摘要
1.通过将人血浆Exosomes小体、重组Wnt分子分别与人外周血CD4+CD25+CD1271ow调节性T细胞(Regulatory T Cells, Treg)作用,观察Treg细胞在体外的存活情况及其Wnt信号通路的变化。
2.通过将同种异基因人外周血B淋巴细胞体外扩增Treg细胞,观察Treg细胞扩增前后在数量上的变化及表型和功能是否改变,建立体外扩增Treg细胞的有效方法,并与树突细胞(dendritic cell, DC)扩增Treg细胞的方法相比较。
1.应用免疫磁珠分离方法分离纯化人外周血CD4+CD25+CD1271owTreg细胞,将Wnt分子和从多个健康供者血浆中分离得到的Exosomes小体分别加入到Treg细胞中,加入人白介素2(IL-2,50U/ml)。利用RT-PCR检测共培养12h后Wnt信号通路涉及的凋亡相关基因的改变,同时采用流式技术检测Wnt信号通路中磷酸化β-catenin水平的改变,并在共培养14天后采用流式技术检测各组细胞的凋亡情况。设立未经处理的Treg细胞组为对照组。
2.将异体健康供者外周血分离得到的B淋巴细胞与磁珠分选得到的Treg细胞共孵育,用IL-2(IOOU/ml)和CD28单抗(500ng/ml)共同刺激,在培养14天的过程中通过流式检测Treg细胞表型的变化,同时与树突状细胞扩增Treg细胞的方法相比较(培养条件相同)。14天培养结束后,将细胞与CFSE处理过的经强刺激(加入CD3单抗500ng/ml,IL-2300U/ml)的CD4+效应性T细胞共培养,在第五天通过流式检测Treg细胞对CD4+T细胞的增殖抑制,设立只经强刺激的CFSE处理过的CD4+T细胞为对照组。
1.通过RT-PCR分析纯化的Treg细胞上的Frizzled(FRZ)受体及低密度脂蛋白相关蛋白5(LRP5)和LRP6的基因表达,结果显示Treg细胞表达FRZ2、3、4,LRP6基因,通过Western Blotting技术检测血浆中Exosomes小体上Wnt3a和Wnt5a的蛋白表达,均有条带出现。共培养12h后,RT-PCR检测凋亡相关基因Bcl-xL、Bcl-2、c-Myc的表达,结果显示Treg细胞与血浆exosomes样小体或Wnt分子共培养时,抗凋亡基因Bcl-2相对于对照组均有明显的上调,另一抗凋亡基因Bcl-xL和诱导凋亡的基因c-Myc无明显变化。培养14天后,通过流式检测凋亡,加入Exosomes样小体或Wnt分子的Treg细胞生存率均比对照组高。
2.经B细胞联合IL-2和CD28单抗刺激的Treg细胞在培养14天后流式检测Foxp3和CD4/CD25,阳性率均很高,此时计数Treg细胞为最初数量的20倍。利用CFSE检测技术,将扩增14天的Treg细胞与CD3单抗和IL-2强刺激的CD4+T细胞共培养,5天后流式检测,扩增后的Treg细胞能够有效地抑制CD4+T细胞的增殖功能。
1.初步判断Wnt信号通路能够影响Treg细胞的生存状态,血浆Exosomes小体能够延长Treg细胞的生存时间,其中Exosomes小体携带Wnt分子与Treg细胞上的Frizzled受体作用可以活化Treg细胞的Wnt信号通路,在提高Treg细胞的生存率方面起了一定的作用,即血浆Exosomes小体可能通过Wnt信号通路途径影响Treg细胞的存活。
2.同种异基因B淋巴细胞能够在体外有效的扩增人外周血Treg细胞,扩增效率可达20倍以上,并且能显著抑制CD4+T细胞的增殖。
1. Through the effect of human plasma exosomes-like vesicles or recombinant Wnt molecules on CD4+ CD25+ CD127low regulatory T cells (Treg), observed the survival of Treg cells in vitro and the changes of the Wnt signaling pathway.
