和尾根部皮下注射初次致敏;第7天(d7)脂质体抗原制剂0. 1 ml,同部位加强致敏;采集wistar大鼠骨髓,采用经典的GM-CSF+IL-4方案制备imDC,经imDCIFN-γ刺激培养,以合成多肽pCol (28-40)多肽作为外源性抗原处理imDCIFN-γ,流式细胞仪检测表面MHC II类分子,及其共刺激分子CD80, CD86和DC表面相对特异性标记OX62的表达情况,在体外与脾脏分离的CD4+T共培养,采用MTT比色法检测CD4+T增殖情况,Annexin-V法检测CD4+T凋亡情况,酶联免疫吸附法检测脾细胞培养液中IL-4、IL-13、IFN-γ的含量,观察体外抗原特异性T细胞功能活性状态;并于首次致敏前7天(-d7)过继转入抗-GBM肾炎模型,间隔7天随机选取相关样本进行生化指标(24小时尿白蛋白,血清肌酐,血尿素氮)肾脏病理学、免疫学指标,病理学、免疫学检查,观察对抗-GBM肾炎肾小球病变抑制效果及抗原特异性免疫耐受的相关机制.
结果:(1)光镜下观察imDC IFN-γ细胞呈圆形或卵圆形,细胞膜略不规则,有少量的短小突起或伪足。流式细胞仪检测imDC和imDC IFN-γ表面低表达的MHC II类分子(14.01%)及共刺激分子CD80(4.78%)、CD86(19.79%),同样低表达DC表面相对特异性标记OX62( 15.31%)(p<0.05, n=4)的水平都显著低于mDC;
(2)抗-GBM肾炎大鼠模型组大鼠尿蛋白,血肌酐,血尿素氮短期内明显升高(P < 0.05,n=10)、HE染色肾小球系膜细胞数增多,炎细胞浸润,PAS染色均有细胞性新月体形成(占肾小囊>50%)、免疫荧光可见大量IgG线样沉积;电镜可见足突融合,基底膜增厚,内皮细胞增生,免疫复合物沉积。
(3)体外共培养的脾淋巴细胞,imDC组与对照组增殖无显著性差异;而imDCIFN-γ组与对照组增殖明显受抑制,对照组OD值(0.1950±0.0160.);imDC组OD值(0.1974±0.017);imDCIFN-γ组为OD值(0.836±0.012)(p<0.05, n=5);imDCIFN-γ组凋亡率为(12.98+0.02),与对照组和imDC组相比凋亡均明显增高(p<0.05, n=5);上调了Th2型细胞因子IL一4及IL-13的表达,而下调Th1型细胞因子IFN-γ;Th1型细胞因子明显向Th2型细胞因子偏移。进一步体内实验发现,过继转入imDC IFN-γ后均能明显降低尿蛋白,血肌酐,血尿素氮水平,同时期亦减少炎细胞浸润,细胞新月体形成比率和速度减少,免疫荧光检测发现IgG沉积减少,荧光强度减弱;过继转入imDCIFN-γ能够抑制细胞性新月体形成,能明显缓解抗-GBM肾炎病情的进展,对抗-GBM肾炎具有保护作用
结论:
1. imDC IFN-γ对外源性抗原多肽pCol(28-40)摄取及提呈能力明显下降;可抑制T细胞增殖,促进T细胞凋亡;实验还表明IFN-γ刺激培养的未成熟树突状细胞促使Th1型细胞因子向Th2型细胞因子偏移,诱导形成特异性抗原免疫耐受。
2.成功建立了抗-GBM肾炎大鼠模型
3. IFN-γ刺激培养的imDC过继转入模型大鼠,可诱导形成抗原特异性免疫耐受,能明显抑制抗-GBM肾炎病情的进展,对抗-GBM肾炎具有保护作用。
Objective: The imDC was stimulated by IFN-γand adoptive transferred into the anti-GBM model. The inhibition effect of the stimulated imDC to glomerular change of anti-GBM nephritis was observed.
