调节性T细胞对NK细胞体外杀伤乳腺癌细胞的影响
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摘要
【目的】分析T-reg细胞在乳腺癌病人中的表达情况及其与NK细胞、T细胞亚群之间的关系;探讨体外富集的T-reg对NK细胞杀伤乳腺癌细胞的影响;探讨NKG2D-MICA在NK细胞杀伤乳腺癌中的作用;观察T-reg对NKG2D-MICA作用的影响。
【方法】1、流式细胞术检测乳腺癌患者外周血T-reg的比例;探讨乳腺癌患者TGF-β1的变化。2、应用LDH法检测NK细胞对四种乳腺癌细胞株杀伤率活性。3、ELISA检测培养上清液中IFN-γ和TGF-β1的水平4、荧光定量PCR检测四种乳腺癌细胞株MICA-mRNA的表达。5、应用流式细胞术检测四种乳腺癌细胞株表面及胞内MICA的表达;6、应用免疫组化技术和免疫荧光技术检测四种乳腺癌细胞株MICA的分布情况。7、应用FCM检测NK细胞表面的NKG2D的变化。
【结果】1、乳腺癌病人T-reg的表达:乳腺癌病人和健康志愿者外周血中T-reg占CD4+T细胞总数的百分比分别是(7.5±3.0)%和(5.1±1.5)%,前者高于后者(P<0.01)。2、当NK细胞与乳腺癌细胞效靶比为10:1时,对MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231的杀伤活性分别为(40.7±3.0)%、(15.5±3.1)%、(29.7±1.8)%以及59.1±2.3)%;加入T-reg的数量为1/5的NK细胞时,杀伤活性分别降至(1.9±0.2)%、(5.7±0.4)%、(3.6±2.8)%以及(3.9±0.2)%;加入T-reg的数量为1/10的NK细胞时,杀伤活性分别降至(7.6±1.6)%、(7.3±1.0)%、(6.6±2.5)%以及(19.7±1.1)%。3、ELISA检测NK细胞分泌的IFN-γ结果显示:在无T-reg存在时,NK细胞与MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231共培养时IFN-γ浓度分别为(221.5±2.6)pg/ml、(264.4±5.5)pg/ml、(754.6±19.0)pg/ml以及(423.0±14.6)pg/ml;加入T-reg的数量为1/5的NK细胞时,IFN-γ浓度分别为(70.7±8.5)pg/ml、(14.6±0.9)pg/ml、(128.3±4.0)pg/ml以及(6.0±1.0)pg/ml;加入T-reg的数量为1/10的NK细胞时,IFN-γ浓度分别为(216.6±4.8)pg/ml、(251.7±2.2)pg/ml、(535.0±8.5)pg/ml以及(52.57±3.4)pg/ml。4、ELISA检测培养上清液中T-reg分泌的TGF-β1结果显示:在无T-reg存在时,NK细胞与MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231共培养时TGF-β1浓度分别为(13.4±0.2)pg/ml、(11.3±0.8)pg/ml、(62.9±1.3)pg/ml以及(6.2±0.5)pg/ml;加入T-reg的数量为1/5的NK细胞时,TGF-β1浓度分别为(27.2±0.3)ng/ml、(12.2±1.0)ng/ml、(77.6±1.0)ng/ml以及(9.1±0.1)ng/ml;加入T-reg的数量为1/10的NK细胞时,TGF-β1浓度分别为(46.8±1.6)ng/ml、(15.8±0.9)ng/ml、(87.8±2.3)ng/ml以及(10.9±0.6)ng/ml。5、MICA的表达与分布:(1)荧光定量PCR结果显示:MDA-MB-231、SKBr-3、MCF-7和MDA-MB-435s四种细胞株MICAmRNA相对于GAPDH的相对含量分别为5.56×10-3、2.53×10-3、1.68×10-3和5.99×10-5。(2)流式细胞术检测4种乳腺癌细胞株MICA表达,结果显示MDA-MB-231表达量为(61.5±4.5)%、MCF-7为(56.5±4.7)%,而SKBr-3和MDA-MB-435s几乎不表达MICA分子;穿孔后MDA-MB-231和MCF-7这两株细胞株MICA的表达量与穿孔前相比增加不明显,而MDA-MB-435s和SKBr-3这两株细胞株MICA的表达量分别为(26.4±2.5)%和(20.7±1.3)%。(3)免疫组化证实MDA-MB-231细胞和SKBr-3细胞MICA在细胞膜和细胞浆都有表达,MCF-7细胞MICA在细胞浆和核膜都有表达,MDA-MB-435s细胞MICA主要在细胞浆表达;免疫荧光证实MDA-MB-231细胞和SKBr-3细胞MICA在细胞膜和细胞浆都有表达,MCF-7细胞MICA主要表达在细胞膜上,MDA-MB-435s细胞MICA主要在细胞浆表达。NK对不同的乳腺癌细胞株的杀伤结果表明,乳腺癌细胞株MICA表达部位不同,NK细胞对其杀伤率不同。
【结论】T-reg细胞对NK细胞杀伤乳腺癌的作用其机制可能与T-reg分泌的细胞因子TGF-β1有关。MICA在乳腺癌细胞膜上表达越多,NK细胞对该细胞的杀伤率越高。
Objective To analyze the expression of T-reg in peripheral blood from patients with breast cancer and its relationship with NK cell and the subset of T cell; explore the effect of T-reg cell on cytotoxicity of NK cell against breast cancer cell in vitro and analyze the possible underlying mechanism; explore the role of NKG2D-MICA on cytotoxicity of NK cell against breast cancer cell; observe the effect on the NKG2D-MICA by T-reg cell.
