人GL50基因转染细胞的构建及功能性鼠抗人GL50单克隆抗体的研制
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摘要
GL50(CD275, inducible costimulator ligand)分子是B7家族的新成员,与其特异性受体ICOS(CD278, inducible costimulator)分子结合所提供的共刺激信号在T细胞的活化、增殖、细胞因子的分泌以及B细胞的增殖、分化、抗体产生等方面发挥着重要的调节作用。GL50信号参与了机体的移植排斥反应、抗肿瘤免疫、抗感染免疫、自身免疫性疾病和过敏反应等众多免疫过程。转染人GL50基因细胞及鼠抗人GL50单克隆抗体(monoclonal antibody, mAb)的研制能够为进一步研究GL50在机体免疫应答中的作用以及与其他共刺激信号通路之间的关系提供有力的工具,从而深入探讨机体免疫应答的本质,同时也为临床相关的疾病诊断、治疗和预防提供了新的手段。
第一部分转染人GL50基因细胞株的建立及其对T细胞体外增殖与活化的促进作用
从人B淋巴瘤细胞株Daudi中抽提总RNA,采用RT-PCR的方法,把总RNA逆转录成cDNA并大量扩增目的基因。将目的基因插入pIRES2-EGFP载体中并测序,通过脂质体转染技术将重组载体pIRES2-EGFP-GL50转染L929细胞。用G418筛选并通过流式细胞术与RT-PCR的方法反复鉴定,最终获得了稳定高表达GL50分子的L929/GL50基因转染细胞。功能分析表明L929/GL50基因转染细胞能够促进T细胞增殖,并可以显著地促进T细胞分泌细胞因子IL-10、IL-4和IL-17,而不影响IL-2的分泌。
第二部分功能性鼠抗人GL50单克隆抗体的研制
以L929/GL50为免疫原,免疫Balb/c小鼠,采用经典杂交瘤技术,将免疫后鼠的脾脏细胞与小鼠骨髓瘤细胞株SP2/0进行融合并经HAT培养基选择培养。以L929/GL50细胞作为阳性筛选细胞株,经流式细胞术分析,对抗体分泌阳性孔内细胞反复筛选并经多次的克隆化培养,最终获得2株稳定、持续分泌鼠抗人GL50单克隆抗体的杂交瘤细胞株,分别命名为2B4、4D11。这两株杂交瘤细胞株经体外连续传代(50代)培养,在液氮冻存半年后复苏,仍然生长状态良好,分泌抗体性能稳定。
采用本科室建立的腹水诱生的方法制备单克隆抗体,腹水的平均产量为3~4ml/只小鼠。经Protein G亲和层析柱分离纯化,两株单克隆抗体纯化后蛋白浓度在3~4mg/ml之间,间接免疫荧光标记法分析表明,其效价在1:5000以上,用于间接免疫荧光分析时抗体蛋白的用量为0.2～2μg/1×106细胞。单克隆抗体核型分析的结果显示,两株杂交瘤2B4与4D11的染色体数目都超过小鼠B细胞与SP2/0的染色体数目,约为100条左右,表明这两株杂交瘤2B4与4D11为融合体。
使用快速定性试纸条鉴定,单克隆抗体2B4与4D11的重链分别为IgG1、IgG2a,两者轻链同为κ链。流式细胞术,Dot-blot与酶联吸附的方法分析表明,单克隆抗体2B4与4D11均能够与人GL50分子特异性结合。抗体的抗原表位竞争抑制实验结果表明,单克隆抗体2B4与4D11识别相同的抗原表位,与商品化抗体MIH12识别的抗原表位不同。
经单抗2B4与4D11鉴定表明,人GL50分子高表达于B淋巴瘤细胞系中的Daudi和Raji,卵巢癌细胞株HO8910,单核细胞来源细胞株THP-1,血管内皮细胞株ECV等。但是,健康人外周血中的B淋巴细胞上基本无GL50分子的表达。功能分析表明,特异性GL50mAb 2B4和4D11均可阻断GL50与ICOS结合从而抑制T细胞在体外的增殖。
GL50 is a new member of B7 family. The interaction of GL50 with ICOS, the specific receptor for GL50, is critically involved in the activation, proliferation, differentiation and cytokine production of T cells as well as in the antibody secretion of B cells during the secondary immune responses. It is well-accepted that the ICOS/GL50 signal pathway plays a pivotal role in the regulation of inflammatory responses, in the prevention of transplantation rejection and tumor development, as well as in the modulation of autoimmune diseases and allergic response. As an important regulatory factor in the signaling pathways, mouse anti-human GL50 monoclonal antibodies provide powerful tools for us to further study the co-stimulatory signal pathway and cross-talking in immune response. So we can investigate the essence of immune response. Thus, establishment of human GL50 transfected cell line and monoclonal antibodies against human GL50 may provide a valuable and potential way for the clinical diagnosis, treatment and prevention of diseases.