Methods: The synthesize Peptide sequence pCol(28-40) was enwrapped into lipid for antigen preparation, the anti-GBM nephritis model was established. Steps are as below: day 0 (d0) 0.25ml liposome antigen was prepared, feet and the root of the tail were initially subcutaneous injected the allergens. The 7th day, 0.1ml liposome antigen were injected in the same parts for enhancement. Based on the ability of imDC to induce immune tolerance, we collected the bone marrow of wistar rat, and prepared imDC with the classic GM-CSF + IL-4 plan. And the imDCIFN-γwas cultured with synthetic peptide pCol (28-40) peptide as an antigen stimulus. We detected the expression of surface MHC II molecules, other costimulatory molecules CD80, CD86 and DC-specific marker OX62 by flow cytometry. And we co-cultured the isolated spleen CD4 + T in vitro. The CD4 + T proliferation were detected by the MTT ratio color assay, the CD4 + T apoptosis were detected by Annexin-V assay, the spleen cell culture medium level of IL-4,IL-13 and IFN-γwere detected by ELISA. We observed the function active state of in vitro antigen-specific T-cell. 7 days before sensitization (-d7), adoptive transfer into anti-GBM nephritis model. Every other 7 days we randomly selected sample for related biochemical indicators (24-hour urinary albumin, serum creatinine, blood urea nitrogen) renal pathology, immunology parameters, serum IgG antibodies and pathology and immunohistochemistry testing to observe the inhibitory effect for glomerular change of anti-GBM nephritis and study the mechanisms of antigen-specific immune tolerance.
Results:
(1) optical microscope showed that the imDC IFN-γcells were round or oval, the cell membrane were slightly irregular with a small number of short protrusions or pseudopodia. The result of flow cytometry show low level of MHC II molecules (14.01%), costimulatory molecules CD80 (4.78%), CD86 (19.79%) on the surface of imDC and imDC IFN-γ. The level of DC specific markers OX62 (15.31%) (p <0.05, n = 4) were significantly lower than imDC;
(2) The urine protein, serum creatinine, blood urea nitrogen of anti-GBM nephritis rats were significantly increased during a short term (P <0.05, n = 10). HE staining showed the increase of mesangial, stromal cells and the infiltration of inflammatory cells. PAS staining showed the formation of crescent (accounting for more than 50% in renal cysts). Immune fluorescence showed that IgG linear deposited. Electron microscopy showed fusion of foot processes, basement membrane thickening, endothelial cell proliferation and immune complex deposition.
(3) The in vitro co-culture of spleen lymphocytes of imDC group and the control group had no significant difference in proliferation, however the proliferation of imDCIFN-γgroup and control group were inhibited significantly. OD values of the control group was 0.1950±0.0160, imDC group was 0.1974±0.017, imDCIFN-γgroup was 0.836±0.012 (p <0.05, n = 5). The apoptosis rate of imDCIFN-γgroup is 12.98 +0.02 which was significantly increased compared with the control group and imDC group (p <0.05, n = 5); The spleen cell supernatants level of IL-4, IL-13, IFN-γand the Thl/Th2 cytokines ratio were detected by ELISA. The Th1 type cytokines IFN-γwere down-regulated while the Th2 type cytokines IL-4 and IL-13 were up-regulated. Th1 type cytokines clearly shifted to Th2 type cytokines. Further in vivo experiments found that adoptive transfer of imDC IFN-γcould significantly reduce proteinuria, serum creatinine, BUN level and crescent formation rate and decrease inflammatory cell infiltration at the same time. Immune fluorescence showed the IgG deposition was reduced. High-dose and multiple-turn in of imDCIFN-γcould inhibit cellular crescent formation and significantly alleviate the development of anti-GBM nephritis.
Conclusion:
1.The uptake and presentation capability of the imDCIFN-γto exogenous antigen pCol (28-40) peptide decreased significantly, the T cell proliferation were inhibited and the T cell apoptosis were promoted. Experiments also showed that the IFN-γstimulated immature dendritic cells promote shift of Th1 type cytokines to Th2 type cytokine and induced the specific antigen immune tolerance.
2. The anti-GBM nephritis rat model was successfully established.
3. IFN-γstimulated imDC adoptive transferred into the rat model could induce immune tolerance and inhibit the development of anti-GBM nephritis which shows its protection to anti-GBM nephritis.
引文
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1. Ya?Huan Lou. Anti-GBM glomerulonephritis: a T cell-mediated autoimmune disease. [J] Arch Immunol Ther Exp.2004,52: 96–103.
2. Borillo J, Wu J, Arends J, et al. Characterization of the Peptide sequencethat causes anti-GBM glomerulo- nephritis. [J] Kidney Int. 2005, 68(3):1061-1070.