2. Through the expansion of Treg cells by allogeneic human peripheral blood B lymphocytes in vitro, observed the changes of Treg cells in the number, phenotype and function after the expansion, established an effective way of the expansion of Treg cells in vitro, and compared with the expansion method by regulatory dendritic cells (rDC).
1.CD4+CD25+CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) using Magnetic cell sorting, Wnt molecules and human plasma exosomes-like vesicles isolated from several healthy donors were cocultured with the Treg cells respectively, with interleukin 2 (IL-2,50U/ml). Analyzed the changes of apoptosis related genes what Wnt signaling pathway involved in after 12h by RT-PCR, detected the changes of phosphorylatedp-catenin level in Wnt signaling pathway and the apoptosis of co-culture cells in every group after 14 days by flow cytometry. The untreated Treg cells were the control group.
2. The purified Treg cells handled by B-lymphocyte isolated from allogeneic peripheral blood with IL-2 (100U/ml) and CD28 monoclonal antibody (500ng/ml), detected the changes of phenotype of Treg cells in the 14 days co-culture. On the 14th day, the expanded Treg cells were cultured with CD4+ effector T cells which were stimulated strongly (addition of 500ng/ml CD3 monoclonal antibody and 300U/ml IL-2), detected the inhibition of the proliferation of CD4+ effector T cells by flow cytometry on the 5th day. The CD4+ effector T cells stimulated strongly only were established as the control group.
1. Analyzed the Frizzled receptors (FRZ) and LDL receptor related protein 5 (LRP5) and LRP6 gene expression on Treg cells by RT-PCR, the results showed that the Treg cells expressed FRZ2,3,4, LRP6 genes. Detected the protein expression of Wnt3a and Wnt5a in Plasma Exosomes by Western Blotting, both appeared the bands. Detection of apoptosis-related genes Bcl-xL, Bcl-2, c-Myc expression after 12h co-culture by RT-PCR showed that anti-apoptosis gene Bel-2 increased significantly in Treg cells cultured with plasma exosomes-like vesicles or the Wnt molecules, the expression of Bcl-xL and c-Myc had no significant change. On the 14th day, the survival rate of Treg cells which added exosomes-like vesicles or the Wnt molecules were higher than the control group by detecting the apoptosis with flow cytometry.
2. After 14 days of coculturation with B cells combined with IL-2 and CD28 monoclonal antibody, the positive rate of Foxp3 and CD4/CD25 of the Treg cells were both high, and the absolute number of Treg cells were amplified 20 times. The expanded Treg cells cultured with the CD4+ T cells which were stimulated by CD3 monoclonal antibody and IL-2, the results detected by flow cytometry using CFSE technology after 5 days showed that the expanded Treg cells can effectively inhibit the proliferation of CD4+ effector T cells.
1. We can initially judge that the Wnt signaling pathway can influence the survival status of Treg cells, plasma Exosomes-like vesicles were able to prolong the survival time of the Treg cells, in which the interaction between Wnt molecules carried by Exosomes-like vesicles and the Frizzled receptors on Treg cells can activate the Wnt signaling pathway of the Treg cells, which played a certain role in improving the survival rate of the Treg cells. The plasma Exosomes-like vesicles can affect the survival of Treg cells in vitro through the Wnt singaling pathway.
2. Allogeneic B lymphocytes can effectively amplify human peripheral blood Treg cells in vitro, the expansion efficiency can be up to 20 times compared to the initial, and the expanded Treg cells can inhibit the proliferation of CD4+ effector T cell significantly.
