CTLA4Ig基因修饰人肝细胞株L02诱导免疫耐受的实验研究
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摘要
肝细胞移植是治疗肝衰竭和代谢性肝病的重要手段之一,相对于肝脏移植,肝细胞移植具有来源较广、费用相对低廉、创伤较小等优点,可提供暂时性肝功能支持,为肝脏重建创造机会。但同肝移植一样,排斥反应影响移植肝细胞的长期存活和远期疗效。目前解决排斥反应的主要策略是使用免疫抑制剂,但此类药物毒副作用较大,需长期甚至终身使用,且增加感染和肿瘤的发生率。在此背景下,诱导移植物免疫耐受成为研究热点。
细胞毒性T淋巴细胞相关抗原4免疫球蛋白(cytotoxic T-lymphocyte associated antigen 4-immunoglobulin, CTLA4Ig)是CTLA4分子胞外功能区与免疫球蛋白IgG恒定区相结合的融合性可溶性蛋白,可与T淋巴细胞表面的CD28分子竞争性结合抗原递呈细胞(antigen presenting cell, APC)表面的B7分子,阻断T细胞活化的共刺激通路,抑制T细胞活化,诱导对特异性抗原的免疫耐受。大量实验已经证实CTLA4Ig可以有效抑制排斥反应,延长移植物存活时间,肝细胞移植中应用CTLA4Ig同样被证实可有效诱导免疫耐受。其主要应用方法为CTLA4Ig蛋白的全身应用或各种载体介导CTLA4Ig基因的全身转染。但此类方法同全身使用环孢霉素等传统免疫抑制剂一样,可能干扰机体整体免疫功能并导致非特异性免疫抑制。在此背景下,本研究利用重组腺病毒载体携带CTLA4Ig基因,在体外转染正常人肝细胞株L02,使L02细胞表达具有免疫抑制生物学活性的CTLA4Ig,以期建立一种通过移植肝细胞自身表达CTLA4Ig诱导免疫耐受的方法。实验方法和结果如下:
一、腺病毒介导CTLA4Ig基因转染的L02细胞生物学特性的研究
以重组人CTLA4Ig腺病毒载体(Ad-CTLA4Ig-EGFP)转染体外培养的正常人肝细胞株L02,观察转染效率、转染后L02细胞对CTLA4Ig的表达以及自身生物学特性的变化。结果显示:
1.自转染后6小时开始L02细胞内可见绿色荧光表达,48-72小时荧光强度达峰值,可持续较强表达2周,随后逐渐衰减,28天时尚余微弱荧光;经流式细胞仪检测,重复感染度MOI为600时,转染效率约为88.0%;免疫细胞化学及Western Blotting检测提示转染后L02细胞胞浆内有大量CTLA4Ig表达,且可分泌至细胞外。ELISA(enzyme linked immunosorbent assay)法检测转染后1周时上清中CTLA4Ig浓度达峰值,约为(16.55±1.20)ng/ml。
2.经绘制细胞生长曲线,发现转染后初期细胞生长速度稍减慢,但无统计学意义;经检测培养上清中尿素含量,发现转染后L02细胞合成分泌尿素的能力较转染前明显升高(P<0.01)。
二、CTLA4Ig基因修饰的L02细胞(CTLA4-L02)体外、体内诱导免疫耐受的研究
1. CTLA4-L02与SD大鼠淋巴细胞混合培养后,以MTT法检测淋巴细胞增殖,发现增值率明显受到抑制(P<0.05),抑制率约为36.8%;经CTLA4-L02抑制后的大鼠脾脏细胞再接受正常L02细胞刺激时仅发生轻度增殖,增值率明显低于接受Hela细胞刺激的脾细胞(P<0.01)。
2.将CTLA4-L02经脾脏注射移植给行2/3肝叶切除术的SD大鼠,术后第3、4周检测鼠肝组织可见表达人白蛋白阳性的细胞存活,而注射生理盐水及正常L02细胞的对照组为阴性;检测术后1周的大鼠外周血发现:1)转染组大鼠(注射CTLA4-L02)外周血CD4+T细胞比例及激活的CD4+T细胞(CD4+CD69+T细胞)比例均较未转染组(注射L02细胞)降低(P<0.01);2)转染组大鼠外周血IL-2水平较未转染组低(P<0.01)。
结论:
1.腺病毒载体可介导CTLA4Ig基因高效率转染人肝细胞株L02,转染后的L02细胞可在胞浆内有效表达CTLA4Ig蛋白并分泌至细胞外,表达持续时间超过4周;而自身生物学特性并未受到明显负面影响。
2.转染后的L02细胞在体外表现出明确的免疫抑制效应,可明显抑制异种的大鼠淋巴细胞增殖,诱导自身发生免疫耐受,且这种耐受具有一定程度的抗原特异性;移植给大鼠后,可抑制大鼠CD4+T细胞的活化和增殖,抑制IL-2的分泌,诱导自身在大鼠肝脏内的免疫耐受和存活。
Hepatocytes transplantation has become an important treatment of patients with end stage liver failure and cirrhosis. Potential advantages of cell transplantation include a simpler, safer, less invasive and costly procedure, and more efficient use of donor organs compared to liver transplantation for treatment of liver diseases. However, hepatocytes transplantation still face immunological processes in liver transplantation.