Methods 1. Flow cytometry(FCM)were evaluated the proportion of T-reg、NK cell and the subset of T cell;and detect the expression of Membrane-bound TGF-β1 on T-reg. 2. LDH assay was manipulated to test the cytotoxicity of NK cell on breast cancer cells.3. ELISA was performed to examine the level of IFN-γsecreted by NK cell in supernatant and TGF-β1 secreted by T-reg cell.4. RQ-PCR was used to identify the expression of MICA-mRNA on breast cancer cell lines. 5. FCM were performed to identify the expression of MICA on the cell surface and intracell of breast cancer cell lines、Immunohistochemistry(IH) and immunofluorescence (IF) were used to detect the distribution of MICA on breast cancer cell lines. 6. FCM were used to detect the expression of NKG2D on NK cells.
Results 1. The expression of T-reg in patients with breast cancer:The proportion of T-reg in patients with breast cancer was higher than that of volunteers ((7.5±3.0)% Vs(5.1±1.5)% ) P<0.01. 2. The effect of T-reg on cytotoxicity of NK cells:when the ratio of NK and breast cancer cell was 10:1,the cytotoxicity of NK cell was (40.7±3.0)%、(15.5±3.1)%、(29.7±1.8)% and (59.1±2.3)% on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively;the cytotoxicity of NK cell decreased to(1.9±0.2)%、(5.7±0.4)%、(3.6±2.8)% and(3.9±0.2)% respectively while co-culture with 1/5 T-reg cell;and (7.6±1.6)%、(7.3±1.0)%、(6.6±2.5)% and(19.7±1.1)% respectively while co-culture with 1/10 T-reg cell.3.ELISA detect IFN-γsecreted by NK cell display: when the ratio of NK and breast cancer cell was 10:1, the concentration of IFN-γwas (221.5±2.6)pg/ml、(264.4±5.5)pg/ml、(754.6±19.0)pg/ml and(423.0±14.6)pg/ml on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively; the concentration of IFN-γdecreased to (70.7±8.5)pg/ml、(14.6±0.9)pg/ml、(128.3±4.0)pg/ml and(6.0±1.0)pg/ml respectively while co-culture with 1/5 T-reg cell;the concentration of IFN-γchange to(216.6±4.8)pg/ml、(251.7±2.2)pg/ml、(535.0±8.5)pg/ml and(52.57±3.4)pg/ml respectively while co-culture with 1/10 T-reg cell.4. ELISA detect TGF-β1 secreted by T-reg cell in the supernatant display: when the ratio of NK and breast cancer cell was 10:1, the concentration of TGF-β1 was(13.4±0.2)pg/ml、(11.3±0.8)pg/ml、(62.9±1.3)pg/ml and(6.2±0.5)pg/ml on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively; the concentration of TGF-β1 increased to (27.2±0.3)ng/ml、(12.2±1.0)ng/ml、(77.6±1.0)ng/ml and(9.1±0.1)ng/ml respectively while co-culture with 1/5 T-reg cell; the concentration of TGF-β1 increse to(46.8±1.6)ng/ml、(15.8±0.9)ng/ml、(87.8±2.3)ng/ml and(10.9±0.6)ng/ml respectively while co-culture with 1/10 T-reg cell.5.The distribution and expressin of MICA:(1)RQ-PCR discover: Relative content of MICA mRNA of MDA-MB-231、SKBr-3、MCF-7 and MDA-MB-435s was 5.56×10-3、2.53×10-3、1.68×10-3and 5.99×10-5 respectively;(2) MICA was not detected on the surface of MDA-MB-435s and SKBr-3,(61.5±4.5)% on MDA-MB-231 and(56.5±4.7)% on MCF-7 by FCM;after cell perforating,the percentage of MICA was (26.4±2.5)% and(20.7±1.3)% on the MDA-MB-435s and SKBr-3 respectively, but the percentage of MICA had not changes for the MDA-MB-231 and MCF-7; (3)Immunohistochemistry(IH) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express in the cytoplasm and nuclear membrane of the MCF-7, and in the cytoplasm of the MDA-MB-435s;Immunofluorescence(IF) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express on the membrane of the MCF-7,and in the cytoplasm of the MDA-MB-435s.With the different location of MICA,cytotoxicity of NK cells is different.
Conclusion T-reg cells may inhibit the cytotoxicity of NK cell, which was associated with the secretion of TGF-β1. The membrane MICA could increase the cytotoxicity of NK cell on breast cancer cell.