引文
[1]Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science, 2003,299(5609):1057-1061
[2]Vukmanovic-Stejic M, Zhang Y, Cook JE, et al. Human CD4+CD25hi Foxp3 regulatory T cells are derived by rapid turnover of memory populations in vivo[J]. J Clin Invest,2006,116 (9):2423-2433
[3]Liu W, Putnam AL, Xu-Yu Z, et al. CD127expression inversely correlates with Foxp3 and suppressive function of human CD4+Treg cells[J]. J Exp Med,2006,203(7):1701-1711
[4]Bienz M. beta-Catenin:a pivot between cell adhesion and Wnt signalling[J]. Curr Biol,2005,15(2):R64·R67
[5]Yi Ding, Shiqian Shen, Andreia C Lino, et al. Beta-catenin stabilization extendsregulatory T cell survivaland induce sanergyin nonregulatory T cells[J]. NATURE MEDICINE,2008,14(2):162-169.
[6]Johnstone RM, Adam M, Hammond JR, et al. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicle(exosomes) [J]. J Biol Chem,1987,262:9412-20.
[7]Keller S, Rupp C, Stoeck A, et al. CD24 is a marker of exosomes secreted into urine and amniotic fluid[J]. Kidney Int,2007,72(9):1095-102.
[8]Admyre C, Johansson SM, Qazi KR, et al. Exosomes with immune modulatory features are present in human breast milk [J]. J Immunol,2007,179:1969-1978.
[9]Gui-Jun Wang, Yuelong Liu, Aijian Qin, et al. Thymus Exosomes-like particles induce regulatory T cells[J]. The Journal of Immunology,2008,181: 5242-5248.
[10]Jiang M, Wang BY, Wang CQ, et, al. Inhibition of Hypoxia-Inducible Factor-1 α and Endothelial Progenitor Cell Differentiation by Adenoviral Transfer of Small Interfering RNA in vitro[J]. Journal of Vascular Research,2006; 43:511-521
[1]Taylor PA, Lees CJ, Blazar BR, et al. The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graft-versus-Hostdisease lethality[J]. Blood,2002,99(10):3493-3499
[2]Burchill MA, Yang J, Vogtenhuber C, et al. IL-2 receptor beta-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells [J]. J Immunol,2007,178(1):280-290
[3]Ring S, Thome M, Pretsch L, et al. Expanded murine regulatory T cells: Analysis of phenotype and function in contact hypersensitivity reactions [J]. J Immunol Methods,2007,326(1-2):10-21
[4]Trenado A, Sudres M, Tang Q, et al. Ex vivo-expanded CD4+CD25+ immunoregulatory T cells prevent graft-versus-host-disease by inhibiting activation/differentiation of pathogenic T cells[J]. J Immunol,2006, 176(2):1266-1273
[5]Yamazaki S, Iyoda T, Tarbell K, et al. Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells[J]. Exp Med, 2003,198(2):235-47
[6]Justin S. Ahn, Deepa K. Krishnadas, Babita Agrawal. Dendritic cells partially abrogatethe regulatory activity of CD4+CD25+T cells present in the human peripheral blood[J]. Internationallmmunology,2007,19(3):227-237
[7]Devi K. Baner jee, Madhav V. Dhodapkar, Elyana Matayeva, 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
[8]Leo C.Chen, Julio C. Delgado, Julio C. Delgado, et, al. Direct Expansion of Human Allospecific FoxP3+CD4+ Regulatory T Cells with Allogeneic B Cells for Therapeutic Application[J]. The Journal of Immunology,2009,183: 4094-4102 腺苷结合A2AR后,效应性T细胞的增殖效应消失,且不能生成IL-2和IFN-γ,同时能够通过抑制工L-6的表达且促进TGF-β的分泌使诱导型Treg细胞生成增多。IL-6能够促进Th-17细胞的发育及生成而同时抑制Treg细胞的生成,TGF-β能够诱导iTreg细胞的生成,因此,IL-6的抑制维持了Treg细胞的存活,TGF-β分泌增多加快了iTreg细胞的生成。3.小结
目前已经发现了多种Treg细胞免疫抑制的作用机制,如直接杀死或诱导效应细胞凋亡、分泌抑制性细胞因子、调控树突状细胞、改变效应细胞生存环境等。Treg细胞可能通过一种或几种作用机制共同发挥其免疫调控作用,直接或间接的影响效应性T细胞的作用,建立外周免疫耐受。进一步开展对Treg细胞免疫抑制功能作用机制的研究将为Treg细胞临床应用于各种疾病的治疗奠定坚实的理论基础。
1. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299(5609):1057-1061.