Cytotoxic T-lymphocyte associated with antigen 4-immunoglobulin (CTLA4-Ig) could block the CD28/B7 co-stimulating pathway, inhibit T cell activation, and thus induce the immunological tolerance to specific antigen. There are massive data indicating that tolerance of graft could be induced by systemic administration of CTLA4-Ig protein or transduction with CTLA4-Ig gene in vivo. However, systemic administration of CTLA4Ig has the potential to inhibit the immune system extensively and cause unwanted systemic adverse effects (e.g., susceptibility to infections and malignancy). In this study, we expected to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation. Main methods and results are as follows:
1. Biological characteristics of L02 cells transducted by Ad-CTLA4Ig-EGFP.
Normal liver cells L02 were transducted by Ad-CTLA4Ig-EGFP; gene transferring efficiency was detected through flow cytometry; immunocytochemistry, Western Blotting and ELISA were used to detective the expression of CTLA4Ig by L02 cells; changes in biological characteristics were observed through cell proliferation curve and urea synthesis.
1.1 Green fluorescence could be observed in the endochylema within 6 hours post-transduction, which reached the highest fluorescence intensity at 48~72 hours post-transduction, and lasted for more than 4 weeks. Gene transferring efficiency reaced 88.0% when MOI=600. Immunocytochemistry, and Western Blotting indicated that CTLA4Ig were highly expressed in cytoplasm and could be secreted to culture supernatant. The peak concentration of CTLA4 in supernatant was (16.55±1.20)ng/ml at D7 post-tansducted.
1.2 Cells growth rate stepped down after gene transfer, but with no statistical significance. However, urea synthesis was enhanced after gene transfer(P<0.01).
2. L02 modified by CTLA4Ig gene (CTLA4-L02) induced immunological tolerance both in vitro and vivo.
2.1 Proliferation of rat splenocytes was inhibited when co-cultured with CTLA4-L02 (P<0.05), inhibition rate reaced 36.8%; the inhibited splenocytes kept a low proliferation when co-cultured with normal L02 cells, but showed a high proliferation when co-cultured with Hela cells(P<0.01).
2.2 CTLA4-L02 cells were splenically injected into SD rats which were performed 2/3 hepatic lobectomy, survival L02 cells which expressed human ALB could be observed in experimental group at the 3rd and 4th week post-transplantation, while none was detected in control group. Percentage of CD4+ and CD4+CD69+ T cells in transducted group were (24.48±1.65)% and (45.11±2.83)%, which were significantly decreased compared to (35.30±2.05)% and (55.22±2.79)% in control group at day 7 post-transplantation (P<0.01). Meanwhile, IL-2 level was also in lower level in transducted group (204.15±1.10 pg/ml) than that in control group (223.52±2.76 pg/ml, P<0.01).