【方法】1、流式细胞术检测乳腺癌患者外周血T-reg的比例;探讨乳腺癌患者TGF-β1的变化。2、应用LDH法检测NK细胞对四种乳腺癌细胞株杀伤率活性。3、ELISA检测培养上清液中IFN-γ和TGF-β1的水平4、荧光定量PCR检测四种乳腺癌细胞株MICA-mRNA的表达。5、应用流式细胞术检测四种乳腺癌细胞株表面及胞内MICA的表达;6、应用免疫组化技术和免疫荧光技术检测四种乳腺癌细胞株MICA的分布情况。7、应用FCM检测NK细胞表面的NKG2D的变化。
【结果】1、乳腺癌病人T-reg的表达:乳腺癌病人和健康志愿者外周血中T-reg占CD4+T细胞总数的百分比分别是(7.5±3.0)%和(5.1±1.5)%,前者高于后者(P<0.01)。2、当NK细胞与乳腺癌细胞效靶比为10:1时,对MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231的杀伤活性分别为(40.7±3.0)%、(15.5±3.1)%、(29.7±1.8)%以及59.1±2.3)%;加入T-reg的数量为1/5的NK细胞时,杀伤活性分别降至(1.9±0.2)%、(5.7±0.4)%、(3.6±2.8)%以及(3.9±0.2)%;加入T-reg的数量为1/10的NK细胞时,杀伤活性分别降至(7.6±1.6)%、(7.3±1.0)%、(6.6±2.5)%以及(19.7±1.1)%。3、ELISA检测NK细胞分泌的IFN-γ结果显示:在无T-reg存在时,NK细胞与MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231共培养时IFN-γ浓度分别为(221.5±2.6)pg/ml、(264.4±5.5)pg/ml、(754.6±19.0)pg/ml以及(423.0±14.6)pg/ml;加入T-reg的数量为1/5的NK细胞时,IFN-γ浓度分别为(70.7±8.5)pg/ml、(14.6±0.9)pg/ml、(128.3±4.0)pg/ml以及(6.0±1.0)pg/ml;加入T-reg的数量为1/10的NK细胞时,IFN-γ浓度分别为(216.6±4.8)pg/ml、(251.7±2.2)pg/ml、(535.0±8.5)pg/ml以及(52.57±3.4)pg/ml。4、ELISA检测培养上清液中T-reg分泌的TGF-β1结果显示:在无T-reg存在时,NK细胞与MCF-7、MDA-MB-435s、SKBr-3和MDA-MB-231共培养时TGF-β1浓度分别为(13.4±0.2)pg/ml、(11.3±0.8)pg/ml、(62.9±1.3)pg/ml以及(6.2±0.5)pg/ml;加入T-reg的数量为1/5的NK细胞时,TGF-β1浓度分别为(27.2±0.3)ng/ml、(12.2±1.0)ng/ml、(77.6±1.0)ng/ml以及(9.1±0.1)ng/ml;加入T-reg的数量为1/10的NK细胞时,TGF-β1浓度分别为(46.8±1.6)ng/ml、(15.8±0.9)ng/ml、(87.8±2.3)ng/ml以及(10.9±0.6)ng/ml。5、MICA的表达与分布:(1)荧光定量PCR结果显示:MDA-MB-231、SKBr-3、MCF-7和MDA-MB-435s四种细胞株MICAmRNA相对于GAPDH的相对含量分别为5.56×10-3、2.53×10-3、1.68×10-3和5.99×10-5。(2)流式细胞术检测4种乳腺癌细胞株MICA表达,结果显示MDA-MB-231表达量为(61.5±4.5)%、MCF-7为(56.5±4.7)%,而SKBr-3和MDA-MB-435s几乎不表达MICA分子;穿孔后MDA-MB-231和MCF-7这两株细胞株MICA的表达量与穿孔前相比增加不明显,而MDA-MB-435s和SKBr-3这两株细胞株MICA的表达量分别为(26.4±2.5)%和(20.7±1.3)%。(3)免疫组化证实MDA-MB-231细胞和SKBr-3细胞MICA在细胞膜和细胞浆都有表达,MCF-7细胞MICA在细胞浆和核膜都有表达,MDA-MB-435s细胞MICA主要在细胞浆表达;免疫荧光证实MDA-MB-231细胞和SKBr-3细胞MICA在细胞膜和细胞浆都有表达,MCF-7细胞MICA主要表达在细胞膜上,MDA-MB-435s细胞MICA主要在细胞浆表达。NK对不同的乳腺癌细胞株的杀伤结果表明,乳腺癌细胞株MICA表达部位不同,NK细胞对其杀伤率不同。
【结论】T-reg细胞对NK细胞杀伤乳腺癌的作用其机制可能与T-reg分泌的细胞因子TGF-β1有关。MICA在乳腺癌细胞膜上表达越多,NK细胞对该细胞的杀伤率越高。
Objective To analyze the expression of T-reg in peripheral blood from patients with breast cancer and its relationship with NK cell and the subset of T cell; explore the effect of T-reg cell on cytotoxicity of NK cell against breast cancer cell in vitro and analyze the possible underlying mechanism; explore the role of NKG2D-MICA on cytotoxicity of NK cell against breast cancer cell; observe the effect on the NKG2D-MICA by T-reg cell.