This article consists of two parts:
Part I Establishment of cell line transfected with human GL50 gene and preliminary study on its co-stimulatory effect on T cell activation and proliferation in vitro
Total RNA was extracted from B lymphoma cell line Daudi, which was reverse transcripted into cDNA . The human GL50 gene was then amplified by RT-PCR. The target gene was inserted into vector pIRES2-EGFP and confirmed by sequencing. The recombinant pIRES2-EGFP-GL50 vector was transfected into L929 cells with LipofectamineTM 2000, and the cells was further selected with G418 for a long time. GL50 expressed on L929/GL50 cells was analysed by FCM and RT-PCR. Effect of L929/GL50 cells on T cells proliferation and cytokine production in vitro was studied by methods of CCK-8 and ELISA. In vitro, L929/GL50 cells could obviously promote the proliferation of T cells, while up-regulating the production of IL-4、IL-10 and IL-17.
Part II Generation and characteration of mouse anti-human GL50 monoclonal antibody with biological function
The L929/GL50 transgenic cells were used to immunize Balb/C mice. The immunized splenocytes were fused with murine myeloma cells (SP2/0) by the cell fuse technique. By means of HAT selective culture and repeated screening with L929/GL50 as antibody-screening positive cell and L929/mock as negative control, two hybridoma cell lines (2B4 and 4D11) continuously and steadily secreting specific anti-human GL50 were obtained. The two hybridomas grew well after long-term culture in vitro and storage in liquid nitrogen.
The monoclonal antibody was produced according to ascites-inducing procedure in mouse abdominal cavity. The average yield of ascites was about 3.5 milliliter (ml) per mouse. After the ascites was purified by protein G affinity chromatography, the concentration of the protein was above 4 milligram (mg)/ml and the ascitic titer was over 1:1000 dilution by immunofluorescence analysis. Every 1×106 cells required 0.2～2 microgram (μg) purified monoclonal antibody in indirect immunofluorescence. Karyotype analysis result showed that the number of the chromosomes of the hybridoma was more than these of mouse somatic chromosomes, which suggested that they are fused cells.
Fast-strip method analysis displayed that monoclonal antibodies 2B4 and 4D11 were mouse IgG1 and IgG2a, and the light chain wereκ. Dot-blot and flow cytometry analysis showed that monoclonal antibodies 2B4 and 4D11 exclusively binded GL50 molecules. The mAbs were also identified by ELISA.The competitive inhibition results showed that monoclonal antibodies (2B4、4D11) and MIH12, a commercial anti-GL50 antibody, recognized different epitopes.
Flow cytometry analysis showed that GL50 molecule was highly expressed on cell lines such as Daudi, Raji, HO8910, THP-1 and ECV. B lymphocytes, isolated from peripheral blood hardly express GL50 molecules. L929/GL50 cells could obviously promote the proliferation of T cells. Specific mAbs 2B4 and 4D11 could act as blockers in the proliferation of T cells.
