PA-MSHA刺激的AML-DC对Treg作用的研究
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摘要
目的：CD4+CD25+调节性T细胞(Regulatory T cells,Treg)占正常人体外周CD4+T淋巴细胞的5%-10%,参与体内免疫抑制过程,具有免疫抑制和免疫无能性。在多种肿瘤患者外周血及肿瘤组织局部的数量增多,诱导维持外周免疫耐受。Treg可分为天然性Treg和获得性Treg。在一定的环境条件下由于抗原提呈细胞(antigen-presenting cell,APC)表面共刺激分子表达的改变或细胞因子种类的变化,使CD4+T细胞分化为获得性Treg。Treg通过降低对肿瘤相关抗原的初始T细胞免疫,导致肿瘤免疫逃逸,从而在肿瘤患者的免疫病理中发挥重要作用。有报道表明,减少Treg的数量在肿瘤生物治疗中至关重要。减少Treg的数量就可以减少Treg介导的免疫抑制作用,从而增强T细胞的效能。另有研究证实,未成熟树突状细胞可促使CD4+T细胞向获得性Treg分化,从而促进Treg的产生。目前肿瘤免疫治疗策略的研究重点集中在打破免疫耐受和提高宿主T细胞反应。铜绿假单胞菌注射液(Vaccine of Pseudomonas aeruginosa with Mannose Sensitive Hemagglutination pili,PA-MSHA)是从带甘露糖敏感血凝菌毛的铜绿假单胞菌中制备而来,因其产生免疫调节作用及低毒无副作用而倍受青睐,目前已广泛应用于抗感染和抗炎治疗,甚至作为免疫调节剂用于抗肿瘤治疗。据报道,PA-MSHA作为白血病、恶性淋巴瘤、肺癌、乳腺癌、胃癌、肝癌等恶性肿瘤的辅助治疗可提高临床有效率,其抗肿瘤的可能机制在于通过增加树突状细胞的抗原递呈能力,提高Th1/Th2的比例,调节细胞免疫和体液免疫的不平衡状态,达到有效的治疗肿瘤的目的,但其具体作用机制尚不十分明确。急性髓细胞白血病(Acutemyeloid leukemia,AML)是造血干细胞异常克隆增生导致的一种恶性肿瘤性疾病,伴有严重的免疫系统损伤。与正常人相比,AML患者外周血和骨髓血中均含有更高比例Treg。孙文平报道了PA-MSHA疫苗能明显提高急性白血病患者的免疫功能,增强其自身的免疫杀伤能力。PA-MSHA疫苗治疗组患者其IL-2水平、NK细胞活性、CD4+/CD8+比值均明显高于对照组。但是PA-MSHA疫苗是否可以通过降低AML患者体内的Treg细胞的数量和功能,从而发挥其抗肿瘤作用,还尚未有报道。本研究通过检测经铜绿假单胞菌注射液刺激的急性髓细胞白血病源性树突状细胞(dendritic cells derived from aute myeloid leukemia,AML-DCs)对Treg作用的影响,探讨PA-MSHA对AML的免疫调节作用。
方法：用来源于初诊未治急性髓细胞白血病患者的经由外周血细胞单采术得到的富集白细胞层与PBS液1：1稀释,采用淋巴细胞分离液进行淋巴细胞分离,3h后弃悬浮细胞,加入重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF),重组人白细胞介素-4(rhIL-4)和RPMI1640完全培养基体外培养至第7天,分为三组：第一组为对照组,不给予任何处理；第二组加入PA-MSHA;第三组加入TNF-a。继续培养24小时。每天取三组DC在倒置显微镜下观察细胞形态。第8天用流式细胞仪检测其细胞表型,并行混合淋巴细胞反应检测AML-DCs对淋巴细胞的增殖作用。将第8天的三组AML-DCs分别与MACS法分离得到的正常人外周血CD4+T细胞共培养7天,然后ELISA法分别测量三组上清液IL-10、TGF-β水平,流式细胞仪检测CD4、CD25的表达,RT-PCR测量Foxp3mRNA水平。
结果：PA-MSHA组和TNF-a组的DC呈现明显的树突状形态,这两组DC的CD1a、CD80、CD83、CD86和HLA-DR表达较对照组明显升高(P<0.05),且这两组DC诱导的T细胞增殖指数随刺激细胞数量的增加而增加,并明显高于对照组。此外,PA-MSHA组和TNF-a组DC诱导Treg产生后所测的IL-10、TGF-β水平,CD4、CD25的表达及Foxp3 mRNA水平均较对照组明显降低(P<0.05)。
结论：PA注射液可以促进AML-DC的进一步成熟,并抑制初始T细胞向Treg方向分化,从而增强对急性髓细胞白血病细胞的抗肿瘤活性。
Objective:CD4+CD25+ Treg cells which have the nature of immune suppression and immune incompetence are about 5%-10% of peripheral CD4+ T cells in healthy people, and participate in process of immune suppression. The numbers of Treg cells are higher in peripheral blood and tumor tissues of a variety of cancer patients and are responsible for inducing and maintaining peripheral tolerance. Treg cells have two subsets, natural Treg and adaptive Treg. Adaptive Treg cells can be differentiated from CD4+ T by the change of expression on the costimulatory molecules of APC or the change of the cytokines. Treg have been suggested to play an important immunopathological role in human cancer by lowering the intrinsic T-cell immunity towards tumor-associated antigen, resulting in tumor immune evasion. Some reports have indicated that Treg depletion can be useful in tumor biotherapy. Treg depletion would eliminate immune-suppression mediated by Treg leading to enhance T-cell activity. Another study has confirmed that immature DC can induced CD4+T cells to adaptive Treg. The current strategies of cancer immunotherapy focus on breaking immune tolerance and enhance the host T cell response. Pseudomonas aeruginosa-mannose sensitive hamemagglutination vaccine (PA-MSHA vaccine) is prepared from Pseudomonas aeruginosa which contains mannose-sensitive hemagglutinin pili. It has been widely used for anti-inflammatory treatment, even for anti-tumor treatment as immune modulator. According to reports, PA-MSHA vaccine could improve clinical efficiency as adjuvant therapy for a variety of cancer such as leukemia, malignant lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer and so on. This vaccine can increase the antigen presenting function of dendritic cells (DCs) or Th1/Th2 proportion, and regulate unbalance between cellular immune and humoral immune to achieve the purpose of effective treatment of tumors. However, the specific mechanism is still not very clear. Acute myeloid leukemia (AML) is a malignant hematopoietic disorder characterized by proliferation of immature myeloid precursors with considerable impairment of the immune system. Compared with healthy controls, AML patients had a higher proportion of CD4+CD25+ T cells in peripheral blood.Professor Sun reported PA-MSHA vaccine can improve immune function in patients with acute leukemia. The level of IL-2, the activity of NK cells, the ratio of CD4+/CD8+ in PA-MSHA vaccine treatment group are significantly higher than those in control group.But whether PA-MSHA vaccine can take effect on anti-tumor through reducing the number and function of the Treg cells or not, has not been reported. This study will detect the effect of the dendritic cells derived from acute myeloid leukemia and induced by PA-MSHA on CD4+CD25+ regulatory T cells, and is to investigate the immune regulatory fuctions of PA-MSHA to acute myeloid leukemia.