Methods 1. Flow cytometry(FCM)were evaluated the proportion of T-reg、NK cell and the subset of T cell;and detect the expression of Membrane-bound TGF-β1 on T-reg. 2. LDH assay was manipulated to test the cytotoxicity of NK cell on breast cancer cells.3. ELISA was performed to examine the level of IFN-γsecreted by NK cell in supernatant and TGF-β1 secreted by T-reg cell.4. RQ-PCR was used to identify the expression of MICA-mRNA on breast cancer cell lines. 5. FCM were performed to identify the expression of MICA on the cell surface and intracell of breast cancer cell lines、Immunohistochemistry(IH) and immunofluorescence (IF) were used to detect the distribution of MICA on breast cancer cell lines. 6. FCM were used to detect the expression of NKG2D on NK cells.
Results 1. The expression of T-reg in patients with breast cancer:The proportion of T-reg in patients with breast cancer was higher than that of volunteers ((7.5±3.0)% Vs(5.1±1.5)% ) P<0.01. 2. The effect of T-reg on cytotoxicity of NK cells:when the ratio of NK and breast cancer cell was 10:1,the cytotoxicity of NK cell was (40.7±3.0)%、(15.5±3.1)%、(29.7±1.8)% and (59.1±2.3)% on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively;the cytotoxicity of NK cell decreased to(1.9±0.2)%、(5.7±0.4)%、(3.6±2.8)% and(3.9±0.2)% respectively while co-culture with 1/5 T-reg cell;and (7.6±1.6)%、(7.3±1.0)%、(6.6±2.5)% and(19.7±1.1)% respectively while co-culture with 1/10 T-reg cell.3.ELISA detect IFN-γsecreted by NK cell display: when the ratio of NK and breast cancer cell was 10:1, the concentration of IFN-γwas (221.5±2.6)pg/ml、(264.4±5.5)pg/ml、(754.6±19.0)pg/ml and(423.0±14.6)pg/ml on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively; the concentration of IFN-γdecreased to (70.7±8.5)pg/ml、(14.6±0.9)pg/ml、(128.3±4.0)pg/ml and(6.0±1.0)pg/ml respectively while co-culture with 1/5 T-reg cell;the concentration of IFN-γchange to(216.6±4.8)pg/ml、(251.7±2.2)pg/ml、(535.0±8.5)pg/ml and(52.57±3.4)pg/ml respectively while co-culture with 1/10 T-reg cell.4. ELISA detect TGF-β1 secreted by T-reg cell in the supernatant display: when the ratio of NK and breast cancer cell was 10:1, the concentration of TGF-β1 was(13.4±0.2)pg/ml、(11.3±0.8)pg/ml、(62.9±1.3)pg/ml and(6.2±0.5)pg/ml on MCF-7、MDA-MB-435s、SKBr-3 and MDA-MB-231 respectively; the concentration of TGF-β1 increased to (27.2±0.3)ng/ml、(12.2±1.0)ng/ml、(77.6±1.0)ng/ml and(9.1±0.1)ng/ml respectively while co-culture with 1/5 T-reg cell; the concentration of TGF-β1 increse to(46.8±1.6)ng/ml、(15.8±0.9)ng/ml、(87.8±2.3)ng/ml and(10.9±0.6)ng/ml respectively while co-culture with 1/10 T-reg cell.5.The distribution and expressin of MICA:(1)RQ-PCR discover: Relative content of MICA mRNA of MDA-MB-231、SKBr-3、MCF-7 and MDA-MB-435s was 5.56×10-3、2.53×10-3、1.68×10-3and 5.99×10-5 respectively;(2) MICA was not detected on the surface of MDA-MB-435s and SKBr-3,(61.5±4.5)% on MDA-MB-231 and(56.5±4.7)% on MCF-7 by FCM;after cell perforating,the percentage of MICA was (26.4±2.5)% and(20.7±1.3)% on the MDA-MB-435s and SKBr-3 respectively, but the percentage of MICA had not changes for the MDA-MB-231 and MCF-7; (3)Immunohistochemistry(IH) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express in the cytoplasm and nuclear membrane of the MCF-7, and in the cytoplasm of the MDA-MB-435s;Immunofluorescence(IF) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express on the membrane of the MCF-7,and in the cytoplasm of the MDA-MB-435s.With the different location of MICA,cytotoxicity of NK cells is different.
Conclusion T-reg cells may inhibit the cytotoxicity of NK cell, which was associated with the secretion of TGF-β1. The membrane MICA could increase the cytotoxicity of NK cell on breast cancer cell.
引文
[1] 张利宁.调节性 T 细胞与肿瘤[J].中国肿瘤生物治疗杂志,2007,14(3):201-205.
[2] Tyler J Curiel, George Coukos, Linhua Zou et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival [J]. Nature medicine, 2004, 10(9):942-949.
[3] Natacha Ralainirina,Aure′lie Poli,Tatiana Michel et al. Control of natural killer (NK) cell functions by CD4+CD25+ regulatory T cells[J]. J Leukoc Biol.2007,81(1):144-153
[4] Francois Ghiringhelli, Cedric Menard, Magali Terme et al. CD4+CD25+ Regulatory T Cells inhibit Natural killer cell functions in a transforming growth factor-β-dependent manner [J]. J Exp Med, 2005, 202(8):1075-1085.