第一部分转染人GL50基因细胞株的建立及其对T细胞体外增殖与活化的促进作用
从人B淋巴瘤细胞株Daudi中抽提总RNA,采用RT-PCR的方法,把总RNA逆转录成cDNA并大量扩增目的基因。将目的基因插入pIRES2-EGFP载体中并测序,通过脂质体转染技术将重组载体pIRES2-EGFP-GL50转染L929细胞。用G418筛选并通过流式细胞术与RT-PCR的方法反复鉴定,最终获得了稳定高表达GL50分子的L929/GL50基因转染细胞。功能分析表明L929/GL50基因转染细胞能够促进T细胞增殖,并可以显著地促进T细胞分泌细胞因子IL-10、IL-4和IL-17,而不影响IL-2的分泌。
第二部分功能性鼠抗人GL50单克隆抗体的研制
以L929/GL50为免疫原,免疫Balb/c小鼠,采用经典杂交瘤技术,将免疫后鼠的脾脏细胞与小鼠骨髓瘤细胞株SP2/0进行融合并经HAT培养基选择培养。以L929/GL50细胞作为阳性筛选细胞株,经流式细胞术分析,对抗体分泌阳性孔内细胞反复筛选并经多次的克隆化培养,最终获得2株稳定、持续分泌鼠抗人GL50单克隆抗体的杂交瘤细胞株,分别命名为2B4、4D11。这两株杂交瘤细胞株经体外连续传代(50代)培养,在液氮冻存半年后复苏,仍然生长状态良好,分泌抗体性能稳定。
采用本科室建立的腹水诱生的方法制备单克隆抗体,腹水的平均产量为3~4ml/只小鼠。经Protein G亲和层析柱分离纯化,两株单克隆抗体纯化后蛋白浓度在3~4mg/ml之间,间接免疫荧光标记法分析表明,其效价在1:5000以上,用于间接免疫荧光分析时抗体蛋白的用量为0.2～2μg/1×106细胞。单克隆抗体核型分析的结果显示,两株杂交瘤2B4与4D11的染色体数目都超过小鼠B细胞与SP2/0的染色体数目,约为100条左右,表明这两株杂交瘤2B4与4D11为融合体。
使用快速定性试纸条鉴定,单克隆抗体2B4与4D11的重链分别为IgG1、IgG2a,两者轻链同为κ链。流式细胞术,Dot-blot与酶联吸附的方法分析表明,单克隆抗体2B4与4D11均能够与人GL50分子特异性结合。抗体的抗原表位竞争抑制实验结果表明,单克隆抗体2B4与4D11识别相同的抗原表位,与商品化抗体MIH12识别的抗原表位不同。
经单抗2B4与4D11鉴定表明,人GL50分子高表达于B淋巴瘤细胞系中的Daudi和Raji,卵巢癌细胞株HO8910,单核细胞来源细胞株THP-1,血管内皮细胞株ECV等。但是,健康人外周血中的B淋巴细胞上基本无GL50分子的表达。功能分析表明,特异性GL50mAb 2B4和4D11均可阻断GL50与ICOS结合从而抑制T细胞在体外的增殖。
GL50 is a new member of B7 family. The interaction of GL50 with ICOS, the specific receptor for GL50, is critically involved in the activation, proliferation, differentiation and cytokine production of T cells as well as in the antibody secretion of B cells during the secondary immune responses. It is well-accepted that the ICOS/GL50 signal pathway plays a pivotal role in the regulation of inflammatory responses, in the prevention of transplantation rejection and tumor development, as well as in the modulation of autoimmune diseases and allergic response. As an important regulatory factor in the signaling pathways, mouse anti-human GL50 monoclonal antibodies provide powerful tools for us to further study the co-stimulatory signal pathway and cross-talking in immune response. So we can investigate the essence of immune response. Thus, establishment of human GL50 transfected cell line and monoclonal antibodies against human GL50 may provide a valuable and potential way for the clinical diagnosis, treatment and prevention of diseases.
This article consists of two parts:
Part I Establishment of cell line transfected with human GL50 gene and preliminary study on its co-stimulatory effect on T cell activation and proliferation in vitro
Total RNA was extracted from B lymphoma cell line Daudi, which was reverse transcripted into cDNA . The human GL50 gene was then amplified by RT-PCR. The target gene was inserted into vector pIRES2-EGFP and confirmed by sequencing. The recombinant pIRES2-EGFP-GL50 vector was transfected into L929 cells with LipofectamineTM 2000, and the cells was further selected with G418 for a long time. GL50 expressed on L929/GL50 cells was analysed by FCM and RT-PCR. Effect of L929/GL50 cells on T cells proliferation and cytokine production in vitro was studied by methods of CCK-8 and ELISA. In vitro, L929/GL50 cells could obviously promote the proliferation of T cells, while up-regulating the production of IL-4、IL-10 and IL-17.
Part II Generation and characteration of mouse anti-human GL50 monoclonal antibody with biological function
The L929/GL50 transgenic cells were used to immunize Balb/C mice. The immunized splenocytes were fused with murine myeloma cells (SP2/0) by the cell fuse technique. By means of HAT selective culture and repeated screening with L929/GL50 as antibody-screening positive cell and L929/mock as negative control, two hybridoma cell lines (2B4 and 4D11) continuously and steadily secreting specific anti-human GL50 were obtained. The two hybridomas grew well after long-term culture in vitro and storage in liquid nitrogen.