3. Robertson J, Wu J, Arends J, et al. Activation of glomerular basement membrane specific B cells in the renal draining lymph node after T cell–mediated glomerular injury. [J]. Am Soc Nephrol. 2005,16:3256–3263.
4. Wu, J, J. Hicks and Y.-H. Lou. Glomerulonephritis induced by recombinant collagen IV_3 chain noncollagen domain 1 is not associated with glomerular basement membrane antibody: a potential T cell-mediated mechanism. [J]. Immunol. 2001,167:2388.
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14. Kathryn J. Wood1and Birgit Sawitzki. Interferon-γ: a crucial role in the function of induced regulatory T cells in vivo. [J].TRENDS in Immunology,,2006,27(4):183-186
15. HoriS,NomuraT,SakaguchiS.ControlofregulatoryTceldevelopmentbythetranscriptionfactorFoxp3[J].Science,2003,299(5609):1057-1061.
16. KelchtermansH,DeKlerckB,MiteraT,etal.DefectivCD4+CD25+regulatoryTcellfunctioningincollagen2inducearthritis:animportantfactorinpathogenesis,counterregulaedbyendogenousIFN-gamma[J].ArthritisResTher,2005 7(2):R402-R415.
17. NishikawaH,KatoT,TawaraI,etal.IFN - gamma Controlthegeneration Pactivation of CD4+CD25+regulatoryTcellsin antitumor immunere sponse.[J]. Immunol ,2005,175(7): 4433-4440.
18. HarringtonLE,HattonRD,ManganPR,etal.Interleuki172producingCD4+effector T cell develop via alineagedis tinctfromtheThelpertype1and2lineages[J].NatImmu-nol,2005, 6(11):1123-1132.
19. Tang Q, Adams J, Bi M, et al. Visualizing regulatory T cell control of autoimmune response in nonobese diabetic mice. Nat Immunol, 2006;7(1):83-92.
20. 4Shevach E M, Mchugh R S, Piccrillo C A, et al. Control of T-cell activationby CD4+CD25+ suppressor T cells. Immunol Rev, 2001;182:58-67.
21. Thornton A M, Shevach E M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2production. J Exp Med, 1998;188(2):287-296.
22. 6Hontsu S, Yoneyama H, Ueha S, et al. Visualization of naturally occurring Foxp3+ regulatory T cells in normal and tumor-bearing mice. Int Immunol,2004;4(14): 1785-1793.
23. Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+)regulatory cells that control intestinal inflammation. J Exp Med, 2000;192(2):295-302.
24. 8Taylor P A, Lees C J, Fournier S, et al. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T T interactions.J Immunol, 2004;172(1):34-39.
25. Brusko T M, Wasserfall C H, Agarwal A, et al. An integral role for heme一l and carbon monoxide in maintaining peripheral tolerance by CD4+CD25+ regulatory T cells. J Immunol, 2005;174(9):5181-5186.
26. Saito S, Sasaki Y, Sakai M. CD4+CD25high regulatory T cells in human J Reprod Immunol, 2005; 65(2):111-120.
27. Shevach E M, Mchugh R S, Piccrillo C A, et al. Control of T-cell activation by CD4+CD25+ suppressor T cells. Immunol Rev, 2001;182:58-67.
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30. Zhou, X. Y., S. Bailey-Bucktrout, et al. "Plasticity of CD4(+) FoxP3(+) T cells." Current Opinion in Immunology,2009,21(3): 281-285.
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1. Ya?Huan Lou. Anti-GBM glomerulonephritis: a T cell-mediated autoimmune disease. [J] Arch Immunol Ther Exp.2004,52: 96–103.
2. Borillo J, Wu J, Arends J, et al. Characterization of the Peptide sequencethat causes anti-GBM glomerulo- nephritis. [J] Kidney Int. 2005, 68(3):1061-1070.
3. Robertson J, Wu J, Arends J, et al. Activation of glomerular basement membrane specific B cells in the renal draining lymph node after T cell–mediated glomerular injury. [J]. Am Soc Nephrol. 2005,16:3256–3263.
4. Wu, J, J. Hicks and Y.-H. Lou. Glomerulonephritis induced by recombinant collagen IV_3 chain noncollagen domain 1 is not associated with glomerular basement membrane antibody: a potential T cell-mediated mechanism. [J]. Immunol. 2001,167:2388.
5. Thornton A M, Shevach E M. 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. Hontsu S, Yoneyama H, Ueha S, et al. Visualization of naturally occurring Foxp3+ regulatory T cells in normal and tumor-bearing mice. Int Immunol, 2004;4(14): 1785-1793.
7. Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med, 2000; 192(2):295-302.
8. Taylor P A, Lees C J, Fournier S, et al. B7 expression on T cells down- regulates immune responses through CTLA-4 ligation via T T interactions. J Immunol, 2004;172(1):34-39.
9. Herman A E, Freeman G J, Mathis D, et al. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med, 2004;199(11):1479-1489.
10. von Boehmer H. Mechanisms of suppression by suppressor T cells. Nat Immunol, 2005;6:338-344.
11. Richard Kitchen. : Experimental Autoimmune Anti–Glomerular Basement Mem- braneGlomerulonephritisA Protective Role for IFN-γ.[J] Am Soc Nephrol ,2004,15: 1764–1774,
12. [2]P'ukushima A, Iamanuchi T, Ishida W, ec rel. Mice lackinn the IP'N-namma receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immuneresponses[J].Immunonenetics, 2005, 57( 5):337-343
13. Nishihori T,Tanahe Y,Su L, et al. Impaired development of CD4+ CD25+ regulatory T cells in the absence of STAT1:increased susceptibility to autoimmune disease「J」.J Exp Med 2004 ,199(1):25一34
14. Kathryn J. Wood1and Birgit Sawitzki. Interferon-γ: a crucial role in the function of induced regulatory T cells in vivo. [J].TRENDS in Immunology,,2006,27(4):183-186
15. HoriS,NomuraT,SakaguchiS.ControlofregulatoryTceldevelopmentbythetranscriptionfactorFoxp3[J].Science,2003,299(5609):1057-1061.
16. KelchtermansH,DeKlerckB,MiteraT,etal.DefectivCD4+CD25+regulatoryTcellfunctioningincollagen2inducearthritis:animportantfactorinpathogenesis,counterregulaedbyendogenousIFN-gamma[J].ArthritisResTher,2005 7(2):R402-R415.
17. NishikawaH,KatoT,TawaraI,etal.IFN - gamma Controlthegeneration Pactivation of CD4+CD25+regulatoryTcellsin antitumor immunere sponse.[J]. Immunol ,2005,175(7): 4433-4440.
18. HarringtonLE,HattonRD,ManganPR,etal.Interleuki172producingCD4+effector T cell develop via alineagedis tinctfromtheThelpertype1and2lineages[J].NatImmu-nol,2005, 6(11):1123-1132.
19. Tang Q, Adams J, Bi M, et al. Visualizing regulatory T cell control of autoimmune response in nonobese diabetic mice. Nat Immunol, 2006;7(1):83-92.
20. 4Shevach E M, Mchugh R S, Piccrillo C A, et al. Control of T-cell activationby CD4+CD25+ suppressor T cells. Immunol Rev, 2001;182:58-67.
21. Thornton A M, Shevach E M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2production. J Exp Med, 1998;188(2):287-296.
22. 6Hontsu S, Yoneyama H, Ueha S, et al. Visualization of naturally occurring Foxp3+ regulatory T cells in normal and tumor-bearing mice. Int Immunol,2004;4(14): 1785-1793.
23. Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+)regulatory cells that control intestinal inflammation. J Exp Med, 2000;192(2):295-302.
24. 8Taylor P A, Lees C J, Fournier S, et al. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T T interactions.J Immunol, 2004;172(1):34-39.
25. Brusko T M, Wasserfall C H, Agarwal A, et al. An integral role for heme一l and carbon monoxide in maintaining peripheral tolerance by CD4+CD25+ regulatory T cells. J Immunol, 2005;174(9):5181-5186.
26. Saito S, Sasaki Y, Sakai M. CD4+CD25high regulatory T cells in human J Reprod Immunol, 2005; 65(2):111-120.
27. Shevach E M, Mchugh R S, Piccrillo C A, et al. Control of T-cell activation by CD4+CD25+ suppressor T cells. Immunol Rev, 2001;182:58-67.
28. Thornton A M, Shevach E M. 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.
29. Reynolds, J., J. Haxby, et al.. "Identification of a nephritogenic immunodominant B and T cell epitope in experimental autoimmune glomerulonephritis." Clin Exp Immunol, 2009,155(2): 311-9.
30. Zhou, X. Y., S. Bailey-Bucktrout, et al. "Plasticity of CD4(+) FoxP3(+) T cells." Current Opinion in Immunology,2009,21(3): 281-285.