[5] Strauss L,Bergmann C,Szczepanski M et al.A Unique Subset of CD4+CD25high Foxp3+T Cells Secreting Interleukin-10 and Transforming growth factor 1 Mediates Suppression in the Tumor Microenvironment[J]. Clin Cancer Res,2007,13(15):4345-4354.
[6] 孙卫民,王慧琴.细胞因子研究方法学.北京:人民卫生出版社,2000,108-109.
[7] Gershon, R.K, Carter, R.L. and Kondo, K. On concomitant immunity in tumour-bearing hamsters[J].Nature 1967,213:674–676
[8] Miyara,M. and Sakaguchi,S. Natural regulatory T cells: mechanisms of suppression [J]. Trends Mol Med. 2007,13(3): 108–116
[9] 刘俊田,岳杰,任秀宝等.乳腺癌患者外周血 CD4+CD25+调节性 T 细胞的检测及意义[J ].中华肿瘤杂志,2005,27(7):423-425
[10] 浦立勇,王学浩,李相成等.免疫磁珠两步法分离大鼠脾脏 CD4 + CD25 + 调节性 T 细胞[J]. 中国普外基础与临床杂志,2006,13(3):275-278
[11] Shohei Hori,Takashi Nomura,Shimon Sakaguchi.Control of Regulatory T Cell Development by the Transcription FactorFoxp3[J].Science,2003,299(14):1057-1061
[12] Weihong Liu,Amy L Putnam, Zhou xu-yu et al. CD127 expression inversely correlates with Foxp3 and suppressive function of human CD4+ T-reg cells[J].J Exp Med,2006,203(7):1701-1711.
[13] 徐勇, 霍梅. 免疫磁珠分离及流式细胞仪分选纯化外周血 CD34+ /CD90+ 干细胞[J].临床检验杂志,2004,22(4):246.
[14] Kawaida H , Kono K, Takahashi A et al.Distribution of CD4+CD25high regulatory T-cells in tumor-draining lymph nodes in patients with gastric cancer [J].J Surgical Research ,2005 ,124(1):151-157.
[15] William L G, Daniel A H. The EWS-WT1 gene fusion in desmoplastic small round cell tumor[J].Seminars in Cancer Biology,2005,15 (3) :197 -205.
[16]曾冬竹,郑峻松,王自强等. 体外富集 T-reg 细胞对 CTL 杀伤 MN K45胃癌细胞活性的影响及机制研究[J]. 消化外科,2006,5(3):197-200.
[17] Udaya K. Liyanage, Todd T. Moore,et al. Prevalence of Regulatory T Cells Is Increased in Peripheral Blood and Tumor Microenvironment of Patients with Pancreas or Breast Adenocarcinoma [J].J Immunol,2002,169(5):2756–2761.
[18] PeterE.Fecci, AlisonE.Sweeney, PeterM.Grossi et al. Systemic Anti-CD25 Monoclonal AntibodyAdministration Safely Enhances Immunity in Murine Glioma without Eliminating Regulatory T Cells[J]. Clin Cancer Res 2006,12(14):4294-4305.
[19] 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.
[20] Aoshang chen , Shanrong Liu , David Park 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.
[21] Tyler J Curiel, George Coukos, Linhua Zou et al. Specificrecruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival [J]. Nature medicine, 2004, 10(9):942-949.
[22] Takashi Ishida and Ryuzo Ueda.CCR4 as a novel molecular target for immunotherapy of cancer[J]. Cancer Sci ,2006, 97(11): 1139-1146
[23] Veronika Groh,Alexander Steinle,Stenfan Bauer et al.Recognition of Stress-Induced MHC Molecules by Intestinal Epithelial γδT Cells[J].Science,1998,279(13):1737-1740.
[24]肖艳梅,付道林,李安生.激光扫描共聚焦显微镜(LSCM )及其生物学应用[J].激光生物学报,1999,8(4):305-311.
[25] 周智锋,叶韵斌,陈强等.MICA 分子在 NK 细胞杀伤乳腺癌中的作用.免疫学杂志[J].2008,24(2):208-212.
[26] Kobayashi, Urabe, Winder et al.Tyrosinase related protein 1(TRP1) functions as a DHICA oxidase in melanin biosynthesis[J].EMBO , 1994 ,13(24):5818-25.
[27] Zahra Madjd, Ian Spendlove, Robert Moss et al.Upregulation of MICA on high-grade invasive operable breast carcinoma [J]. Cancer Immunity, 2007,7(17):1-10
[28] Pende D,Rivera P,Marcenaro S,et al .Major histocompatibility complex class Ⅰ-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity[J]. Cancer Res,2002,62(21):6178-6186
[29] 李楠主编. 激光扫描共聚焦显微术[ M].人民军医出版社, 1997:1-11.
[30] 张彩,冯进波,田志刚等.膜型 分泌型 MICA 对 NK 细胞受体 NKG2D 的相反调节效应及其对 NK 细胞受体谱的影响[J].中华微生物和免疫学杂志,2004,24(2):107-111.