Conclusions
1. CTLA4Ig gene could be transferred into L02 cells high efficaciously mediated by adenovirus vector. After transducted, L02 cells could express CTLA4Ig in cytoplasm and secreted to culture supernatant, which lasted for more than 4 weeks. No obvious negative change of biological characteristics was observed.
2. CTLA4-L02 cells appeared immunological inhibition activity in vitro, which could inhibit the proliferation of rat splenocytes, and induce immunological tolerance, and this tolerance appeared antigen specificity to some extent. After transplanted into rat, CTLA4-L02 could inhibit the proliferation and activation of CD4+T cells, decrease the level of IL-2 and induce immunological tolerance of itselves to survive in rat liver.
细胞毒性T淋巴细胞相关抗原4免疫球蛋白(cytotoxic T-lymphocyte associated antigen 4-immunoglobulin, CTLA4Ig)是CTLA4分子胞外功能区与免疫球蛋白IgG恒定区相结合的融合性可溶性蛋白,可与T淋巴细胞表面的CD28分子竞争性结合抗原递呈细胞(antigen presenting cell, APC)表面的B7分子,阻断T细胞活化的共刺激通路,抑制T细胞活化,诱导对特异性抗原的免疫耐受。大量实验已经证实CTLA4Ig可以有效抑制排斥反应,延长移植物存活时间,肝细胞移植中应用CTLA4Ig同样被证实可有效诱导免疫耐受。其主要应用方法为CTLA4Ig蛋白的全身应用或各种载体介导CTLA4Ig基因的全身转染。但此类方法同全身使用环孢霉素等传统免疫抑制剂一样,可能干扰机体整体免疫功能并导致非特异性免疫抑制。在此背景下,本研究利用重组腺病毒载体携带CTLA4Ig基因,在体外转染正常人肝细胞株L02,使L02细胞表达具有免疫抑制生物学活性的CTLA4Ig,以期建立一种通过移植肝细胞自身表达CTLA4Ig诱导免疫耐受的方法。实验方法和结果如下:
一、腺病毒介导CTLA4Ig基因转染的L02细胞生物学特性的研究
以重组人CTLA4Ig腺病毒载体(Ad-CTLA4Ig-EGFP)转染体外培养的正常人肝细胞株L02,观察转染效率、转染后L02细胞对CTLA4Ig的表达以及自身生物学特性的变化。结果显示:
1.自转染后6小时开始L02细胞内可见绿色荧光表达,48-72小时荧光强度达峰值,可持续较强表达2周,随后逐渐衰减,28天时尚余微弱荧光;经流式细胞仪检测,重复感染度MOI为600时,转染效率约为88.0%;免疫细胞化学及Western Blotting检测提示转染后L02细胞胞浆内有大量CTLA4Ig表达,且可分泌至细胞外。ELISA(enzyme linked immunosorbent assay)法检测转染后1周时上清中CTLA4Ig浓度达峰值,约为(16.55±1.20)ng/ml。
2.经绘制细胞生长曲线,发现转染后初期细胞生长速度稍减慢,但无统计学意义;经检测培养上清中尿素含量,发现转染后L02细胞合成分泌尿素的能力较转染前明显升高(P<0.01)。
二、CTLA4Ig基因修饰的L02细胞(CTLA4-L02)体外、体内诱导免疫耐受的研究
1. CTLA4-L02与SD大鼠淋巴细胞混合培养后,以MTT法检测淋巴细胞增殖,发现增值率明显受到抑制(P<0.05),抑制率约为36.8%;经CTLA4-L02抑制后的大鼠脾脏细胞再接受正常L02细胞刺激时仅发生轻度增殖,增值率明显低于接受Hela细胞刺激的脾细胞(P<0.01)。
2.将CTLA4-L02经脾脏注射移植给行2/3肝叶切除术的SD大鼠,术后第3、4周检测鼠肝组织可见表达人白蛋白阳性的细胞存活,而注射生理盐水及正常L02细胞的对照组为阴性;检测术后1周的大鼠外周血发现:1)转染组大鼠(注射CTLA4-L02)外周血CD4+T细胞比例及激活的CD4+T细胞(CD4+CD69+T细胞)比例均较未转染组(注射L02细胞)降低(P<0.01);2)转染组大鼠外周血IL-2水平较未转染组低(P<0.01)。
结论:
1.腺病毒载体可介导CTLA4Ig基因高效率转染人肝细胞株L02,转染后的L02细胞可在胞浆内有效表达CTLA4Ig蛋白并分泌至细胞外,表达持续时间超过4周;而自身生物学特性并未受到明显负面影响。
2.转染后的L02细胞在体外表现出明确的免疫抑制效应,可明显抑制异种的大鼠淋巴细胞增殖,诱导自身发生免疫耐受,且这种耐受具有一定程度的抗原特异性;移植给大鼠后,可抑制大鼠CD4+T细胞的活化和增殖,抑制IL-2的分泌,诱导自身在大鼠肝脏内的免疫耐受和存活。
Hepatocytes transplantation has become an important treatment of patients with end stage liver failure and cirrhosis. Potential advantages of cell transplantation include a simpler, safer, less invasive and costly procedure, and more efficient use of donor organs compared to liver transplantation for treatment of liver diseases. However, hepatocytes transplantation still face immunological processes in liver transplantation.
