鼠抗人4-1BBL分子功能性单克隆抗体的研制及其生物学特性的研究
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摘要
4-1BBL(CD137L)是Ⅱ型跨膜糖蛋白分子，TNF超家族成员，表达于各种活化的抗原提呈细胞(APC)表面，如：IFN-γ活化的巨噬细胞、CD40配体活化的B细胞、单核细胞、树突状细胞(DC)、T细胞、肿瘤细胞等。编码人4-1BBL的基因位于19p13.3。其受体4-1BB(CD137)为TNF受体(TNFR)超家族成员，分子量为30KD的Ⅰ型跨膜糖蛋白表达于活化的CD4~+和CD8~+T细胞表面。编码人4-1BB的基因位于1p36。4-1BB／4-1BBL介导的共刺激信号可以诱导T细胞的活化，增殖及抗凋亡作用，同时还可维持T细胞的长期生存及提高细胞因子的分泌。4-1BB／4-1BBL可以协同CD28／B7促进T细胞IL-2的分泌，4-1BBL亦可独立于CD28传递共刺激信号联合TCR信号参与T细胞的活化。CD28／B7主要在初次免疫应答中起作用而4-1BB／4-1BBL信号在初次免疫应答的后期，二次免疫应答及免疫记忆中发挥效应。有趣的是4-1BBL还可介导的逆向共刺激信号诱导单核细胞、肿瘤细胞的增殖及促进其细胞因子的分泌。这种逆向信号可能肿瘤免疫应答中可能起至关重要的作用。4-1BBL分子的激动剂或拮抗剂的研制及其在4-1BBL／4-1BB信号传导调节作用的研究在肿瘤、移植排斥反应、自身免疫性疾病中具有重要的理论研究和应用价值。
1．本研究以人多发性骨髓瘤(multiple myeloma，MM)细胞转入4-1BBL基因细胞株XG1-4-1BBL为免疫原，免疫BALB／C小鼠，采用B淋巴细胞杂交瘤技术，将免疫后小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2／0进行细胞融合，以XG1-4-1BBL细胞株作
鼠抗人4，IBBL分子功能性单克隆抗体的研制及其生物学特性的研究
中文摘要
为阳性细胞株，以转基因细胞的母本细胞XGI作为阴性对照细胞株，经免疫荧光标
记分析及抗体分泌阳性孔内细胞的反复筛选并经多次的克隆化培养，最终获得1株
持续、稳定分泌鼠抗人4一IBBL单克隆抗体的杂交瘤细胞株，命名为IFI。经快速定
性试纸分析法鉴定，它分泌的Ig类别为IgGI。经体外长期传代培养和液氮冻存后复
苏，杂交瘤细胞生长良好，分泌抗体的性能稳定。
2.继而采用本室建立的腹水诱生和纯化方案，腹水形成的阳性率为95%，腹水的产
量平均为75mL/只小鼠。经ProteinG亲和层析法纯化抗体，纯化后腹水mAb蛋白
的平均得率为3.50mg/mL。免疫荧光法分析表明，纯化后腹水型单抗的效价为1:20()0
以上，纯化后抗体蛋白用于间接免疫荧光分析的用量为0.25一2.0 09/1 xl护细胞。
3.为了研究单克隆抗体对4一IBBL分子表达细胞的识别和结合，选择表达4一IBBL
膜分子的SUpT、Jurkat、HepGZ、Raji、Daudi、HL60、U266和新鲜分离的单核细
胞通过间接免疫荧光标记法及流式细胞仪(FCM)分析，结果显示，单克隆抗体11:1
能特异性的识别不同细胞表达的4一IBBL分子。
4.进一步中和/阻断实验中表明，IFI能特异性阻断rh4一IBBIj分子介导的协同刺激
信号，从而抑制rh4一IBBL联合CD3激发型单抗对人外周血T淋巴细胞的增殖激发
效应。
5.经过3H一TdR掺入法、细胞记数、光镜观察及FCM分析证实IFI能介导逆向共
刺激信号促进表达4一BBL的人外周血来源的单核细胞增殖(3一9倍)，其作用明显
强于th4一IBB对单核细胞促增殖效应。经IFI诱导扩增的单核细胞，经GM一CSF、IL一东
TNF一Q诱导培养后能分化发育成树突状细胞(DCs)初步分析证实该诱导的DCs不
论在表型还是在激发T细胞的功能较常规方法诱导的DCs无显著差异。同时我们还
发现IFI也能促进DCs的增殖，提示IFI有可能成为一种更有效的体外培养大量单
核细胞及DCs的诱导剂，在肿瘤免疫治疗中具有潜在的临床应用价值。更令人感兴
趣的是IFI能取代GM一CSF联合IL一4诱导单核细胞分化发育成DCs。4一IBBL的信
号传导通路及单核细胞、DCs的分化发育的机制至今尚不明确，值得进一步探讨玄，
同时运用mAb IFI我们还发现，4一1 BBL分子特征性表达于呱、MS白血病新鲜标
本细胞上，而且mAb 1 Fll一10“留ml时，即可促进天然表达4一1 BBL分子的单核细
胞来源的肿瘤胞株HL60、SHI一1(MS型白血病细胞株)及MS白血病新鲜标本细胞
鼠抗人4一1 BBL分子功能性单克隆抗体的研制及其生物学特性的研究
中文摘要
生长，且这种对肿瘤细胞生长的促进作用随培养时间及单克隆抗体浓度的增加而增
强。结果表明IFI在研究髓性白血病的生物学行为中具有重要的价值。
6，为了研究活化T细胞上4一IBBL的表达及其功能，采用常规方法成功研制了一株
抗人CD28分子的激发型单抗(1 0F3)。该单抗的亚类为IgGI，腹水形成的阳性率为
95%，腹水的产量平均为8.0 mL/只小鼠，纯化后腹水mAb蛋白的平均得率为
7.0m留mL，抗体效价在1:5000以上。该单抗可联合CD3激发型单抗活化人外周血
T细胞并促进其增殖(约5倍)。研究发现CD3+l 0F3活化的T细胞明显上调4一IBBL
的表达。提示共表达于T细胞表面的4一IBBL和4一IBB可能参与了T细胞自身的调
节，其机制有待于进一步的探讨。
综上所述，本研究成功的研制成稳定和特异分泌抗人4一IBBL及CD28的功能
性单克隆抗体的二株杂交瘤，首次证实所获的抗人4一IBBL单克隆抗体(IFI)通过