Methods:The buffy coat separated from peripheral blood cells of untreated newly diagnosed patients with acute myeloid leukemia by leukapheresis was diluted 1:1 with phosphate buffered saline(PBS). Lymphocytes were isolated using Ficoll lymphocyte separating medium, and the cells were incubated in RPMI1640 complete mediums for 3h. Then after the suspended cells were removed, recombinant human granulocyte-macrophage colony-stimulating factor(rhGM—CSF)and recombinant human interleukin-4(rhlL-4) were added into medium and the culture were incubated for 7 days. Then the cells were devided into three groups. As the control group, the first group was not given any treatment. The second group was added with PA-MSHA. Tumor necrosis factor-a(TNF-a) added in the third group, which continued to incubat 24 hours. The morphological feature was observed with inverted microscope everyday. The phenotypes of cells were detected by flow cytometry, and the proliferation effect of three groups of AML-DCs on Allogeneic T lymphocytes was investigated by a mixed lymphocyte reaction system which was analyzed by MTT assay. In the 8th day, the CD4+ T cells separated from peripheral blood cells of healthy adult by magnetic cell sorting (MACS) system were respectively incubated with the DCs from the three groups for 7 days.Then the supernatant concentration of IL-10, TGF-p was detected by ELISA kit. The cells of all the groups were harvested respectively and further tested for the expression of CD4+CD25+ T cells by flow cytometry. The RT-PCR method was used to examine Foxp3mRNA expression.
Results:The morphology of dendritic cells in PA-MSHA group and TNF-a group were irregular with slender synapses on their surface, and the expressions of CDla,CD80, CD83, CD86and HLA-DR on mature dendritic cells in PA-MSHA group and TNF-a group were significantly higher on day 8 than the control group, and proliferation indexes of the two groups was also higher than the control group as the more numbers of stimulating cells. In addition, the levels of IL-10 and TGF-β, the expressions of CD4 and CD25 on Treg, and the expression of Foxp3mRNA in PA-MSHA group and TNF-a group are all higher than the control group.
Conclusion:PA-MSHA can further promote the maturation of AML-DC, and thus inhibit the differentiation from CD4+ T cells to Treg. So PA-MSHA can enhance anti-tumor activity for acute myeloid leukemia patients.
方法：用来源于初诊未治急性髓细胞白血病患者的经由外周血细胞单采术得到的富集白细胞层与PBS液1：1稀释,采用淋巴细胞分离液进行淋巴细胞分离,3h后弃悬浮细胞,加入重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF),重组人白细胞介素-4(rhIL-4)和RPMI1640完全培养基体外培养至第7天,分为三组：第一组为对照组,不给予任何处理；第二组加入PA-MSHA;第三组加入TNF-a。继续培养24小时。每天取三组DC在倒置显微镜下观察细胞形态。第8天用流式细胞仪检测其细胞表型,并行混合淋巴细胞反应检测AML-DCs对淋巴细胞的增殖作用。将第8天的三组AML-DCs分别与MACS法分离得到的正常人外周血CD4+T细胞共培养7天,然后ELISA法分别测量三组上清液IL-10、TGF-β水平,流式细胞仪检测CD4、CD25的表达,RT-PCR测量Foxp3mRNA水平。
结果：PA-MSHA组和TNF-a组的DC呈现明显的树突状形态,这两组DC的CD1a、CD80、CD83、CD86和HLA-DR表达较对照组明显升高(P<0.05),且这两组DC诱导的T细胞增殖指数随刺激细胞数量的增加而增加,并明显高于对照组。此外,PA-MSHA组和TNF-a组DC诱导Treg产生后所测的IL-10、TGF-β水平,CD4、CD25的表达及Foxp3 mRNA水平均较对照组明显降低(P<0.05)。
结论：PA注射液可以促进AML-DC的进一步成熟,并抑制初始T细胞向Treg方向分化,从而增强对急性髓细胞白血病细胞的抗肿瘤活性。
Objective:CD4+CD25+ Treg cells which have the nature of immune suppression and immune incompetence are about 5%-10% of peripheral CD4+ T cells in healthy people, and participate in process of immune suppression. The numbers of Treg cells are higher in peripheral blood and tumor tissues of a variety of cancer patients and are responsible for inducing and maintaining peripheral tolerance. Treg cells have two subsets, natural Treg and adaptive Treg. Adaptive Treg cells can be differentiated from CD4+ T by the change of expression on the costimulatory molecules of APC or the change of the cytokines. Treg have been suggested to play an important immunopathological role in human cancer by lowering the intrinsic T-cell immunity towards tumor-associated antigen, resulting in tumor immune evasion. Some reports have indicated that Treg depletion can be useful in tumor biotherapy. Treg depletion would eliminate immune-suppression mediated by Treg leading to enhance T-cell activity. Another study has confirmed that immature DC can induced CD4+T cells to adaptive Treg. The current strategies of cancer immunotherapy focus on breaking immune tolerance and enhance the host T cell response. Pseudomonas aeruginosa-mannose sensitive hamemagglutination vaccine (PA-MSHA vaccine) is prepared from Pseudomonas aeruginosa which contains mannose-sensitive hemagglutinin pili. It has been widely used for anti-inflammatory treatment, even for anti-tumor treatment as immune modulator. According to reports, PA-MSHA vaccine could improve clinical efficiency as adjuvant therapy for a variety of cancer such as leukemia, malignant lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer and so on. This vaccine can increase the antigen presenting function of dendritic cells (DCs) or Th1/Th2 proportion, and regulate unbalance between cellular immune and humoral immune to achieve the purpose of effective treatment of tumors. However, the specific mechanism is still not very clear. Acute myeloid leukemia (AML) is a malignant hematopoietic disorder characterized by proliferation of immature myeloid precursors with considerable impairment of the immune system. Compared with healthy controls, AML patients had a higher proportion of CD4+CD25+ T cells in peripheral blood.Professor Sun reported PA-MSHA vaccine can improve immune function in patients with acute leukemia. The level of IL-2, the activity of NK cells, the ratio of CD4+/CD8+ in PA-MSHA vaccine treatment group are significantly higher than those in control group.But whether PA-MSHA vaccine can take effect on anti-tumor through reducing the number and function of the Treg cells or not, has not been reported. This study will detect the effect of the dendritic cells derived from acute myeloid leukemia and induced by PA-MSHA on CD4+CD25+ regulatory T cells, and is to investigate the immune regulatory fuctions of PA-MSHA to acute myeloid leukemia.
Methods:The buffy coat separated from peripheral blood cells of untreated newly diagnosed patients with acute myeloid leukemia by leukapheresis was diluted 1:1 with phosphate buffered saline(PBS). Lymphocytes were isolated using Ficoll lymphocyte separating medium, and the cells were incubated in RPMI1640 complete mediums for 3h. Then after the suspended cells were removed, recombinant human granulocyte-macrophage colony-stimulating factor(rhGM—CSF)and recombinant human interleukin-4(rhlL-4) were added into medium and the culture were incubated for 7 days. Then the cells were devided into three groups. As the control group, the first group was not given any treatment. The second group was added with PA-MSHA. Tumor necrosis factor-a(TNF-a) added in the third group, which continued to incubat 24 hours. The morphological feature was observed with inverted microscope everyday. The phenotypes of cells were detected by flow cytometry, and the proliferation effect of three groups of AML-DCs on Allogeneic T lymphocytes was investigated by a mixed lymphocyte reaction system which was analyzed by MTT assay. In the 8th day, the CD4+ T cells separated from peripheral blood cells of healthy adult by magnetic cell sorting (MACS) system were respectively incubated with the DCs from the three groups for 7 days.Then the supernatant concentration of IL-10, TGF-p was detected by ELISA kit. The cells of all the groups were harvested respectively and further tested for the expression of CD4+CD25+ T cells by flow cytometry. The RT-PCR method was used to examine Foxp3mRNA expression.