Cytotoxic T-lymphocyte associated with antigen 4-immunoglobulin (CTLA4-Ig) could block the CD28/B7 co-stimulating pathway, inhibit T cell activation, and thus induce the immunological tolerance to specific antigen. There are massive data indicating that tolerance of graft could be induced by systemic administration of CTLA4-Ig protein or transduction with CTLA4-Ig gene in vivo. However, systemic administration of CTLA4Ig has the potential to inhibit the immune system extensively and cause unwanted systemic adverse effects (e.g., susceptibility to infections and malignancy). In this study, we expected to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation. Main methods and results are as follows:
1. Biological characteristics of L02 cells transducted by Ad-CTLA4Ig-EGFP.
Normal liver cells L02 were transducted by Ad-CTLA4Ig-EGFP; gene transferring efficiency was detected through flow cytometry; immunocytochemistry, Western Blotting and ELISA were used to detective the expression of CTLA4Ig by L02 cells; changes in biological characteristics were observed through cell proliferation curve and urea synthesis.
1.1 Green fluorescence could be observed in the endochylema within 6 hours post-transduction, which reached the highest fluorescence intensity at 48~72 hours post-transduction, and lasted for more than 4 weeks. Gene transferring efficiency reaced 88.0% when MOI=600. Immunocytochemistry, and Western Blotting indicated that CTLA4Ig were highly expressed in cytoplasm and could be secreted to culture supernatant. The peak concentration of CTLA4 in supernatant was (16.55±1.20)ng/ml at D7 post-tansducted.
1.2 Cells growth rate stepped down after gene transfer, but with no statistical significance. However, urea synthesis was enhanced after gene transfer(P<0.01).
2. L02 modified by CTLA4Ig gene (CTLA4-L02) induced immunological tolerance both in vitro and vivo.
2.1 Proliferation of rat splenocytes was inhibited when co-cultured with CTLA4-L02 (P<0.05), inhibition rate reaced 36.8%; the inhibited splenocytes kept a low proliferation when co-cultured with normal L02 cells, but showed a high proliferation when co-cultured with Hela cells(P<0.01).
2.2 CTLA4-L02 cells were splenically injected into SD rats which were performed 2/3 hepatic lobectomy, survival L02 cells which expressed human ALB could be observed in experimental group at the 3rd and 4th week post-transplantation, while none was detected in control group. Percentage of CD4+ and CD4+CD69+ T cells in transducted group were (24.48±1.65)% and (45.11±2.83)%, which were significantly decreased compared to (35.30±2.05)% and (55.22±2.79)% in control group at day 7 post-transplantation (P<0.01). Meanwhile, IL-2 level was also in lower level in transducted group (204.15±1.10 pg/ml) than that in control group (223.52±2.76 pg/ml, P<0.01).