The monoclonal antibody was produced according to ascites-inducing procedure in mouse abdominal cavity. The average yield of ascites was about 3.5 milliliter (ml) per mouse. After the ascites was purified by protein G affinity chromatography, the concentration of the protein was above 4 milligram (mg)/ml and the ascitic titer was over 1:1000 dilution by immunofluorescence analysis. Every 1×106 cells required 0.2～2 microgram (μg) purified monoclonal antibody in indirect immunofluorescence. Karyotype analysis result showed that the number of the chromosomes of the hybridoma was more than these of mouse somatic chromosomes, which suggested that they are fused cells.
Fast-strip method analysis displayed that monoclonal antibodies 2B4 and 4D11 were mouse IgG1 and IgG2a, and the light chain wereκ. Dot-blot and flow cytometry analysis showed that monoclonal antibodies 2B4 and 4D11 exclusively binded GL50 molecules. The mAbs were also identified by ELISA.The competitive inhibition results showed that monoclonal antibodies (2B4、4D11) and MIH12, a commercial anti-GL50 antibody, recognized different epitopes.
Flow cytometry analysis showed that GL50 molecule was highly expressed on cell lines such as Daudi, Raji, HO8910, THP-1 and ECV. B lymphocytes, isolated from peripheral blood hardly express GL50 molecules. L929/GL50 cells could obviously promote the proliferation of T cells. Specific mAbs 2B4 and 4D11 could act as blockers in the proliferation of T cells.
引文
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1. Wang S ,Zhu G,Chapoval A I et al . Costimulation of T cells by B7-H2 ,a B7-like molecule that binds ICOS. Blood. 2000; 96 : 2808-2813.
2 Yoshinanga S K,Whoriskey J S ,Khare S D, et al . T-cell co-stimulation through B7RP -1 and ICOS. Nature .1999; 402: 827-832.
3 Kadkhoda K, Wang S, Joyee AG, et al. Th1 Cytokine Responses Fail to Effectively Control Chlamydia Lung Infection in ICOS Ligand Knockout Mice. J Immunol. 2010; 184: 3780 -3788.
4邱玉华,张学光等.鼠抗人CD28分子单克隆抗体的研制及生物学特性研究.细胞与分子免疫学杂志. 2001; 17: 368-370.
5 Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 2005;1:175:3-4.
6 Chung Y, Yang X, Chang SH, et al. Expression and regulation of IL-22 in the IL-17-producing CD4+ T lymphocytes. Cell Res. 2006;16: 902-907.
7 Wilson NJ, Boniface K, Chan JR, et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol. 2007; 8: 950-957.
8 Yu JJ, Gaffen SL. Interleukin-17: a novel inflammatory cytokine that bridges innate and adaptive immunity. Front Biosci. 2008; 13:170-177.
9 Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity. 2004; 21: 467-476.
1. Lanzavecchia A, Lezzi G, Viola A: From TCR engagement to T cell activation: a kinetic view of T cell behavior. Cell. 1999;96:1–4.
2. Aicher A, Hayden-Ledbetter M, Brady WA, et al. Characterization of Human Inducible Costimulator Ligand Expression and Function. J Immunol. 2000; 164:4689-4696.
3. Yoshinaga SK, Whoriskey JS, Khare SD, et al. T-cell co-stimulation through B7RP-1 and ICOS. Nature. 1999; 402:827-836.
4. Ling V, Wu PW, Finnerty HF, et al. Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor. J Immunol. 2000;164:1653-1657.
5. Swallow MM, Wallin JJ, Sha WC.B7h:a novel costimulatory homolog of B7.1 and B7.2, is induced by TNF-α. Immunity. 1999;11:423-432.
6. Richter G, Mollweide A, Uhlmann E, et al. Therapeutic Blockade of ICOS:ICOSL Interaction Alleviates Acute Graft Versus Host Disease. Blood. 2005;106:5237.
7. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immuno.l 1996;14:233–258.
8. Taylor P, Panoskaltsis-Mortari A, Freeman G, et al. The ICOS:ICOSL Costimulatory Pathway Plays an Important Role in GVHD and Bone Marrow (BM) Graft Rejection. Blood. 2004;104:591-602.