2. Vukmanovic-Stejic M, Zhang Y, Cook JE, et al. Human CD4+CD25hi Foxp3 regulatory T cells are derived by rapid turnover of memory populations in vivo[J]. J Clin Invest,2006,116:2423.
3. Fontenot J D, Gavin M A, Rudensky A Y. Foxp3 programs the development and function of CD4+CD25+regulatory T cell[J]. Nat Immunol,2003,
4.(4):330-336.
4.郝世勇,聂华,等.CD4+CD25+T调节性T细胞与肿瘤免疫[J].临床血液学杂志,2007,4(6)：285-288.
5. Karakhanova S, Munder M, Schneider M, et al. Highly efficient expansion of human CD4+CD25+ regulatory T cells for cellular immunotherapy in patients with graft-versus-host disease. J Immunother,2006,29(3):336-349.
6.Seidel MG, Ernst U, Printz D, et aL Expression of the putatively regulatory T-cell marker FOXP3 by CD4'CD25' T cells after pediatric hematopoietic stem cell transplantation. Haematologica,2006, 91(4):566-569.
7. Liu W, Putnam AL, Xu-Yu Z, et al. CD127expression inversely correlates with Foxp3 and suppressive function of human CD4+Treg cells[J]. J Exp Med,2006,203(7):1701-1711.
8.Morelli AE,7homson AW. Dendritic cells:regulators d alloimmunity and opportunities for tolerance induction[J]. ImmurmRev,2003,196: 125-146
9. Bluestone JA, Tang Q. How do CD4+ CD25+ regulatory T cell, control autoirmrunity? [J]. Cure Opin Immunol,2005,17(6):638-642.
10.Wang RF, Peng G, Wang HY. Regulatory T cells and Toll — like receptors in tumor immunity [J].Semin Immuml,2006,18(2):136-142.
11. Cohen JL, Trenado", Vasey D, et al. CD4+ CD25+irnmuno-regulatory T cells; New therapeutics for graft-versus-host disease [J]. J Exp Med, 2002,196(3):401-406.
12.高波,熊思东.CD4+CD25+调节性T细胞作用机制的双模式[J].生命的化学,2006,26(2)：131-133.
13. Weber SE, Harbertson J, Godebu E, et al. Adaptive islet-specific regulatory CD4 T cells control autoimmune diabetes and mediate the disappearance of pathogenic Thl cells in vivo[J]. J Immunol 2006, 176:4730-9.
14.Nadir Askenasy, Ayelet Kaminitz, Shai Yarkoni. Mechanisms of T regulatory cell function[J]. Autoimmunity Reviews,2008,03,001:1-6.
15. Esma S Yolcu, Shifra Ash, Ayelet Kaminitz, et al. Apoptosis as a mechanism of T-regulatory cell homeostasis and suppression[J]. Immunology and Cell Biology,2008,86:650-658.
16. von Herrath MG et al. Nat Rev Immunol,2003,3:223-232
17. Hawrylowicz, C. M. & O'Garra, A. Potential role of interleukin_10_secreting regulatory T cells in allergy and asthma[J]. Nature Rev. Immunol.2005,5:271-283
18. Kearley, J., Barker, J. E., Robinson, D. S. & Lloyd, C. M. Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+regulatory T cells is interleukin 10 dependent [J]. Exp. Med. 2005,202:1539-1547
19.Rubtsov, Y. P. et al. Regulatory T cell-derived interleukin_10 limits inflammation at environmental interfaces [J]. Immunity,2008,28: 546-558
20.Shoba Amarnath, Li Dong, Jun Li, Yuntao Wu et al. Endogenous TGF-β activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected human CD4+CD25-T cells[J]. Retrovirology,2007,4:57-73.