[31] Andreas Busche, Torsten Goldmann, Ulrike Naumann,et al .Natural Killer Cell-Mediated Rejection of Experimental Human Lung Cancer by Genetic Overexpression of Major Histocompatibility Complex Class IChain-Related Gene A[J]. Hum Gene Ther,2006,17(2):135-146
[32] Nicholas F.S. Watson,Ian Spendlove,Zahra Madjd,et al. Expression of the stress-related MHC class I chain-related protein MICA is an indicator of good prognosis in colorectal cancer patients[J].Int.J.Cancer,2006,118(6):1445–1452
[1] Andrea Iellem, Margherita Mariani, Rosmarie Lang et al. Unique Chemotactic Response Profile and Specific Expression of Chemokine Receptors CCR4 and CCR8 by CD4+CD25+ Regulatory T Cells[J].J.Exp.Med, 2001,194( 6): 847–853.
[2] Shohei Hori,Takashi Nomura, Shimon Sakaguchi. Control of Regulatory T Cell Development by the Transcription Factor Foxp3 [J]. Science,2003,299(5609):1057-1061.
[3] Weihong Liu,Amy L Putnam, Zhou xu-yu et al. CD127 expression inversely correlates with Foxp3 and suppressive function of human CD4+ T-reg cells[J].J Exp Med,2006,203(7):1701-1711.
[4] Helmut Jonuleit, Edgar Schmitt, Hacer Kakirman et al. Infectious Tolerance: Human CD25+ Regulatory T Cells Convey Suppressor Activity to Conventional CD4+ T Helper Cells [J]. J Exp Med,2002,196(2):255-260.
[5] Kathryn J. Wood and Birgit Sawitzki .Interferonγ: a crucial role in the function of induced regulatory T cells in vivo [J]. TRENDS in Immunology,2006,27(4): 183-187.
[6] Ciriaco A. Piccirillo and Ethan M. Shevach. Cutting Edge: Control of CD8+ T Cell Activation by CD4+CD25+ Immunoregulatory Cells[J].J Immunol ,2001,167(3): 1137–1140.
[7] Piotr Trzonkowski, Ewa Szmit, Jolanta Mys liwska et al .CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cell-to-cell interaction [J]. Clinical Immunology,2004,112(3): 258–267.
[8] Wim Janssens, Vincent Carlier, Bo Wu et al. CD4+CD25+ T Cells Lyse Antigen-Presenting B Cells by Fas-Fas Ligand Interaction in an Epitope-Specific Manner[J]. J Immunol,2003,171(9): 4604–4612.
[9] Hyung W. Lim, Peter Hillsamer and Chang H. Kim. Regulatory T cellscan migrate to follicles upon T cell activation and suppress GC-Th cells and GC-Th cell–driven B cell responses [J]. J Clin Invest,2004,114(11): 1640–1649.
[10] Hyung W. Lim, Peter Hillsamer, Allison H. Banham et al. Cutting Edge: Direct Suppression of B Cells by CD4+CD25+ Regulatory T Cells [J]. J Immunol,2005,175(7): 4180–4183.
[11] Ceoilia Oderup, Lukas Cederbom, Anna Makowska 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]. J Immunol,2006,118(2): 240-249.
[12] Francesca Fallarino, Ursula Grohmann, Kwang Woo Hwang et al. Modulation of tryptophan catabolism by regulatory T cells [J]. Nature immunology,2003,4(12): 1206-1212.
[13] Francois Ghiringhelli, Cedric Menard, Magali Terme et al. CD4+CD25+ Regulatory T Cells inhibit Natural killer cell functions in a transforming growth factor-β-dependent manner [J]. J Exp Med, 2005, 202(8): 1075-1085.
[14] Takeshi Azuma, Tsuyoshi Takahashi, Atsushi Kunisato et al. Human CD4+ CD25+Regulatory T Cells Suppress NKT Cell Functions [J].Cancer research, 2003,63(15): 4516–4520.
[15] Antonio La Cava, Luc Van Kaer and Fu-Dong-Shi. CD4+CD25+Tregs and NKT cells: regulators regulating regulators [J].TRENDS in Immunology,2006,27(7 ): 322-327.
[16] Venet F, Pachot A, Debard AL et al. Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism [J]. J Immunol.2006,177(9):6540-6547.
[17] Tyler J Curiel, George Coukos, Linhua Zou et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival [J]. Nature medicine, 2004, 10(9): 942-949.
[18] William J. Grossman, James W. Verbsky, Winfried Barchet et al. Human T Regulatory Cells Can Use the Perforin Pathway to Cause Autologous Target Cell Death [J]. Immunity,2004,21(4): 589–601.
[19] David C. Gondek, Li-Fan Lu, Sergio A. Quezada et al. Cutting Edge: Contact-Mediated Suppression by CD4 + CD25 + Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism [J]. J Immunol,2005,174(4): 1783-1786.
[20] Maurus de la Rosa, Sascha Rutz, Heike Dorninger et al. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function [J].Eur J Immunol,2004,34(9): 2480-2488.
[2] Tyler J Curiel, George Coukos, Linhua Zou et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival [J]. Nature medicine, 2004, 10(9):942-949.
[3] Natacha Ralainirina,Aure′lie Poli,Tatiana Michel et al. Control of natural killer (NK) cell functions by CD4+CD25+ regulatory T cells[J]. J Leukoc Biol.2007,81(1):144-153
[4] Francois Ghiringhelli, Cedric Menard, Magali Terme et al. CD4+CD25+ Regulatory T Cells inhibit Natural killer cell functions in a transforming growth factor-β-dependent manner [J]. J Exp Med, 2005, 202(8):1075-1085.