9. Yoshinaga SK, Zhang M, Pistillo J, et al. Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS. Int Immunol. 2000;12:1439-1447.
10. Wang SD, Zhu GF, Chapoval AI, et al. Costimulation of T cells by B7-H2 ,a B7-like molecule that binds ICOS. Blood. 2000;96 :2808-2813.
11. Ling V, Wu PW, Miyashiro JS, et al. expression of inducible costimulator-ligand splices variants: lymphoid regulation of mouse GL50-B and human GL50 molecules.Immunology. 2001;166:730-736.
12. Wang YG, Zhu MZ, Yu P, et al. Promoting Immune Responses by LIGHT in the Face of Abundant Regulatory T Cell Inhibition. J Immunol. 2010;184:1589– 1595.
13. Qiu YH, Sun ZW, Shi Q, et al. Apoptosis of multiple myeloma cells induced by agonist monoclonal antibody against human CD28. Cell Immunol. 2005;236:154–160.
14. Xingluo Liu XL, Bai XF, et al. B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8+ T lymphocytes in vivo. J Exp Med. 2001;194:1339-1348.
15. Iwai H, Kozono Y, Hirose S, et al. Amelioration of Collagen-Induced Arthritis by Blockade of Inducible Costimulator-B7 Homologous Protein Costimulation, J Immunol. 2002;169: 4332- 4339.
16. Kosuge H, Suzuki J, Gotoh R, et al. Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway. Transplantation. 2003;75:1374-1380.
17. Villegas EN, Lieberman LA, Mason N, et al. A role for inducible costimulatorprotein in the CD28-independent mechanism of resistance to Toxoplasma gondii. J Immunol. 2002;169:937-943.
18. Liang LD, Porter EM, Sha WC. Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be reduced by CD40 signaling. J Exp Med. 2002;196 :97-108.
19. Sporici RA, Beswick RL, von Allmen C, et al. ICOS ligand costimulation is required for T-cell encephalitogenicity. Clin Immunol. 2001;100:277-288.
20. Akbari O, Freeman GJ, Meyer EH, et al. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med. 2002;8:1024-1032.
21.邱玉华,张学光.一种显著提高小鼠腹水产量的新方法.中国免疫学杂志.1995; 11:366-372.
2 Yoshinaga SK, Whoriskey JS, Khare SD, et a1. T-cell co-stimulation through B7RP-1 and ICOS. Nature. 1999; 402: 827-832.
3 Mages H W, Hutloff A, Heuck G, et a1. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS lisand. Eur J Immunol. 2000; 30: l040-1047.
4 Ling Y, Wu PW, Finnerty HF, et al. Differential expression of inducible costimulator-ligand splice variants:lymphoid regulation of mouse GL50-B and human GL50 molecules.Immunology. 2001; 166: 730-736.
5 Guo Y, Wu Y, Zhao M, et al.Mutational analysis and an alternatively spliced product of B7 defined its CD28/CTLA-4-binding site on immunoglubolin G-like domain.J Exp Med. 1995; 181: 1345.
6许雪青,白云,王丰,等.人ICOSIg的真核表达及其对MLR反应的影响.免疫学杂志. 2005; 21, 1-5.
7 Rutitzky LI, zkaynak E,. Rottman JB, et al. Disruption of the ICOS-B7RP-1 Costimulatory Pathway Leads to Enhanced Hepatic Immunopathology and Increased Gamma Interferon Production by CD4 T Cells inMurine Schistosomiasis. The Journal of Clinical Investigation. 2003; 54: 234-243.
8 Yu XZ, Yaming L, Nurieva RI, et al. Opposing Effects of ICOS on Graft-versus-Host DiseaseMediated by CD4 and CD8 T Cell. The Journal of Immunology. 2006; 176: 7394–7401.
9 Ozkaynak E , Gao W, Shemmeri N, et al. Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection. Nat Immunol. 2001; 2:591-596.
10 Katsumata Y, Harigai M, Sugiura T, et al. Attenuation of Experimental Autoimmune Myositis by Blocking ICOS-ICOS Ligand Interaction. The Journal of Immunology. 2007; 179: 3772- 3779.
11 Zheng Y, Jost M, Gaughan JP. et al. ICOS-B7 Homologous Protein Interactions Are Necessary for Mercury-Induced Autoimmunity. The Journalof Immunology. 2005; 174: 3117–3121.