21. Kazuhiko Nakamura, Atsushi Kitani, Ivan Fuss, et al. TGF-1 Plays an Important Role in the Mechanism of CD4+CD25+Regulatory T Cell Activity in Both Humans and Mice. J Immunol,2004,172:834-842
22. Strauss, L. et al. A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-β1 mediates suppression in the tumor microenvironment[J]. Clin. Cancer Res,2007, 13:4345-4354
23. Hilchey, S. P., De, A., Rimsza, L. M., Bankert, R. B.&Bernstein, S. H. Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+[J]. Immunol,2007,178:4051-4061
24. Massimo C Fantini, Sabine Dominitzki, Angelo. Et al. In vitro generation of CD4+CD25+regulatory cells from murine naive T cells[J]. Nature Protocols,2007,2(7):1789-1794
25. Dat Q. Tran, Heather Ramsey, and Ethan M. Shevach. Induction of FOXP3 expression in naive human CD4+FOXP3-T cells by T-cell receptor stimulation is transforming growth factor-β-dependent but does not confer a regulatory phenotype[J]. Immunobiology,2007,110(8): 2983-2990.
26. Collison, L. W. et al. The inhibitory cytokine IL 35 contributes to regulatory T_cell function[J]. Nature,2007,450:566-569
27. Dario A. A. Vignali, Lauren W. Collison, Creg J. How regulatory T cells work[J]. Nature Reviews,2008,8:523-532.
28. Tang, Q. et al. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice[J]. Nature Immunol,2006,7:83-92
29. Takahashi, T. et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells:induction of autoimmune disease by breaking their anergic/suppressive state[J]. Int. Immunol,1998,10:1969-1980
30. Serra, P. et al. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells[J]. Immunity,2003, 19:877-889
31.Mellor, A. L. & Munn, D. H. IDO expression by dendritic cells: tolerance and tryptophan catabolism[J]. Nature Rev. Immunol,2004,4: 762-774
32. Cederbom, L., Hall, H. & Ivars, F. CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur. [J]. Immunol,2000,30:1538-1543
33. Taams, L. S. et al. Modulation of monocyte/macrophage function by human CD4+CD25+regulatory T cells [J]. Hum. Immunol,2005,66:222-230
34. Workman, C. J. & Vignali, D. A. A. Negative regulation of T cell homeostasis by LAG-3 (CD223) [J]. Immunol,2004,174:688-695
35. Liang, B. et al. Regulatory T cells inhibit dendritic cells by LAG-3 engagement of MHC class Ⅱ[J]. Immunol,2008,180:5916-5926
36. Sarris, M., Andersen, K. G., Randow, F., Mayr, L. & Betz, A. G. Neuropilin_1 expression on regulatory T cells enhances their interactions with dendritic cells during antigen recognition[J]. Immunity,2008,28:402-413
37. de la Rosa, M., Rutz, S., Dorninger, H. & Scheffold, A. Interleukin_2 is essential for CD4+CD25+ regulatory T cell function. Eur[J]. Immunol,2004,34:2480-2488
38. Chinen J et al. Advances in asthma, allergy and immunology series 2004:basic and clinical immunology [J]. J Allergy Clin Immunol,2004, 114(2):398-405
39. Oberle, N., Eberhardt, N., Falk, C. S., Krammer, P. H. & Suri-Payer, E. Rapid suppression of cytokine transcription in human CD4+CD25-T cells by CD4+Foxp3+ regulatory T cells:independence of IL-2 consumption, TGF-β, and various inhibitors of TCR signaling[J]. Immunol,2007,179:3578-3587
40. Deaglio, S. et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression [J]. Exp. Med,2007,204:1257-1265
41. Borsellino, G. et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells:hydrolysis of extracellular ATP and immune suppression [J]. Blood,2007,110:1225-1232
42. Zarek, P. E. et al. A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells [J]. Blood,2008,111:251-259
43. Oukka, M. Interplay between pathogenic Th17 and regulatory T cells. Ann[J]. Rheum. Dis,2007,66 (Suppl 3):87-90