Results:The morphology of dendritic cells in PA-MSHA group and TNF-a group were irregular with slender synapses on their surface, and the expressions of CDla,CD80, CD83, CD86and HLA-DR on mature dendritic cells in PA-MSHA group and TNF-a group were significantly higher on day 8 than the control group, and proliferation indexes of the two groups was also higher than the control group as the more numbers of stimulating cells. In addition, the levels of IL-10 and TGF-β, the expressions of CD4 and CD25 on Treg, and the expression of Foxp3mRNA in PA-MSHA group and TNF-a group are all higher than the control group.
Conclusion:PA-MSHA can further promote the maturation of AML-DC, and thus inhibit the differentiation from CD4+ T cells to Treg. So PA-MSHA can enhance anti-tumor activity for acute myeloid leukemia patients.
引文
[1]Wiemik PH, Canellos GP, Dutcher J P, et al. Neoplastic diseases of the blood[M].3th Ed. Churchill Livingstone:a division of Harcourt Brace&Company.1996.1.
[2]National Cancer Institute. Acute myeloid leukemia. December 2, 2008.
[3]林壵,李承博,侯东泽.肿瘤生物治疗新进展[J].中国实用医药,2009,4(001)：225-226.
[4]Iannello A, Ahmad A. Role of antibody-dependent cell-mediated cytotoxicity in the efficacy of therapeutic anti-cancer monoclonal antibodies[J]. Cancer Metastasis Rev,2005,24(4):487-99.
[5]Banchereau J, Palucka A K. Dendritic cells as therapeutic vaccines against cancer[J]. Nat Rev Immunol,2005, (5):296-306.
[6]Berzofsky J A, terabe M, Oh S, et al, Progress on new vaccine strategies for the immunotherapy and prevention of cancer[J].J clin Invest,2004,113(11):1515-1525.
[7]彭枫,魏于全.肿瘤疫苗的临床研究进展[J].癌症,2006,25(8)：1059-1062.
[8]Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I morphology, quantitation, tissue distribution[J].Exp Med,1973,137(5):1142-1162.
[9]罗道升,戴宇平.树突状细胞疫苗在肾癌免疫治疗中的前景[J].国外医学泌尿系统分册,2004,24(1)：54-58.
[10]Inaba KM, Inaba N, Romani H, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor [J].Exp Med, 1992,176(6):1693-1702.
[11]Romani NS, Grunerm D, Brangm E, et al. Proliferating dendritic cell progenitors in human blood[J].Exp Med 1994,180(1):83-93.
[12]Figdor CG.. Molecular characterization of dendritic cells operating at the interface of innate or acquired immunity [J].Pathol Biol,2003,51(2):61-63.
[13]Troy AJ, Davidson PJ, Atkinson CH, el al.CDla dendritic cells predominate in transitional cell carcinoma of bladder and kidney but are minimally activated[J]. J Urol,1999,161(6):1962-1967.
[14]Mailliard RB, Dallal RM, Son YI, el al. Dendritic cells promote T-cell survival or death depending upon their maturation state and presentation of antigen[J]. Immunol Invest,2000,29(2),177-185.
[15]Old LJ. Cancer vaccines:an overview [J]. Cancer Immun,2008,8 (1):1.
[16]Osada T, Clav TM, Woo CY, et al. Dendritic cell-based immunotherapy [J]. Int Rev Immunol,2006,25(5-6):377-413.
[17]Yamanaka R, Honma J. Tumor lysate and IL-18 loaded dendritic cells elicits Th 1 response, tumor specific CD8+ cytotoxic T cells in patients with malignant glioma[J]. Neurooncel,2005,72(2):107-113.
[18]Hock BD, Roberts G, McKenzie JL, et al. Exposure to the electrofusion Process can increase the immunogenicity of human cells[J]. Cancer Immunol Immunother,2005,54(9):880-890.
[19]Maurizio Chiriva-Internati, Yong Liu, Paul L, et al. Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno-associated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells [J]. Eur J Immunol, 2002,32(1):30-38.
[20]Choudhury A, Liang JC, Thomas EK,et al. Dendritic cells derived in vitro from acutemyelogenous leukemia cells stimulate autologus, antileukemia T cell responses[J]. Blood,1999,93(3):780-786.
[21]KaterAP, van OersMH, Kipps TJ. Cellular immune therapy for chronic lymphocytic leukemia[J]. Blood,2007,110(8):2811-2818.
[22]童向民,金洁,李敏伟,等.白血病细胞和正常细胞生成树突状细胞的比较研究[J].实用肿瘤杂志,2005,20(1)：49-52.
[23]Marta S, Labeur, Berthold Roters, Birgit Pers, et al. Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage [J]. The Journal of Immunology, 1999,162(1):168-175.
[24]Kim KD, Lee HG, Kim JK, et al. Enhanced antigen presenting activity and tumur necrosis factor-alpha independent activation of dendritic cells following treatment witll mycobacterium bovis bacillus Calmette Guerin, Immunology,1999,97(4):626-633.
[25]Brunner C, Seiderer J, Schlamp A,et al. Enhanced dendritic cell maturation by TNF-a or CpG DNA drives T cell activation in vitro and therapeutic antitumor immune responses in vivo[J].J Immunol,2000,165(11):62-78.
[26]Romani N, Reider D, Heuer M, et al, Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability[J]. J Immunol Methods,1996,196 (2):137-151.
[27]Wang C, Al Omar HM, Radvanyi L, et al.Clonal heterogeneity of dendritic cells derived from patients with chronic myeloid leukemia and enhancement of their T cells stimulatory activity by IFN-α.Exp Hematol,1999,27(7):1176-1184.
[28]Brossart P, Grunebach F, Stuhler G, et al. Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte macrophage colony stimulating factor[J]. Blood,1998,92(11):4238-4247.
[29]Bedrosian I, Roros JO, Xu Setal. Granulocyte macrophage colony stimulating factor interleukin2, and interleukin 12 synergize with calciumionophore to enhance dendritic cell function[J]. J lmmunother,2000,23(3):311-320.
[30]Koski GK, Schwartz GN, Weng DE et al. Calciumionophore treated myeloid cells acquire many dendritic cell characteristics independent of prior differentiation state, transformation status, or sensitivity to biologicagents[J]. Blood 1999,94 (4):1359-1371.
[31]Waclavicek M, Berer A, Oehler L et al.Calciumionophore:a single reagent for the differentiation of primary human acute myelogenous leukaemia cells towards dendritic cells[J].Br J Haematol,2001, 114(2):466-473.
[32]陈国安,肖希斌,袁利亚,等.当归多糖促进慢性粒细胞自血病细胞性树突状细胞的诱导生成[J].中草药,2004,35(5)：135-137.
[33]张在云,吴金民,潘宏铭,等.参麦注射液对树突状细胞免疫活性的影响[J].科技通报,2003,19(3)：241-243.
[34]Mukherji B, Wilhelm SA, Guha A, et al. Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro eytotoxic immune response[J]. J Immunol,1986,136(5):1888-1892.
[35]Sakaguehi 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 sine mechanism of self-tolerance causes various autoimmune diseases[J]. J Immunol,1995,155 (3): 1151-1164.
[36]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.
[37]Fontenot JD, Dcoley JL, Farr AG, et al. Developmental regulation of Foxp3 expression during ontogeny[J]. J Exp Med,2005,202(7):901-906.
[38]Hartigan-O' Connor J, Poon C, Sinclair, et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorring of live cells[J].J Immunol Methods,2007,319(1-2):41-52.