Conclusions
1. CTLA4Ig gene could be transferred into L02 cells high efficaciously mediated by adenovirus vector. After transducted, L02 cells could express CTLA4Ig in cytoplasm and secreted to culture supernatant, which lasted for more than 4 weeks. No obvious negative change of biological characteristics was observed.
2. CTLA4-L02 cells appeared immunological inhibition activity in vitro, which could inhibit the proliferation of rat splenocytes, and induce immunological tolerance, and this tolerance appeared antigen specificity to some extent. After transplanted into rat, CTLA4-L02 could inhibit the proliferation and activation of CD4+T cells, decrease the level of IL-2 and induce immunological tolerance of itselves to survive in rat liver.
引文
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1. Janeway C A, Jr., Bottomly K. Signals and signs for lymphocyte responses[J]. Cell. 1994 Jan 28;76(2):275-285.
2. Guerder S, Flavell R A. Costimulation in tolerance and autoimmunity[J]. Int Rev Immunol. 1995;13(2):135-146.
3. Lenschow D J, Walunas T L, Bluestone J A. CD28/B7 system of T cell costimulation[J]. Annu Rev Immunol. 1996;14(233-258.
4. June C H, Ledbetter J A, Linsley P S, et al. Role of the CD28 receptor in T-cell activation[J]. Immunol Today. 1990 Jun;11(6):211-216.
5. Waterhouse P, Penninger J M, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4[J]. Science. 1995 Nov 10;270(5238):985-988.
6. Sayegh M H, Turka L A. The role of T-cell costimulatory activation pathways in transplant rejection[J]. N Engl J Med. 1998 Jun 18;338(25):1813-1821.
7. Reiser H, Stadecker M J. Costimulatory B7 molecules in the pathogenesis of infectious and autoimmune diseases[J]. N Engl J Med. 1996 Oct 31;335(18):1369-1377.
8. Sharpe A H, Freeman G J. The B7-CD28 superfamily[J]. Nat Rev Immunol. 2002 Feb;2(2):116-126.
9. Sansom D M, Manzotti C N, Zheng Y. What's the difference between CD80 and CD86?[J]. Trends Immunol. 2003 Jun;24(6):314-319.
10. Gause W C, Halvorson M J, Lu P, et al. The function of costimulatory molecules and the development of IL-4-producing T cells[J]. Immunol Today. 1997 Mar;18(3): 115-120.
11. Brunet J F, Denizot F, Luciani M F, et al. A new member of the immunoglobulin superfamily--CTLA-4[J]. Nature. 1987 Jul 16-22;328(6127):267-270.
12. Dariavach P, Mattei M G, Golstein P, et al. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains[J]. Eur J Immunol. 1988 Dec;18(12):1901-1905.
13. Ling V, Wu P W, Finnerty H F, et al. Complete sequence determination of the mouse and human CTLA4 gene loci: cross-species DNA sequence similarity beyond exon borders[J]. Genomics. 1999 Sep 15;60(3):341-355.
14. Lindsten T, Lee K P, Harris E S, et al. Characterization of CTLA-4 structure and expression on human T cells[J]. J Immunol. 1993 Oct 1;151(7):3489-3499.
15. Linsley P S, Nadler S G, Bajorath J, et al. Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4 (CTLA-4). A disulfide-linked homodimer binds two CD86 molecules[J]. J Biol Chem. 1995 Jun 23;270(25):15417-15424.
16. Darlington P J, Kirchhof M G, Criado G, et al. Hierarchical regulation of CTLA-4 dimer-based lattice formation and its biological relevance for T cell inactivation[J]. J Immunol. 2005 Jul 15;175(2):996-1004.