逆向信号传导，非常显著地促进人外周血单核细胞的增殖，诱导单?
Human 4-1BBL(CD137L), a type II transmembrane glycoprotein, which is encoded by a gene located at 19p13.3, belongs to TNF Ligand superfamily . 4-1BBL is expressed on many types of cells, including IFN-activated macrophages, CD40 ligand-activated B cells, dendritic cells, monocytes, T cells or tumor cells. Its receptor, 4-1 BB(CD137), a member of the TNFR superfamily, is a 30 KDa type I transmembrane glycoprotein, which is encoded by a gene located at 1p36, and expressed on activated CD4 and CD8 T cells. Ligation of 4-1BB by anti-4-1BB Abs or 4-1BBL promotes T cells activation, proliferation, cytolytic effector function, cytokine secretion, and prevents activation-induced cell death. Morever CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells. When given in conjunction with a strong signal through the TCR, 4-1BBL can endow T cells costimulation independently of the CD28 costimulatory pathway. CD28 is important for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects
T cell numbers much later in the initial response and secondary response, and is essential for the survival and/or responsiveness of the memory CD8 T cell pool. Interestingly, human 4-1BBL may be involved in reverse signaling in APC, which can induce proliferation of monocytes and some tumor cells,and cytokine production. This reverse signal may play a critical role in immune response. For these reason, blocking 4-1BB signal or 4-1BBL reverse signal could result in the immune tolerance specific to T cells and contributes to a new way for intervention of autoimmune disease, hypersensitivity and allogenetic graft rejection, paradoxically, enhancing costimulatory signals is available for antitumor immunity. Therefore,
transduction may have significant theoretic and clinical value.
In this study, a multiple myeloma (MM) cell line transfected with human 4-1BBL gene XG1-4-1BBL was used to immunize Balb/c mice. The immunized spleencytes were fused with mouse myeloma cells (SP2/0) by using polyethylene glycol (PEG), and then they were cultured in HAT selection medium. With XG1-4-1BBL cells, the hybridomas secreting specific mAbs were screened by immunoflurescence assay. Through repeatedly cloning and screening, one hybridoma cell line(1F1) continuously and steadily secreting specific anti-human 4-1BBL was obtained. This hybridoma grew well after long-term culture in vitro and storage in liquid nitrogen.