[39]McHngh RS, Whitters MJ, Piceirillo CA, et al. 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.
[40]Read S, Malmstrom V, Powrie F.Cytotoxie 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.
[41]Shevach EM. CD4+CD25+ suppressor T cells:more questions than answers[J]. Nat Rev Immunol,2002,2(6):389-400.
[42]Ramsdell F, Ziegler SF. Transcription factors in autoimmunity [J]. Current Opinion in Immunology,2003,15(6):718-724.
[43]Sakaguchi S, Sakaguchi N, Shimizu J, et al. Immunologic tolerance maintained by CD4+CD25+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance[J]. Immunol Rev,2001,182:18-32.
[44]Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells[J].Nat rev Immunol 2003,3(3):253-257.
[45]Sakaguchi S. Naturally arising Foxp3- experessing CD4+CD25+ regulatory T-cells in immunological tolerance to self[J]. Nat Immunol,2005,699(4):345-352.
[46]H Groux, A O'Garra, Bigler M, et al.A CD4+T-cell subset inhibits antigenspecific T-cell responses and prevents colitis [J]. Nature, 1997,389 (6652):737-742.
[47]Jonuleit H, E Schmitt, K Steinbrink, et al. Dendritic Cells as a Tool to Induce Anergic and Regulatory T Cells [J]. Trends in immunology, 2001,22(7):394-400.
[48]Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies and suppressive function of CD4+CD25+ high regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine[J]. Blood,2005,106(6):2018-2025.
[49]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood,2004,103(5):1755-1762.
[50]Gray CP, Amsio P, Hersey P. Association of increased levels of heavy-chain ferritin with increased CD4+CD25+ regulatory T cell levels in patients with melanoma[J]. Clin Cancer Res,2003,9 (7):2551-2559.
[51]Liynage UK, Moere TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenviroment of patients with pancreas or breast adenocarcinoma[J]. Immunol,2002, 169(5):2756-2761.
[52]Sasada T, Kimura 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.
[53]Kono K, Kawaida H, Takahashi A, et al.CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers[J]. Cancer Immunol Immunother,2006,55 (9):1064-1071.
[54]Hiraoka N, Onozao K, Kosuge T, et al. Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions[J]. Clin Cancer Res,2006,12(18):5423-5434.
[55]Kobayashi N, Hiraoka N, Yamagami W, et al. FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis [J].Clin Cancer Res,2007,13(3):902-911.
[56]Woo EY,Chu CS.Goletz TJ, et al. Regulatory CD4(+)CD25 (+)T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61(12):4766-4772.
[57]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood,2004,103(5):1755-1762.
[58]Beyer M, Schultze JL. Regulatory T cels In cancer[J], Blood,2006, 108(3):804-811.
[59]Zou W. Immunosuppressive networks in the tumor environment and their therapeutic relevance [J]. Nat Rev Cancer,2005,5 (4):263-274.
[60]Shevach EM, Dipaolo RA, Andersson J, et al. The lifestyle of naturally occuring CD4+CD25+Foxp3 regulatory T cells[J]. Immunol Rev,2006,212:60-73.
[61]Siddiqui SA, Frigola X,Bonne-Annee S, et al. Tumor-infiltrating Foxp3- CD4+CD25+ T cells predict poor survival in renal cell carcinoma[J].Clin Cancer Res,2007,13(7):2075-2081.
[62]Ono M, Shimizu J, Miyachi Y, et al. Control of autoimmune myocarditis and multiorgan inflammation by glucocorticoid-induced TNF receptor family related protein (high), Foxp3-expressing CD25+ and CD25- regulatory T cells[J].J Immunol,2006,176(8):4748-4756.
[63]Mihalyo MA, Doody AD, McAleer JP, et al. In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization:implications for adoptive immunotherapy[J]. J Immunol,2004,172(9):5338-5345.
[64]Oderup C, Cederbom L, Makowska A, et al.Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+CD25+ regulatory T-cell-mediated suppression[J]. Immunology,2006,118 (2):240-249.
[65]Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells[J]. Am J Transplant,2006,6 (6):1297-1311.
[66]Watanabe N, Wang YH, Lee HK, et al.Hassall's corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus[J]. Nature,2005,436(7054):1181-1185.
[67]Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[J]. NatImmunol,2003, 4(4):330-336.
[68]Wang X, J Zheng, J Liu, et al. Increased Population of CD4(+)CD25(High), Regulatory T Cells with Their Higher Apoptotic and Proliferating Status in Peripheral Blood of Acute Myeloid Leukemia Patients[J], Eur J Haematol,2005,75(6):468-476.
[69]Nair S, Boczkowski D, FassnachtM, et al. Vaccination against the forkhead family transcription factor Foxp3 enhances tumor immunity[J]. Cancer Res,2007,67(1):371-380.
[70]Chen A, Liu S, Park D, et al. Depleting intratumoral CD4+CD25+ regulatory T cells via FasL protein transfer enhances the therapeutic efficacy of adoptive T cell transfer [J]. Cancer Res,2007,67 (3):1291-1298.
[71]Ghiringhelli F, Menard C, Puig PE, et al. Metronomic cyclophosphamideregimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer ImmunolImmunother,2007,56(5):641-648.
[72]Chu Y, Wang LX, Yang G, et al. Efficacy of GM-CSF-producing tumor vaccine after docetaxel chemotherapy in mice bearing established Lewis lung carcinoma[J]. J Immunother,2006,29(4):367-380.
[73]Beyer M, Kochan ek M, Darabi K, et al. Reduced frequencies and supperssive function of CO4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine [J], Blood,2005,106(6):2018-2025.
[74]Lopez M, Aguilera R, Perez C, et al.The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines [J].Immunobiology,2006,211(1-2):127-136.
[75]Peng G, Guo Z, Kiniwa Y, et al. Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function [J]. Science,2005,309 (5739):1380-1384.
[76]李志平,郝德治,杨玉琼,等.绿脓杆菌制剂辅助治疗恶性淋巴瘤临床研究[J].重庆医学,2000,29(2)：170-172.
[77]李志平,郝德治,张洪,等.绿脓杆菌制剂辅助治疗恶性淋巴瘤和肺癌的临床实验研究[J].华西医科大学学报,2000,31(3)：334-337.
[78]朱亚芳,顾洪兵,魏金芝,等.绿脓杆菌制剂治疗恶性体腔积液34例[J].南通大学学报(医学版),2007,27(2)：141-142.
[79]郏琴,孙依萍,蔡蓉,等.绿脓杆菌制剂辅助治疗肺癌临床研究[J].实用医学杂志,2007,23(16)：2487-2489.
[80]刘妮,王琪.绿脓杆菌MSHA菌毛株菌苗对恶性淋巴瘤的治疗及其机制初探[J].中国肿瘤临床,1997,24(7)：520-523.
[81]宋希双,姜涛,吴东军.绿脓杆菌制剂膀胱灌注预防膀胱癌术后复发的研究[J].中华泌尿外科杂志,2001,22(10)：597-598.
[82]赵宜良,杨巍.绿脓杆菌制剂胃癌腹水治疗体会[J].辽宁医学杂志,2004,18(5)：248-250.
[83]L I Z P, HAO D Z, REN L, et al.A clinical study of PA-MSHA vaccine in adjuvant treatment of lung cancer[J]. SichuanMed J(四川医学),2000,21(3)：193-195.
[84]SUN W P, FU H W, LIU N, et al. Clinical investigation of PA-MSHA strain vaccine on the improvement of immune functions of cancer patients [J].Chin J Microbiol Immunol(中华微生物学和免疫学杂志),2000,20(4)：373-376.