12 Mcadam A J, Chang T, Lumelsky A L, et a1. Mouse inducible costinmla-torv molecule(ICOS)expression is enhanced by CD28 costinmlation and regulates differentiation of CD4+T cells.J Immunol. 2000; 165: 5035-5040.
13 Iwai H, Kozono Y, Hirose S, et al. Amelioration of collagen-induced arthritis by blockade of inducible costinmlator-B7 homologous protein costimulation. J Immuno1. 2002; l69: 4332-4339.
14 Liang L ,Porter EM, Sha WC. Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be reduced by CD40 signaling. J Exp Med. 2002; 196:97-108.
15 Hawiger D, Tran E, Du W, et al. ICOS Mediates the Development of Insulin-Dependent Diabetes Mellitus in Nonobese Diabetic Mice.The Journal of Immunology. 2008; 180: 3140-3147.
16 Khayyamian S ,Hutloff A ,Buchner K, et al . ICOS-ligand , expressed on humanendothelial cells ,costimulates Th1 and Th2 cytokine secretion by memory CD4 + T cells. Proc Natl Acad Sci U. S. A.2002; 99:6198-6203.
17 Wallin J J ,Liang L ,Bakardjiev A, et al . Enhancement of CD8 + T cell responses by ICOS/B7h costimulation. J Immunol. 2001; 167:132-139.
18 Ogasawara K,Yoshinaga S K,Lanier L L. Inducible costimulator costimulates cytotoxic activity and IFN-gamma production in activated murine NK cells. J Immunol. 2002; 169:3676-3685.
19 Tesciuba A G,Subudhi S ,Rother R P, et al. Inducible costimulator regulates Th2-mediated inflammation ,but not Th2 differentiation ,in a model of allergic airway disease. J Immunol. 2001; 167:1996-2003.
20 Iwai H ,Kozono Y,Hirose S, et al. Amelioration of collagen-induced arthritis by blockade of inducible costimulator-B7 homologous protein costimulation. J Immunol. 2002; 169:4332-4339.
21 Aicher A, Hayden LM, Brady WA, et al. Characterization of human inducible costimulator ligand expression and function. J. Immunol. 2000;164: 4689-4696.
22 Ling V, Wu PW, Finnerty HF, et al. Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor. J. Immunol. 2000;164: 1653-1657.
23 Swallow MM, Wallin JJ, Sha WC, et al. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFalpha. Immunity. 1999; 11: 423-432.
24 Brodie D, Collins AV, Iaboni A, et al. LICOS, a primordial costimulatory ligand. Curr. Biol. 2000; 10: 333-336.
25 Yoshinaga SK, Zhang M, Pistillo J, et al. Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS. Int. Immunol. 2000;12:1439-1447.
26 Tang G, Qin Q, Zhang P, et al. Reverse signaling using an inducible costimulator to enhance immunogenic function of dendritic cells. Cell Mol Life Sci. 2009; 66: 3067-80.
27 Bonhagen K, Liesenfeld O, Stadecker MJ, et al. ICOS+ Th cells produce distinct cytokines in different mucosal immune responses. Eur. J. Immunol. 2003;33:392-401.
28 Riley JL, Blair PJ, Musser JT, et al. ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement. J. Immunol. 2001;166:4943-4948.
29 Gonzalo JA, Delaney T, Corcoran J, et al. Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation. J.Immunol. 2001; 166:1-5.
30 Smith KM, Brewer JM, Webb P, et al. Inducible costimulatory molecule-B7-related protein 1 interactions are important for the clonal expansion and B cell helper functions of naive, Th1, and Th2 T cells. J.Immunol. 2003; 170:2310-2315.
31 Ara G, Baher A, Storm N, et al. Potent activity of soluble B7RP-1-Fc in therapy of murine tumors in syngeneic hosts. Int. J. Cancer. 2003; 103: 501-507.
32 Wong SC, Oh E, Ng CH, et al. Impaired germinal center formation and recall T cell-dependent immune responses in mice lacking the co-stimulatory ligand B7-H2. Blood. 2003; 102:1381-1388.
33 Mak TW, Shahinian A, Yoshinaga SK, et al. Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell dependent B cell responses. Nat. Immunol. 2003; 4:765-772.
34 Mollweide A, Staege MS, Hoeschen C, et al. Only therapeutic ICOS:ICOSL blockade alleviates acute graft versus host disease. Klin Padiatr. 2009; 221: 344-50.