17. Ostrov D A, Shi W, Schwartz J C, et al. Structure of murine CTLA-4 and its role in modulating T cell responsiveness[J]. Science. 2000 Oct 27;290(5492):816-819.
18. Metzler W J, Bajorath J, Fenderson W, et al. Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28[J]. Nat Struct Biol. 1997 Jul;4(7):527-531.
19. Baroja M L, Luxenberg D, Chau T, et al. The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation[J]. J Immunol. 2000 Jan 1;164(1):49-55.
20. Baroja M L, Vijayakrishnan L, Bettelli E, et al. Inhibition of CTLA-4 function by the regulatory subunit of serine/threonine phosphatase 2A[J]. J Immunol. 2002 May 15;168(10):5070-5078.
21. Thompson C B, Allison J P. The emerging role of CTLA-4 as an immune attenuator[J]. Immunity. 1997 Oct;7(4):445-450.
22. Collins A V, Brodie D W, Gilbert R J, et al. The interaction properties of costimulatory molecules revisited[J]. Immunity. 2002 Aug;17(2):201-210.
23. van der Merwe P A, Bodian D L, Daenke S, et al. CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics[J]. J Exp Med. 1997 Feb 3;185(3):393-403.
24. van der Merwe P A, Davis S J. Molecular interactions mediating T cell antigenrecognition[J]. Annu Rev Immunol. 2003;21(659-684.
25. Stamper C C, Zhang Y, Tobin J F, et al. Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses[J]. Nature. 2001 Mar 29;410(6828):608-611.
26. 0Zhang X, Schwartz J C, Almo S C, et al. Crystal structure of the receptor-binding domain of human B7-2: insights into organization and signaling[J]. Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2586-2591.
27. Harper K, Balzano C, Rouvier E, et al. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location[J]. J Immunol. 1991 Aug 1;147(3):1037-1044.
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11. Lu S, Yu Y, Gao Y, et al. Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats[J]. Hepatobiliary Pancreat Dis Int. 2008 Jun;7(3):258-263.
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26. Kita Y, Li X K, Ohba M, et al. Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene[J]. Transplantation. 1999 Sep 27;68(6):758-766.
27. Kita Y, Nogimura H, Ida M, et al. Time course of gene expression after the injection of adenoviral vectors containing CTLA4IG gene[J]. Transplant Proc. 2004 Oct;36(8): 2443-2445.
28. Benigni A, Tomasoni S, Turka L A, et al. Adeno-associated virus-mediated CTLA4Ig gene transfer protects MHC-mismatched renal allografts from chronic rejection[J]. J Am Soc Nephrol. 2006 Jun;17(6):1665-1672.
29. Miao G, Ito T, Uchikoshi F, et al. Development of donor-specific immunoregulatory T-cells after local CTLA4Ig gene transfer to pancreatic allograft[J]. Transplantation. 2004 Jul 15;78(1):59-64.
30.代飞,吴军,许建中, et al.基因修饰人骨髓间充质干细胞作为骨组织工程种子细胞异种移植的实验研究[J].中华创伤杂志. 2005;21(11):838-844.
31.朱斌,杨甲梅,钱其军, et al.同种肝移植大鼠转染融合基因人CTLA4-Ig抑制急性排斥反应[J].中华器官移植杂志. 2005;26(4):199-202.
32.莫朝晖,王维,刘涛, et al. AAV体外介导hCTLA4-Ig在新生猪胰岛细胞中的表达与生物学活性[J].中南大学学报(医学版). 2007;32(1):36-40.
1. Janeway C A, Jr., Bottomly K. Signals and signs for lymphocyte responses[J]. Cell. 1994 Jan 28;76(2):275-285.
2. Guerder S, Flavell R A. Costimulation in tolerance and autoimmunity[J]. Int Rev Immunol. 1995;13(2):135-146.
3. Lenschow D J, Walunas T L, Bluestone J A. CD28/B7 system of T cell costimulation[J]. Annu Rev Immunol. 1996;14(233-258.