[85]谢普耀,富丽,毛裕芳.“绿脓杆菌MSHA菌毛菌苗”对白血病患者抗感染效应的初步观察[J].大连医学院学报,1994：16(3)：198-200.
[86]张明策,牟希亚,郭雁群,PA-MSHA菌毛株菌苗的抗菌转移瘤和免疫调节机制的研究[J].上海免疫学杂志,1994：11(5)：268-270.
[87]Li Z, Hao D, Zhang H, et al. A clinical study on PA-MSHA vaccine used for adjuvant therapy of lymphoma and lung cancer[J].Hua Xi Yi Ke Da Xue Xue Bao.2000,31:334-337.
[88]孙文平,付红文,刘妮,等,PA-MSHA菌毛株疫苗对三种癌症患者免疫疗效的临床观察[J].中华微生物学和免疫学杂志,2000,20(4)：373-376.
[89]Wolf AM,D Wolf, M Steurer, et al. Increase of Regulatory T Cells in the Peripheral Blood of Cancer Patients[J], Clinical cancer research,2003,9(2):606-612.
[90]Jonuleit H, E Schmitt, K Steinbrink, et al. Dendritic Cells as a Tool to Induce Anergic and Regulatory T Cells[J]. Trends in immunology,2001,2(7):394-400.
[91]Wang X, J Zheng, J Liu,et al. Increased Population of CD4(+) CD25(High), Regulatory T Cells with Their Higher Apoptotic and Proliferating Status in Peripheral Blood of Acute Myeloid Leukemia Patients[J].Eur J Haematol,2005,75:468-476.
[92]张明策,牟希亚.PA-MSHA菌苗的抗肿瘤活性和免疫调节作用[J].上海免疫学杂志,1994,14(005)：268-271.
[93]张之南,主编,血液病诊断及疗效标准[M].天津：天津科学技术出版社,1998.219.
[94]罗征秀,刘恩梅,邓兵,等.Foxp3表达与CD4^+CD25^+调节性T细胞在儿童哮喘发病中的作用[J],中华儿科杂志,2006,44(004)：267-271.
[95]Cao Z, L Shi,Y Li, et al. Pseudomonas Aeruginosa:Mannose Sensitive Hemagglutinin Inhibits the Growth of Human Hepatocarcinoma Cells Via Mannose-Mediated Apoptosis[J]. Digestive Diseases and Sciences,2009,54(10):2118-2127.
[96]Remmel E, Terracciano L, Noppen C, et al. Modulation of dendritic cell phenoltype and mobility by tumor cells in vitro[J].Hum Immunol,2001,62(1):39-49.
[1]Mukherji B, Wilhelm SA, Guha A, et al. Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro eytotoxic immune response [J]. J Immunol,1986,136(5):1888-1892.
[2]Sakaguehi 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 sine mechanism of self-tolerance causes various autoimmune diseases [J]. J Immunol,1995,155(3): 1151-1164.
[3]Fehervari Z, Sakaguchil S. Development and function of CD25+CD4+ regulatory T ceHs[J].Curt Opin Immunol,2004,16(2):203-208.
[4]Seo N, Hayakawa S, Takigawa M, et al. Interleukin 10 expressed at early tumour sites induces subsequent generation of CD4+ T regulatory cells and systanic collapse of antitumour immunity [J]. Immunology,2001,103(4):449-457.
[5]Schramm C,Huber S, Protschka M, et al. TGF-β regulates the CD4+ CD25+T cell pool and the expression of Foxp3 in vivo[J]. Int Immunol, 2004,16 (9):1241-1249.
[6]Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells [J]. Nat rev Immunol 2003,3(3):253-257.
[7]Sakaguchi S. Naturally arising Foxp3-experessing CD4+CD25+ regulatory T-cells in immunological tolerance to self[J]. Nat Immunol,2005,699 (4):345-352.
[8]Groux H,O, Garra, Bigler M, et al. A CD4+T-cell subset inhibits antigen-specific T-cell responses and prevents colitis[J]. Nature,1997,389 (6652):737-742.
[9]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.
[10]Fontenot JD, Dcoley JL, Farr AG, et al. Developmental regulation of Foxp3 expression during ontogeny [J]. J Exp Med,2005,202(7):901-906.
[11]Hartigan-O'Connor J, Poon C, Sinclair, et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and 8orring of live cells[J].J Immunol Methods,2007,319(1-2):41-52.
[12]McHngh RS, Whitters MJ, Piceirillo CA, et al. 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.
[13]Read S, Malmstrom V, Powrie F. Cytotoxie 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.
[14]Shevach EM. CD4+CD25+ suppressor T cells:more questions than answers[J]. Nat Rev Immunol,2002,2(6):389-400.
[15]Ramsdell F, Ziegler SF. Transcription factors in autoimmunity[J]. Current Opinion in Immunology,2003,15(6):718-724.
[16]Sakaguchi S, Sakaguchi N, Shimizu J, et al. Immunologic tolerance maintained by CD4+ CD25+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance[J]. Immunol Rev,2001,182:18-32.
[17]Oderup C, Cederbom L, Makowska A, et al. Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+ CD25+ regulatory T-cell-mediated suppression[J]. Immunology,2006,118(2):240-249.
[18]Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells [J]. Am J Transplant, 2006,6(6):1297-1311.
[19]Watanabe N, Wang YH, Lee HK, et al. Hassall's corpuscles instruct dendritic cells to induce CD4+ CD25+ regulatory T cells in human thymus[J]. Nature,2005,436(7054):1181-1185
[20]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.
[21]Liynage UK, Moere TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenviroment of patients with pancreas or breast adenocarcinoma [J]. Immunol,2002, 169(5):2756-2761
[22]Sasada T, Kimura 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.
[23]Kono K, Kawaida H, Takahashi A, et al. CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers[J]. Cancer Immunol Immunother,2006,55 (9):1064-1071.
[24]Hiraoka N, Onozao K, Kosuge T, et al. Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions [J]. Clin Cancer Res,2006,12(18):5423-5434.
[25]Kobayashi N, Hiraoka N, Yamagami W, et al. FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis [J]. Clin Cancer Res,2007,13(3):902-911.
[26]Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4(+)CD25 (+)T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61 (12):4766-4772.
[27]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J].Blood2004,103(5):1755-1762.
[28]Gray CP, Amsio P, Hersey P. Association of increased levels of heavy-chain ferritin with increased CD4+CD25+ regulatory T cell levels in patients with melanoma[J]. Clin Cancer Res,2003,9(7): 2551-2559.
[29]Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies and suppressive function of CD4+CD25high regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine[J]. Blood,2005,106(6):2018-2025.
[30]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood,2004,103(5):1755-1762
[31]Beyer M, Schultze JL. Regulatory T cels In cancer[J]. Blood,2006, 108 (3):804-811.
[32]Zou W.Immunosuppressive networks in the tumor environment and their therapeutic relevance [J]. Nat Rev Cancer,2005,5 (4):263-274.
[33]Shevach EM, Dipaolo RA, Andersson J, et al. The lifestyle of naturally occurring CD4+CD25+ Foxp3 regulatory T cells[J], Immunol Rev,2006,212:60-73.
[34]Siddiqui SA, Frigola X, Bonne-Annee S, et al. Tumor-infiltrating Foxp3-CD4+CD25+ T cells predict poor survival in renal cell carcinoma[J]. Clin Cancer Res,2007,13(7):2075-2081.