35 Taylor PA, Panoskaltsis-Mortari A, Freeman GJ, et al. Targeting of inducible costimulator (ICOS) expressed on alloreactive T cells down-regulates graft-versus-host disease (GVHD) and facilitates engraftment of allogeneic bone marrow (BM). Blood. 2005; 105: 3372– 3380.
36 Kosuge H, Suzuki JI, Gotoh R, et al. Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen pathway. Transplantation. 2003; 75:1374-1380.
37 Liu W, Feng W, Wang F, et al. Adenovirus-mediated ICOSIg gene transfer alleviates cardiac remodeling in experimental autoimmune myocarditis. Immunol Cell Biol. 2008; 86: 659-65.
38 Li WM, Liu W, Zhang RH, et al. Effects of adenovirus-mediated gene transferof ICOSIg fusion protein on experimental autoimmune myocarditis in Lewis rats. Zhonghua Xin Xue Guan Bing Za Zhi. 2007; 35: 28-32.
39 Sporici RA, Beswick RL, von Allmen C, et al. ICOS ligand costimulation is required for T-cell encephalitogenicity. Clin. Immunol. 2001;100:277-288.
40 Desbarats, J., Wade, T., Wade, W. F., et al. Dichotomy between naive and memory CD4+ T cell responses to Fas engagement. Proc. Natl. Acad. Sci USA. 1999; 96:8104–8109.
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42 Fossiez F, Djossou O, Chomarat P, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J. Exp. Med. 1996; 183:2593-2603.
43 Okamoto T, Saito S, Yamanaka H, et al. Expression and function of the costimulator H4/ICOS on activated T cells of patients with rheumatoid arthritis. J. Rheumatol. 2003; 30:1157-1163.
44 Kopf M, Coyle AJ, Schmitz N, et al. Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection. J. Exp. Med. 2000;192: 53-61.
45 Bonhagen K, Liesenfeld O, Stadecker MJ, et al. ICOS+ Th cells produce distinct cytokines in different mucosal immune responses. Eur. J. Immunol. 2003; 33:392-401.
46 Gonzalo JA, Delaney T, Corcoran J, et al. Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation. J.Immunol. 2001; 166:1-5.
47 Kadkhoda K, Wang S, Joyee AG, et al. Th1 Cytokine Responses Fail to Effectively Control Chlamydia Lung Infection in ICOS Ligand Knockout Mice. J. Immunol. 2010; 184: 3780-3788.
48 Bonhagen K, Liesenfeld O, Stadecker MJ, et al. ICOS+ Th cells produce distinct cytokines in different mucosal immune responses. Eur. J. Immunol. 2003;33:392-401.
49 Chen L, Ashe S, Brady WA, et al. Costimulation of antitumor immunity by the B7 counte receptor for the T lymphocyte molecules CD28 and CTLA-4. Cell. 1992; 71:1093-1102.
50 Townsend SE, Allison JP. Tumor rejection after direct costimulation of CD8 T cells by B7-transfected melanoma cells. Science. 1993; 259:368-370.
51 Leach DR, Krummel MF, Allison JP, et al. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996; 271: 1734-1736.
52 Kosuge H, Suzuki JI, Gotoh R, et al. Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen pathway. Transplantation. 2003; 75:1374-1380.
53 Xiang J. Expression of co-stimulatory 4-1BB ligand induces significant tumor regression and protective immunity. Cancer Biother. Radiopharm. 1999; 14:353-361.
54 Weinberg AD, Rivera MM, Prell R, et al. Engagement of the OX-40 receptor in vivo enhances antitumor immunity. J. Immunol.2000;30:1040-1047.
55 Liu X, Bai XF, Wen J, et al. B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8+ T lymphocytes in vivo. J. Exp. Med. 2001;194: 1339-1348.
1. Wang S ,Zhu G,Chapoval A I et al . Costimulation of T cells by B7-H2 ,a B7-like molecule that binds ICOS. Blood. 2000; 96 : 2808-2813.
2 Yoshinanga S K,Whoriskey J S ,Khare S D, et al . T-cell co-stimulation through B7RP -1 and ICOS. Nature .1999; 402: 827-832.
3 Kadkhoda K, Wang S, Joyee AG, et al. Th1 Cytokine Responses Fail to Effectively Control Chlamydia Lung Infection in ICOS Ligand Knockout Mice. J Immunol. 2010; 184: 3780 -3788.