[5] Strauss L,Bergmann C,Szczepanski M et al.A Unique Subset of CD4+CD25high Foxp3+T Cells Secreting Interleukin-10 and Transforming growth factor 1 Mediates Suppression in the Tumor Microenvironment[J]. Clin Cancer Res,2007,13(15):4345-4354.
[6] 孙卫民,王慧琴.细胞因子研究方法学.北京:人民卫生出版社,2000,108-109.
[7] Gershon, R.K, Carter, R.L. and Kondo, K. On concomitant immunity in tumour-bearing hamsters[J].Nature 1967,213:674–676
[8] Miyara,M. and Sakaguchi,S. Natural regulatory T cells: mechanisms of suppression [J]. Trends Mol Med. 2007,13(3): 108–116
[9] 刘俊田,岳杰,任秀宝等.乳腺癌患者外周血 CD4+CD25+调节性 T 细胞的检测及意义[J ].中华肿瘤杂志,2005,27(7):423-425
[10] 浦立勇,王学浩,李相成等.免疫磁珠两步法分离大鼠脾脏 CD4 + CD25 + 调节性 T 细胞[J]. 中国普外基础与临床杂志,2006,13(3):275-278
[11] Shohei Hori,Takashi Nomura,Shimon Sakaguchi.Control of Regulatory T Cell Development by the Transcription FactorFoxp3[J].Science,2003,299(14):1057-1061
[12] Weihong Liu,Amy L Putnam, Zhou xu-yu et al. CD127 expression inversely correlates with Foxp3 and suppressive function of human CD4+ T-reg cells[J].J Exp Med,2006,203(7):1701-1711.
[13] 徐勇, 霍梅. 免疫磁珠分离及流式细胞仪分选纯化外周血 CD34+ /CD90+ 干细胞[J].临床检验杂志,2004,22(4):246.
[14] Kawaida H , Kono K, Takahashi A et al.Distribution of CD4+CD25high regulatory T-cells in tumor-draining lymph nodes in patients with gastric cancer [J].J Surgical Research ,2005 ,124(1):151-157.
[15] William L G, Daniel A H. The EWS-WT1 gene fusion in desmoplastic small round cell tumor[J].Seminars in Cancer Biology,2005,15 (3) :197 -205.
[16]曾冬竹,郑峻松,王自强等. 体外富集 T-reg 细胞对 CTL 杀伤 MN K45胃癌细胞活性的影响及机制研究[J]. 消化外科,2006,5(3):197-200.
[17] Udaya K. Liyanage, Todd T. Moore,et al. Prevalence of Regulatory T Cells Is Increased in Peripheral Blood and Tumor Microenvironment of Patients with Pancreas or Breast Adenocarcinoma [J].J Immunol,2002,169(5):2756–2761.
[18] PeterE.Fecci, AlisonE.Sweeney, PeterM.Grossi et al. Systemic Anti-CD25 Monoclonal AntibodyAdministration Safely Enhances Immunity in Murine Glioma without Eliminating Regulatory T Cells[J]. Clin Cancer Res 2006,12(14):4294-4305.
[19] 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.
[20] Aoshang chen , Shanrong Liu , David Park 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.
[21] Tyler J Curiel, George Coukos, Linhua Zou et al. Specificrecruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival [J]. Nature medicine, 2004, 10(9):942-949.
[22] Takashi Ishida and Ryuzo Ueda.CCR4 as a novel molecular target for immunotherapy of cancer[J]. Cancer Sci ,2006, 97(11): 1139-1146
[23] Veronika Groh,Alexander Steinle,Stenfan Bauer et al.Recognition of Stress-Induced MHC Molecules by Intestinal Epithelial γδT Cells[J].Science,1998,279(13):1737-1740.
[24]肖艳梅,付道林,李安生.激光扫描共聚焦显微镜(LSCM )及其生物学应用[J].激光生物学报,1999,8(4):305-311.
[25] 周智锋,叶韵斌,陈强等.MICA 分子在 NK 细胞杀伤乳腺癌中的作用.免疫学杂志[J].2008,24(2):208-212.
[26] Kobayashi, Urabe, Winder et al.Tyrosinase related protein 1(TRP1) functions as a DHICA oxidase in melanin biosynthesis[J].EMBO , 1994 ,13(24):5818-25.
[27] Zahra Madjd, Ian Spendlove, Robert Moss et al.Upregulation of MICA on high-grade invasive operable breast carcinoma [J]. Cancer Immunity, 2007,7(17):1-10
[28] Pende D,Rivera P,Marcenaro S,et al .Major histocompatibility complex class Ⅰ-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity[J]. Cancer Res,2002,62(21):6178-6186
[29] 李楠主编. 激光扫描共聚焦显微术[ M].人民军医出版社, 1997:1-11.
[30] 张彩,冯进波,田志刚等.膜型 分泌型 MICA 对 NK 细胞受体 NKG2D 的相反调节效应及其对 NK 细胞受体谱的影响[J].中华微生物和免疫学杂志,2004,24(2):107-111.