[35]Ono M, Shimizu J, Miyachi Y, et al. Control of autoimmune myocarditis and multiorgan inflammation by glucocorticoid-induced TNF receptor family-related protein (high), Foxp3- expressing CD25+ and CD25-regulatory T cells[J].J Immunol,2006,176(8):4748-4756.
[36]Mihalyo MA, Doody AD, McAleer JP, et al. In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization:implications for adoptive immunotherapy[J]. J Immunol,2004,172(9):5338-5345.
[2]National Cancer Institute. Acute myeloid leukemia. December 2, 2008.
[3]林壵,李承博,侯东泽.肿瘤生物治疗新进展[J].中国实用医药,2009,4(001)：225-226.
[4]Iannello A, Ahmad A. Role of antibody-dependent cell-mediated cytotoxicity in the efficacy of therapeutic anti-cancer monoclonal antibodies[J]. Cancer Metastasis Rev,2005,24(4):487-99.
[5]Banchereau J, Palucka A K. Dendritic cells as therapeutic vaccines against cancer[J]. Nat Rev Immunol,2005, (5):296-306.
[6]Berzofsky J A, terabe M, Oh S, et al, Progress on new vaccine strategies for the immunotherapy and prevention of cancer[J].J clin Invest,2004,113(11):1515-1525.
[7]彭枫,魏于全.肿瘤疫苗的临床研究进展[J].癌症,2006,25(8)：1059-1062.
[8]Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I morphology, quantitation, tissue distribution[J].Exp Med,1973,137(5):1142-1162.
[9]罗道升,戴宇平.树突状细胞疫苗在肾癌免疫治疗中的前景[J].国外医学泌尿系统分册,2004,24(1)：54-58.
[10]Inaba KM, Inaba N, Romani H, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor [J].Exp Med, 1992,176(6):1693-1702.
[11]Romani NS, Grunerm D, Brangm E, et al. Proliferating dendritic cell progenitors in human blood[J].Exp Med 1994,180(1):83-93.
[12]Figdor CG.. Molecular characterization of dendritic cells operating at the interface of innate or acquired immunity [J].Pathol Biol,2003,51(2):61-63.
[13]Troy AJ, Davidson PJ, Atkinson CH, el al.CDla dendritic cells predominate in transitional cell carcinoma of bladder and kidney but are minimally activated[J]. J Urol,1999,161(6):1962-1967.
[14]Mailliard RB, Dallal RM, Son YI, el al. Dendritic cells promote T-cell survival or death depending upon their maturation state and presentation of antigen[J]. Immunol Invest,2000,29(2),177-185.
[15]Old LJ. Cancer vaccines:an overview [J]. Cancer Immun,2008,8 (1):1.
[16]Osada T, Clav TM, Woo CY, et al. Dendritic cell-based immunotherapy [J]. Int Rev Immunol,2006,25(5-6):377-413.
[17]Yamanaka R, Honma J. Tumor lysate and IL-18 loaded dendritic cells elicits Th 1 response, tumor specific CD8+ cytotoxic T cells in patients with malignant glioma[J]. Neurooncel,2005,72(2):107-113.
[18]Hock BD, Roberts G, McKenzie JL, et al. Exposure to the electrofusion Process can increase the immunogenicity of human cells[J]. Cancer Immunol Immunother,2005,54(9):880-890.
[19]Maurizio Chiriva-Internati, Yong Liu, Paul L, et al. Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno-associated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells [J]. Eur J Immunol, 2002,32(1):30-38.
[20]Choudhury A, Liang JC, Thomas EK,et al. Dendritic cells derived in vitro from acutemyelogenous leukemia cells stimulate autologus, antileukemia T cell responses[J]. Blood,1999,93(3):780-786.
[21]KaterAP, van OersMH, Kipps TJ. Cellular immune therapy for chronic lymphocytic leukemia[J]. Blood,2007,110(8):2811-2818.
[22]童向民,金洁,李敏伟,等.白血病细胞和正常细胞生成树突状细胞的比较研究[J].实用肿瘤杂志,2005,20(1)：49-52.
[23]Marta S, Labeur, Berthold Roters, Birgit Pers, et al. Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage [J]. The Journal of Immunology, 1999,162(1):168-175.
[24]Kim KD, Lee HG, Kim JK, et al. Enhanced antigen presenting activity and tumur necrosis factor-alpha independent activation of dendritic cells following treatment witll mycobacterium bovis bacillus Calmette Guerin, Immunology,1999,97(4):626-633.
[25]Brunner C, Seiderer J, Schlamp A,et al. Enhanced dendritic cell maturation by TNF-a or CpG DNA drives T cell activation in vitro and therapeutic antitumor immune responses in vivo[J].J Immunol,2000,165(11):62-78.
[26]Romani N, Reider D, Heuer M, et al, Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability[J]. J Immunol Methods,1996,196 (2):137-151.
[27]Wang C, Al Omar HM, Radvanyi L, et al.Clonal heterogeneity of dendritic cells derived from patients with chronic myeloid leukemia and enhancement of their T cells stimulatory activity by IFN-α.Exp Hematol,1999,27(7):1176-1184.
[28]Brossart P, Grunebach F, Stuhler G, et al. Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte macrophage colony stimulating factor[J]. Blood,1998,92(11):4238-4247.
[29]Bedrosian I, Roros JO, Xu Setal. Granulocyte macrophage colony stimulating factor interleukin2, and interleukin 12 synergize with calciumionophore to enhance dendritic cell function[J]. J lmmunother,2000,23(3):311-320.
[30]Koski GK, Schwartz GN, Weng DE et al. Calciumionophore treated myeloid cells acquire many dendritic cell characteristics independent of prior differentiation state, transformation status, or sensitivity to biologicagents[J]. Blood 1999,94 (4):1359-1371.
[31]Waclavicek M, Berer A, Oehler L et al.Calciumionophore:a single reagent for the differentiation of primary human acute myelogenous leukaemia cells towards dendritic cells[J].Br J Haematol,2001, 114(2):466-473.
[32]陈国安,肖希斌,袁利亚,等.当归多糖促进慢性粒细胞自血病细胞性树突状细胞的诱导生成[J].中草药,2004,35(5)：135-137.
[33]张在云,吴金民,潘宏铭,等.参麦注射液对树突状细胞免疫活性的影响[J].科技通报,2003,19(3)：241-243.
[34]Mukherji B, Wilhelm SA, Guha A, et al. Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro eytotoxic immune response[J]. J Immunol,1986,136(5):1888-1892.
[35]Sakaguehi 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 sine mechanism of self-tolerance causes various autoimmune diseases[J]. J Immunol,1995,155 (3): 1151-1164.
[36]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.
[37]Fontenot JD, Dcoley JL, Farr AG, et al. Developmental regulation of Foxp3 expression during ontogeny[J]. J Exp Med,2005,202(7):901-906.
[38]Hartigan-O' Connor J, Poon C, Sinclair, et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorring of live cells[J].J Immunol Methods,2007,319(1-2):41-52.
[39]McHngh RS, Whitters MJ, Piceirillo CA, et al. 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.
[40]Read S, Malmstrom V, Powrie F.Cytotoxie 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.
[41]Shevach EM. CD4+CD25+ suppressor T cells:more questions than answers[J]. Nat Rev Immunol,2002,2(6):389-400.
[42]Ramsdell F, Ziegler SF. Transcription factors in autoimmunity [J]. Current Opinion in Immunology,2003,15(6):718-724.
[43]Sakaguchi S, Sakaguchi N, Shimizu J, et al. Immunologic tolerance maintained by CD4+CD25+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance[J]. Immunol Rev,2001,182:18-32.