4邱玉华,张学光等.鼠抗人CD28分子单克隆抗体的研制及生物学特性研究.细胞与分子免疫学杂志. 2001; 17: 368-370.
5 Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 2005;1:175:3-4.
6 Chung Y, Yang X, Chang SH, et al. Expression and regulation of IL-22 in the IL-17-producing CD4+ T lymphocytes. Cell Res. 2006;16: 902-907.
7 Wilson NJ, Boniface K, Chan JR, et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol. 2007; 8: 950-957.
8 Yu JJ, Gaffen SL. Interleukin-17: a novel inflammatory cytokine that bridges innate and adaptive immunity. Front Biosci. 2008; 13:170-177.
9 Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity. 2004; 21: 467-476.
1. Lanzavecchia A, Lezzi G, Viola A: From TCR engagement to T cell activation: a kinetic view of T cell behavior. Cell. 1999;96:1–4.
2. Aicher A, Hayden-Ledbetter M, Brady WA, et al. Characterization of Human Inducible Costimulator Ligand Expression and Function. J Immunol. 2000; 164:4689-4696.
3. Yoshinaga SK, Whoriskey JS, Khare SD, et al. T-cell co-stimulation through B7RP-1 and ICOS. Nature. 1999; 402:827-836.
4. Ling V, Wu PW, Finnerty HF, et al. Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor. J Immunol. 2000;164:1653-1657.
5. Swallow MM, Wallin JJ, Sha WC.B7h:a novel costimulatory homolog of B7.1 and B7.2, is induced by TNF-α. Immunity. 1999;11:423-432.
6. Richter G, Mollweide A, Uhlmann E, et al. Therapeutic Blockade of ICOS:ICOSL Interaction Alleviates Acute Graft Versus Host Disease. Blood. 2005;106:5237.
7. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immuno.l 1996;14:233–258.
8. Taylor P, Panoskaltsis-Mortari A, Freeman G, et al. The ICOS:ICOSL Costimulatory Pathway Plays an Important Role in GVHD and Bone Marrow (BM) Graft Rejection. Blood. 2004;104:591-602.
9. Yoshinaga SK, Zhang M, Pistillo J, et al. Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS. Int Immunol. 2000;12:1439-1447.
10. Wang SD, Zhu GF, Chapoval AI, et al. Costimulation of T cells by B7-H2 ,a B7-like molecule that binds ICOS. Blood. 2000;96 :2808-2813.
11. Ling V, Wu PW, Miyashiro JS, et al. expression of inducible costimulator-ligand splices variants: lymphoid regulation of mouse GL50-B and human GL50 molecules.Immunology. 2001;166:730-736.
12. Wang YG, Zhu MZ, Yu P, et al. Promoting Immune Responses by LIGHT in the Face of Abundant Regulatory T Cell Inhibition. J Immunol. 2010;184:1589– 1595.
13. Qiu YH, Sun ZW, Shi Q, et al. Apoptosis of multiple myeloma cells induced by agonist monoclonal antibody against human CD28. Cell Immunol. 2005;236:154–160.
14. Xingluo Liu XL, Bai XF, et al. B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8+ T lymphocytes in vivo. J Exp Med. 2001;194:1339-1348.
15. Iwai H, Kozono Y, Hirose S, et al. Amelioration of Collagen-Induced Arthritis by Blockade of Inducible Costimulator-B7 Homologous Protein Costimulation, J Immunol. 2002;169: 4332- 4339.
16. Kosuge H, Suzuki J, Gotoh R, et al. Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway. Transplantation. 2003;75:1374-1380.
17. Villegas EN, Lieberman LA, Mason N, et al. A role for inducible costimulatorprotein in the CD28-independent mechanism of resistance to Toxoplasma gondii. J Immunol. 2002;169:937-943.
18. Liang LD, Porter EM, Sha WC. Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be reduced by CD40 signaling. J Exp Med. 2002;196 :97-108.
19. Sporici RA, Beswick RL, von Allmen C, et al. ICOS ligand costimulation is required for T-cell encephalitogenicity. Clin Immunol. 2001;100:277-288.
20. Akbari O, Freeman GJ, Meyer EH, et al. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med. 2002;8:1024-1032.
21.邱玉华,张学光.一种显著提高小鼠腹水产量的新方法.中国免疫学杂志.1995; 11:366-372.