[31] Andreas Busche, Torsten Goldmann, Ulrike Naumann,et al .Natural Killer Cell-Mediated Rejection of Experimental Human Lung Cancer by Genetic Overexpression of Major Histocompatibility Complex Class IChain-Related Gene A[J]. Hum Gene Ther,2006,17(2):135-146
[32] Nicholas F.S. Watson,Ian Spendlove,Zahra Madjd,et al. Expression of the stress-related MHC class I chain-related protein MICA is an indicator of good prognosis in colorectal cancer patients[J].Int.J.Cancer,2006,118(6):1445–1452
[1] Andrea Iellem, Margherita Mariani, Rosmarie Lang et al. Unique Chemotactic Response Profile and Specific Expression of Chemokine Receptors CCR4 and CCR8 by CD4+CD25+ Regulatory T Cells[J].J.Exp.Med, 2001,194( 6): 847–853.
[2] Shohei Hori,Takashi Nomura, Shimon Sakaguchi. Control of Regulatory T Cell Development by the Transcription Factor Foxp3 [J]. Science,2003,299(5609):1057-1061.
[3] Weihong Liu,Amy L Putnam, Zhou xu-yu et al. CD127 expression inversely correlates with Foxp3 and suppressive function of human CD4+ T-reg cells[J].J Exp Med,2006,203(7):1701-1711.
[4] Helmut Jonuleit, Edgar Schmitt, Hacer Kakirman et al. Infectious Tolerance: Human CD25+ Regulatory T Cells Convey Suppressor Activity to Conventional CD4+ T Helper Cells [J]. J Exp Med,2002,196(2):255-260.
[5] Kathryn J. Wood and Birgit Sawitzki .Interferonγ: a crucial role in the function of induced regulatory T cells in vivo [J]. TRENDS in Immunology,2006,27(4): 183-187.
[6] Ciriaco A. Piccirillo and Ethan M. Shevach. Cutting Edge: Control of CD8+ T Cell Activation by CD4+CD25+ Immunoregulatory Cells[J].J Immunol ,2001,167(3): 1137–1140.
[7] Piotr Trzonkowski, Ewa Szmit, Jolanta Mys liwska et al .CD4+CD25+ T regulatory cells inhibit cytotoxic activity of T CD8+ and NK lymphocytes in the direct cell-to-cell interaction [J]. Clinical Immunology,2004,112(3): 258–267.
[8] Wim Janssens, Vincent Carlier, Bo Wu et al. CD4+CD25+ T Cells Lyse Antigen-Presenting B Cells by Fas-Fas Ligand Interaction in an Epitope-Specific Manner[J]. J Immunol,2003,171(9): 4604–4612.
[9] Hyung W. Lim, Peter Hillsamer and Chang H. Kim. Regulatory T cellscan migrate to follicles upon T cell activation and suppress GC-Th cells and GC-Th cell–driven B cell responses [J]. J Clin Invest,2004,114(11): 1640–1649.
[10] Hyung W. Lim, Peter Hillsamer, Allison H. Banham et al. Cutting Edge: Direct Suppression of B Cells by CD4+CD25+ Regulatory T Cells [J]. J Immunol,2005,175(7): 4180–4183.
[11] Ceoilia Oderup, Lukas Cederbom, Anna Makowska 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]. J Immunol,2006,118(2): 240-249.
[12] Francesca Fallarino, Ursula Grohmann, Kwang Woo Hwang et al. Modulation of tryptophan catabolism by regulatory T cells [J]. Nature immunology,2003,4(12): 1206-1212.
[13] Francois Ghiringhelli, Cedric Menard, Magali Terme et al. CD4+CD25+ Regulatory T Cells inhibit Natural killer cell functions in a transforming growth factor-β-dependent manner [J]. J Exp Med, 2005, 202(8): 1075-1085.
[14] Takeshi Azuma, Tsuyoshi Takahashi, Atsushi Kunisato et al. Human CD4+ CD25+Regulatory T Cells Suppress NKT Cell Functions [J].Cancer research, 2003,63(15): 4516–4520.
[15] Antonio La Cava, Luc Van Kaer and Fu-Dong-Shi. CD4+CD25+Tregs and NKT cells: regulators regulating regulators [J].TRENDS in Immunology,2006,27(7 ): 322-327.
[16] Venet F, Pachot A, Debard AL et al. Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism [J]. J Immunol.2006,177(9):6540-6547.
[17] Tyler J Curiel, George Coukos, Linhua Zou et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival [J]. Nature medicine, 2004, 10(9): 942-949.
[18] William J. Grossman, James W. Verbsky, Winfried Barchet et al. Human T Regulatory Cells Can Use the Perforin Pathway to Cause Autologous Target Cell Death [J]. Immunity,2004,21(4): 589–601.
[19] David C. Gondek, Li-Fan Lu, Sergio A. Quezada et al. Cutting Edge: Contact-Mediated Suppression by CD4 + CD25 + Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism [J]. J Immunol,2005,174(4): 1783-1786.
[20] Maurus de la Rosa, Sascha Rutz, Heike Dorninger et al. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function [J].Eur J Immunol,2004,34(9): 2480-2488.