[44]Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells[J].Nat rev Immunol 2003,3(3):253-257.
[45]Sakaguchi S. Naturally arising Foxp3- experessing CD4+CD25+ regulatory T-cells in immunological tolerance to self[J]. Nat Immunol,2005,699(4):345-352.
[46]H Groux, A O'Garra, Bigler M, et al.A CD4+T-cell subset inhibits antigenspecific T-cell responses and prevents colitis [J]. Nature, 1997,389 (6652):737-742.
[47]Jonuleit H, E Schmitt, K Steinbrink, et al. Dendritic Cells as a Tool to Induce Anergic and Regulatory T Cells [J]. Trends in immunology, 2001,22(7):394-400.
[48]Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies and suppressive function of CD4+CD25+ high regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine[J]. Blood,2005,106(6):2018-2025.
[49]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood,2004,103(5):1755-1762.
[50]Gray CP, Amsio P, Hersey P. Association of increased levels of heavy-chain ferritin with increased CD4+CD25+ regulatory T cell levels in patients with melanoma[J]. Clin Cancer Res,2003,9 (7):2551-2559.
[51]Liynage UK, Moere TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenviroment of patients with pancreas or breast adenocarcinoma[J]. Immunol,2002, 169(5):2756-2761.
[52]Sasada T, Kimura 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.
[53]Kono K, Kawaida H, Takahashi A, et al.CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers[J]. Cancer Immunol Immunother,2006,55 (9):1064-1071.
[54]Hiraoka N, Onozao K, Kosuge T, et al. Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions[J]. Clin Cancer Res,2006,12(18):5423-5434.
[55]Kobayashi N, Hiraoka N, Yamagami W, et al. FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis [J].Clin Cancer Res,2007,13(3):902-911.
[56]Woo EY,Chu CS.Goletz TJ, et al. Regulatory CD4(+)CD25 (+)T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61(12):4766-4772.
[57]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood,2004,103(5):1755-1762.
[58]Beyer M, Schultze JL. Regulatory T cels In cancer[J], Blood,2006, 108(3):804-811.
[59]Zou W. Immunosuppressive networks in the tumor environment and their therapeutic relevance [J]. Nat Rev Cancer,2005,5 (4):263-274.
[60]Shevach EM, Dipaolo RA, Andersson J, et al. The lifestyle of naturally occuring CD4+CD25+Foxp3 regulatory T cells[J]. Immunol Rev,2006,212:60-73.
[61]Siddiqui SA, Frigola X,Bonne-Annee S, et al. Tumor-infiltrating Foxp3- CD4+CD25+ T cells predict poor survival in renal cell carcinoma[J].Clin Cancer Res,2007,13(7):2075-2081.
[62]Ono M, Shimizu J, Miyachi Y, et al. Control of autoimmune myocarditis and multiorgan inflammation by glucocorticoid-induced TNF receptor family related protein (high), Foxp3-expressing CD25+ and CD25- regulatory T cells[J].J Immunol,2006,176(8):4748-4756.
[63]Mihalyo MA, Doody AD, McAleer JP, et al. In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization:implications for adoptive immunotherapy[J]. J Immunol,2004,172(9):5338-5345.
[64]Oderup C, Cederbom L, Makowska A, et al.Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+CD25+ regulatory T-cell-mediated suppression[J]. Immunology,2006,118 (2):240-249.
[65]Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells[J]. Am J Transplant,2006,6 (6):1297-1311.
[66]Watanabe N, Wang YH, Lee HK, et al.Hassall's corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus[J]. Nature,2005,436(7054):1181-1185.
[67]Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[J]. NatImmunol,2003, 4(4):330-336.
[68]Wang X, J Zheng, J Liu, et al. Increased Population of CD4(+)CD25(High), Regulatory T Cells with Their Higher Apoptotic and Proliferating Status in Peripheral Blood of Acute Myeloid Leukemia Patients[J], Eur J Haematol,2005,75(6):468-476.
[69]Nair S, Boczkowski D, FassnachtM, et al. Vaccination against the forkhead family transcription factor Foxp3 enhances tumor immunity[J]. Cancer Res,2007,67(1):371-380.
[70]Chen A, Liu S, Park D, et al. Depleting intratumoral CD4+CD25+ regulatory T cells via FasL protein transfer enhances the therapeutic efficacy of adoptive T cell transfer [J]. Cancer Res,2007,67 (3):1291-1298.
[71]Ghiringhelli F, Menard C, Puig PE, et al. Metronomic cyclophosphamideregimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer ImmunolImmunother,2007,56(5):641-648.
[72]Chu Y, Wang LX, Yang G, et al. Efficacy of GM-CSF-producing tumor vaccine after docetaxel chemotherapy in mice bearing established Lewis lung carcinoma[J]. J Immunother,2006,29(4):367-380.
[73]Beyer M, Kochan ek M, Darabi K, et al. Reduced frequencies and supperssive function of CO4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine [J], Blood,2005,106(6):2018-2025.
[74]Lopez M, Aguilera R, Perez C, et al.The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines [J].Immunobiology,2006,211(1-2):127-136.
[75]Peng G, Guo Z, Kiniwa Y, et al. Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function [J]. Science,2005,309 (5739):1380-1384.
[76]李志平,郝德治,杨玉琼,等.绿脓杆菌制剂辅助治疗恶性淋巴瘤临床研究[J].重庆医学,2000,29(2)：170-172.
[77]李志平,郝德治,张洪,等.绿脓杆菌制剂辅助治疗恶性淋巴瘤和肺癌的临床实验研究[J].华西医科大学学报,2000,31(3)：334-337.
[78]朱亚芳,顾洪兵,魏金芝,等.绿脓杆菌制剂治疗恶性体腔积液34例[J].南通大学学报(医学版),2007,27(2)：141-142.
[79]郏琴,孙依萍,蔡蓉,等.绿脓杆菌制剂辅助治疗肺癌临床研究[J].实用医学杂志,2007,23(16)：2487-2489.
[80]刘妮,王琪.绿脓杆菌MSHA菌毛株菌苗对恶性淋巴瘤的治疗及其机制初探[J].中国肿瘤临床,1997,24(7)：520-523.
[81]宋希双,姜涛,吴东军.绿脓杆菌制剂膀胱灌注预防膀胱癌术后复发的研究[J].中华泌尿外科杂志,2001,22(10)：597-598.
[82]赵宜良,杨巍.绿脓杆菌制剂胃癌腹水治疗体会[J].辽宁医学杂志,2004,18(5)：248-250.
[83]L I Z P, HAO D Z, REN L, et al.A clinical study of PA-MSHA vaccine in adjuvant treatment of lung cancer[J]. SichuanMed J(四川医学),2000,21(3)：193-195.
[84]SUN W P, FU H W, LIU N, et al. Clinical investigation of PA-MSHA strain vaccine on the improvement of immune functions of cancer patients [J].Chin J Microbiol Immunol(中华微生物学和免疫学杂志),2000,20(4)：373-376.
[85]谢普耀,富丽,毛裕芳.“绿脓杆菌MSHA菌毛菌苗”对白血病患者抗感染效应的初步观察[J].大连医学院学报,1994：16(3)：198-200.
[86]张明策,牟希亚,郭雁群,PA-MSHA菌毛株菌苗的抗菌转移瘤和免疫调节机制的研究[J].上海免疫学杂志,1994：11(5)：268-270.
[87]Li Z, Hao D, Zhang H, et al. A clinical study on PA-MSHA vaccine used for adjuvant therapy of lymphoma and lung cancer[J].Hua Xi Yi Ke Da Xue Xue Bao.2000,31:334-337.