This mAb was produced by in-vivo procedures in mouse peritoneal cavity and the ascitic tker was over 1:2000 dilution by flow cytometry assay, in which every 1 X 106 cells required 0.25-2.00 microgram purified mAb. Fast-strip method analyses displayed that this mAb belonged to mouse IgG1 (1F1).
To further elucidate the recognition ability of the mAb against 4-1BBL molecule, the phenotype analysis was utilized by flow cytometry assay. The result indicated that the mAb could recognize 4-1BBL molecule expressed on SupT, Jurkat, U266, HepG2, HL60, XG1, XG6, Daudi, Raji, monocytes. These results suggested that the antigen-antibody binding 4-1BBL in this particular circumstance to expressing cells had high specificity and affinity.
The monocytes from PBMCs that naturally expressing tansmember molecule 4-1 BBL were cultured in the plates that precoated with the MAb 1F1. The effect of 1F1 on the monocytes was assessed by cell number and tritiated thymidine radioactivity counting. We demonstrated that, compared with rh4-1BB protein, the MAb 1F1 could more dramatically trigger the proliferation of monocytes from PBMCs , which in the context of GM-CSF+IL-4+TNF-a can be induced into mature dendritic cells(DCs). We also found that 1F1 could promoted proliferation of DCs. These results showed a new stratagy for obtaining a larger quantity of monocytes for inducing DCs in vitro. Very interestingly, 1F1 could replace GM-CSF, and induced monocytes into mature dendritic cells in conjugation
of IL-4 and TNF-a. The mechanism is still in darkness, therefore 1F1 may become a new tool for studying the effect of 4-lBB
1．本研究以人多发性骨髓瘤(multiple myeloma，MM)细胞转入4-1BBL基因细胞株XG1-4-1BBL为免疫原，免疫BALB／C小鼠，采用B淋巴细胞杂交瘤技术，将免疫后小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2／0进行细胞融合，以XG1-4-1BBL细胞株作
鼠抗人4，IBBL分子功能性单克隆抗体的研制及其生物学特性的研究
中文摘要
为阳性细胞株，以转基因细胞的母本细胞XGI作为阴性对照细胞株，经免疫荧光标
记分析及抗体分泌阳性孔内细胞的反复筛选并经多次的克隆化培养，最终获得1株
持续、稳定分泌鼠抗人4一IBBL单克隆抗体的杂交瘤细胞株，命名为IFI。经快速定
性试纸分析法鉴定，它分泌的Ig类别为IgGI。经体外长期传代培养和液氮冻存后复
苏，杂交瘤细胞生长良好，分泌抗体的性能稳定。
2.继而采用本室建立的腹水诱生和纯化方案，腹水形成的阳性率为95%，腹水的产
量平均为75mL/只小鼠。经ProteinG亲和层析法纯化抗体，纯化后腹水mAb蛋白
的平均得率为3.50mg/mL。免疫荧光法分析表明，纯化后腹水型单抗的效价为1:20()0
以上，纯化后抗体蛋白用于间接免疫荧光分析的用量为0.25一2.0 09/1 xl护细胞。
3.为了研究单克隆抗体对4一IBBL分子表达细胞的识别和结合，选择表达4一IBBL
膜分子的SUpT、Jurkat、HepGZ、Raji、Daudi、HL60、U266和新鲜分离的单核细
胞通过间接免疫荧光标记法及流式细胞仪(FCM)分析，结果显示，单克隆抗体11:1
能特异性的识别不同细胞表达的4一IBBL分子。
4.进一步中和/阻断实验中表明，IFI能特异性阻断rh4一IBBIj分子介导的协同刺激
信号，从而抑制rh4一IBBL联合CD3激发型单抗对人外周血T淋巴细胞的增殖激发
效应。
5.经过3H一TdR掺入法、细胞记数、光镜观察及FCM分析证实IFI能介导逆向共
刺激信号促进表达4一BBL的人外周血来源的单核细胞增殖(3一9倍)，其作用明显
强于th4一IBB对单核细胞促增殖效应。经IFI诱导扩增的单核细胞，经GM一CSF、IL一东
TNF一Q诱导培养后能分化发育成树突状细胞(DCs)初步分析证实该诱导的DCs不
论在表型还是在激发T细胞的功能较常规方法诱导的DCs无显著差异。同时我们还
发现IFI也能促进DCs的增殖，提示IFI有可能成为一种更有效的体外培养大量单
核细胞及DCs的诱导剂，在肿瘤免疫治疗中具有潜在的临床应用价值。更令人感兴
趣的是IFI能取代GM一CSF联合IL一4诱导单核细胞分化发育成DCs。4一IBBL的信
号传导通路及单核细胞、DCs的分化发育的机制至今尚不明确，值得进一步探讨玄，
同时运用mAb IFI我们还发现，4一1 BBL分子特征性表达于呱、MS白血病新鲜标
本细胞上，而且mAb 1 Fll一10“留ml时，即可促进天然表达4一1 BBL分子的单核细
胞来源的肿瘤胞株HL60、SHI一1(MS型白血病细胞株)及MS白血病新鲜标本细胞