4. June C H, Ledbetter J A, Linsley P S, et al. Role of the CD28 receptor in T-cell activation[J]. Immunol Today. 1990 Jun;11(6):211-216.
5. Waterhouse P, Penninger J M, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4[J]. Science. 1995 Nov 10;270(5238):985-988.
6. Sayegh M H, Turka L A. The role of T-cell costimulatory activation pathways in transplant rejection[J]. N Engl J Med. 1998 Jun 18;338(25):1813-1821.
7. Reiser H, Stadecker M J. Costimulatory B7 molecules in the pathogenesis of infectious and autoimmune diseases[J]. N Engl J Med. 1996 Oct 31;335(18):1369-1377.
8. Sharpe A H, Freeman G J. The B7-CD28 superfamily[J]. Nat Rev Immunol. 2002 Feb;2(2):116-126.
9. Sansom D M, Manzotti C N, Zheng Y. What's the difference between CD80 and CD86?[J]. Trends Immunol. 2003 Jun;24(6):314-319.
10. Gause W C, Halvorson M J, Lu P, et al. The function of costimulatory molecules and the development of IL-4-producing T cells[J]. Immunol Today. 1997 Mar;18(3): 115-120.
11. Brunet J F, Denizot F, Luciani M F, et al. A new member of the immunoglobulin superfamily--CTLA-4[J]. Nature. 1987 Jul 16-22;328(6127):267-270.
12. Dariavach P, Mattei M G, Golstein P, et al. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains[J]. Eur J Immunol. 1988 Dec;18(12):1901-1905.
13. Ling V, Wu P W, Finnerty H F, et al. Complete sequence determination of the mouse and human CTLA4 gene loci: cross-species DNA sequence similarity beyond exon borders[J]. Genomics. 1999 Sep 15;60(3):341-355.
14. Lindsten T, Lee K P, Harris E S, et al. Characterization of CTLA-4 structure and expression on human T cells[J]. J Immunol. 1993 Oct 1;151(7):3489-3499.
15. Linsley P S, Nadler S G, Bajorath J, et al. Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4 (CTLA-4). A disulfide-linked homodimer binds two CD86 molecules[J]. J Biol Chem. 1995 Jun 23;270(25):15417-15424.
16. Darlington P J, Kirchhof M G, Criado G, et al. Hierarchical regulation of CTLA-4 dimer-based lattice formation and its biological relevance for T cell inactivation[J]. J Immunol. 2005 Jul 15;175(2):996-1004.
17. Ostrov D A, Shi W, Schwartz J C, et al. Structure of murine CTLA-4 and its role in modulating T cell responsiveness[J]. Science. 2000 Oct 27;290(5492):816-819.
18. Metzler W J, Bajorath J, Fenderson W, et al. Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28[J]. Nat Struct Biol. 1997 Jul;4(7):527-531.
19. Baroja M L, Luxenberg D, Chau T, et al. The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation[J]. J Immunol. 2000 Jan 1;164(1):49-55.
20. Baroja M L, Vijayakrishnan L, Bettelli E, et al. Inhibition of CTLA-4 function by the regulatory subunit of serine/threonine phosphatase 2A[J]. J Immunol. 2002 May 15;168(10):5070-5078.
21. Thompson C B, Allison J P. The emerging role of CTLA-4 as an immune attenuator[J]. Immunity. 1997 Oct;7(4):445-450.
22. Collins A V, Brodie D W, Gilbert R J, et al. The interaction properties of costimulatory molecules revisited[J]. Immunity. 2002 Aug;17(2):201-210.
23. van der Merwe P A, Bodian D L, Daenke S, et al. CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics[J]. J Exp Med. 1997 Feb 3;185(3):393-403.
24. van der Merwe P A, Davis S J. Molecular interactions mediating T cell antigenrecognition[J]. Annu Rev Immunol. 2003;21(659-684.
25. Stamper C C, Zhang Y, Tobin J F, et al. Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses[J]. Nature. 2001 Mar 29;410(6828):608-611.
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