[88]孙文平,付红文,刘妮,等,PA-MSHA菌毛株疫苗对三种癌症患者免疫疗效的临床观察[J].中华微生物学和免疫学杂志,2000,20(4)：373-376.
[89]Wolf AM,D Wolf, M Steurer, et al. Increase of Regulatory T Cells in the Peripheral Blood of Cancer Patients[J], Clinical cancer research,2003,9(2):606-612.
[90]Jonuleit H, E Schmitt, K Steinbrink, et al. Dendritic Cells as a Tool to Induce Anergic and Regulatory T Cells[J]. Trends in immunology,2001,2(7):394-400.
[91]Wang X, J Zheng, J Liu,et al. Increased Population of CD4(+) CD25(High), Regulatory T Cells with Their Higher Apoptotic and Proliferating Status in Peripheral Blood of Acute Myeloid Leukemia Patients[J].Eur J Haematol,2005,75:468-476.
[92]张明策,牟希亚.PA-MSHA菌苗的抗肿瘤活性和免疫调节作用[J].上海免疫学杂志,1994,14(005)：268-271.
[93]张之南,主编,血液病诊断及疗效标准[M].天津：天津科学技术出版社,1998.219.
[94]罗征秀,刘恩梅,邓兵,等.Foxp3表达与CD4^+CD25^+调节性T细胞在儿童哮喘发病中的作用[J],中华儿科杂志,2006,44(004)：267-271.
[95]Cao Z, L Shi,Y Li, et al. Pseudomonas Aeruginosa:Mannose Sensitive Hemagglutinin Inhibits the Growth of Human Hepatocarcinoma Cells Via Mannose-Mediated Apoptosis[J]. Digestive Diseases and Sciences,2009,54(10):2118-2127.
[96]Remmel E, Terracciano L, Noppen C, et al. Modulation of dendritic cell phenoltype and mobility by tumor cells in vitro[J].Hum Immunol,2001,62(1):39-49.
[1]Mukherji B, Wilhelm SA, Guha A, et al. Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro eytotoxic immune response [J]. J Immunol,1986,136(5):1888-1892.
[2]Sakaguehi 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 sine mechanism of self-tolerance causes various autoimmune diseases [J]. J Immunol,1995,155(3): 1151-1164.
[3]Fehervari Z, Sakaguchil S. Development and function of CD25+CD4+ regulatory T ceHs[J].Curt Opin Immunol,2004,16(2):203-208.
[4]Seo N, Hayakawa S, Takigawa M, et al. Interleukin 10 expressed at early tumour sites induces subsequent generation of CD4+ T regulatory cells and systanic collapse of antitumour immunity [J]. Immunology,2001,103(4):449-457.
[5]Schramm C,Huber S, Protschka M, et al. TGF-β regulates the CD4+ CD25+T cell pool and the expression of Foxp3 in vivo[J]. Int Immunol, 2004,16 (9):1241-1249.
[6]Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells [J]. Nat rev Immunol 2003,3(3):253-257.
[7]Sakaguchi S. Naturally arising Foxp3-experessing CD4+CD25+ regulatory T-cells in immunological tolerance to self[J]. Nat Immunol,2005,699 (4):345-352.
[8]Groux H,O, Garra, Bigler M, et al. A CD4+T-cell subset inhibits antigen-specific T-cell responses and prevents colitis[J]. Nature,1997,389 (6652):737-742.
[9]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.
[10]Fontenot JD, Dcoley JL, Farr AG, et al. Developmental regulation of Foxp3 expression during ontogeny [J]. J Exp Med,2005,202(7):901-906.
[11]Hartigan-O'Connor J, Poon C, Sinclair, et al. Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and 8orring of live cells[J].J Immunol Methods,2007,319(1-2):41-52.
[12]McHngh RS, Whitters MJ, Piceirillo CA, et al. 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.
[13]Read S, Malmstrom V, Powrie F. Cytotoxie 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.
[14]Shevach EM. CD4+CD25+ suppressor T cells:more questions than answers[J]. Nat Rev Immunol,2002,2(6):389-400.
[15]Ramsdell F, Ziegler SF. Transcription factors in autoimmunity[J]. Current Opinion in Immunology,2003,15(6):718-724.
[16]Sakaguchi S, Sakaguchi N, Shimizu J, et al. Immunologic tolerance maintained by CD4+ CD25+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance[J]. Immunol Rev,2001,182:18-32.
[17]Oderup C, Cederbom L, Makowska A, et al. Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+ CD25+ regulatory T-cell-mediated suppression[J]. Immunology,2006,118(2):240-249.
[18]Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells [J]. Am J Transplant, 2006,6(6):1297-1311.
[19]Watanabe N, Wang YH, Lee HK, et al. Hassall's corpuscles instruct dendritic cells to induce CD4+ CD25+ regulatory T cells in human thymus[J]. Nature,2005,436(7054):1181-1185
[20]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.
[21]Liynage UK, Moere TT, Joo HG, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenviroment of patients with pancreas or breast adenocarcinoma [J]. Immunol,2002, 169(5):2756-2761
[22]Sasada T, Kimura 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.
[23]Kono K, Kawaida H, Takahashi A, et al. CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers[J]. Cancer Immunol Immunother,2006,55 (9):1064-1071.
[24]Hiraoka N, Onozao K, Kosuge T, et al. Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions [J]. Clin Cancer Res,2006,12(18):5423-5434.
[25]Kobayashi N, Hiraoka N, Yamagami W, et al. FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis [J]. Clin Cancer Res,2007,13(3):902-911.
[26]Woo EY, Chu CS, Goletz TJ, et al. Regulatory CD4(+)CD25 (+)T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer[J]. Cancer Res,2001,61 (12):4766-4772.
[27]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J].Blood2004,103(5):1755-1762.
[28]Gray CP, Amsio P, Hersey P. Association of increased levels of heavy-chain ferritin with increased CD4+CD25+ regulatory T cell levels in patients with melanoma[J]. Clin Cancer Res,2003,9(7): 2551-2559.
[29]Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies and suppressive function of CD4+CD25high regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine[J]. Blood,2005,106(6):2018-2025.
[30]Marshall NA, Christie LE, Munro LR, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma[J]. Blood,2004,103(5):1755-1762
[31]Beyer M, Schultze JL. Regulatory T cels In cancer[J]. Blood,2006, 108 (3):804-811.
[32]Zou W.Immunosuppressive networks in the tumor environment and their therapeutic relevance [J]. Nat Rev Cancer,2005,5 (4):263-274.
[33]Shevach EM, Dipaolo RA, Andersson J, et al. The lifestyle of naturally occurring CD4+CD25+ Foxp3 regulatory T cells[J], Immunol Rev,2006,212:60-73.
[34]Siddiqui SA, Frigola X, Bonne-Annee S, et al. Tumor-infiltrating Foxp3-CD4+CD25+ T cells predict poor survival in renal cell carcinoma[J]. Clin Cancer Res,2007,13(7):2075-2081.
[35]Ono M, Shimizu J, Miyachi Y, et al. Control of autoimmune myocarditis and multiorgan inflammation by glucocorticoid-induced TNF receptor family-related protein (high), Foxp3- expressing CD25+ and CD25-regulatory T cells[J].J Immunol,2006,176(8):4748-4756.
[36]Mihalyo MA, Doody AD, McAleer JP, et al. In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization:implications for adoptive immunotherapy[J]. J Immunol,2004,172(9):5338-5345.