鼠抗人4一1 BBL分子功能性单克隆抗体的研制及其生物学特性的研究
中文摘要
生长，且这种对肿瘤细胞生长的促进作用随培养时间及单克隆抗体浓度的增加而增
强。结果表明IFI在研究髓性白血病的生物学行为中具有重要的价值。
6，为了研究活化T细胞上4一IBBL的表达及其功能，采用常规方法成功研制了一株
抗人CD28分子的激发型单抗(1 0F3)。该单抗的亚类为IgGI，腹水形成的阳性率为
95%，腹水的产量平均为8.0 mL/只小鼠，纯化后腹水mAb蛋白的平均得率为
7.0m留mL，抗体效价在1:5000以上。该单抗可联合CD3激发型单抗活化人外周血
T细胞并促进其增殖(约5倍)。研究发现CD3+l 0F3活化的T细胞明显上调4一IBBL
的表达。提示共表达于T细胞表面的4一IBBL和4一IBB可能参与了T细胞自身的调
节，其机制有待于进一步的探讨。
综上所述，本研究成功的研制成稳定和特异分泌抗人4一IBBL及CD28的功能
性单克隆抗体的二株杂交瘤，首次证实所获的抗人4一IBBL单克隆抗体(IFI)通过
逆向信号传导，非常显著地促进人外周血单核细胞的增殖，诱导单?
Human 4-1BBL(CD137L), a type II transmembrane glycoprotein, which is encoded by a gene located at 19p13.3, belongs to TNF Ligand superfamily . 4-1BBL is expressed on many types of cells, including IFN-activated macrophages, CD40 ligand-activated B cells, dendritic cells, monocytes, T cells or tumor cells. Its receptor, 4-1 BB(CD137), a member of the TNFR superfamily, is a 30 KDa type I transmembrane glycoprotein, which is encoded by a gene located at 1p36, and expressed on activated CD4 and CD8 T cells. Ligation of 4-1BB by anti-4-1BB Abs or 4-1BBL promotes T cells activation, proliferation, cytolytic effector function, cytokine secretion, and prevents activation-induced cell death. Morever CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells. When given in conjunction with a strong signal through the TCR, 4-1BBL can endow T cells costimulation independently of the CD28 costimulatory pathway. CD28 is important for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects
T cell numbers much later in the initial response and secondary response, and is essential for the survival and/or responsiveness of the memory CD8 T cell pool. Interestingly, human 4-1BBL may be involved in reverse signaling in APC, which can induce proliferation of monocytes and some tumor cells,and cytokine production. This reverse signal may play a critical role in immune response. For these reason, blocking 4-1BB signal or 4-1BBL reverse signal could result in the immune tolerance specific to T cells and contributes to a new way for intervention of autoimmune disease, hypersensitivity and allogenetic graft rejection, paradoxically, enhancing costimulatory signals is available for antitumor immunity. Therefore,
transduction may have significant theoretic and clinical value.
In this study, a multiple myeloma (MM) cell line transfected with human 4-1BBL gene XG1-4-1BBL was used to immunize Balb/c mice. The immunized spleencytes were fused with mouse myeloma cells (SP2/0) by using polyethylene glycol (PEG), and then they were cultured in HAT selection medium. With XG1-4-1BBL cells, the hybridomas secreting specific mAbs were screened by immunoflurescence assay. Through repeatedly cloning and screening, one hybridoma cell line(1F1) continuously and steadily secreting specific anti-human 4-1BBL was obtained. This hybridoma grew well after long-term culture in vitro and storage in liquid nitrogen.
This mAb was produced by in-vivo procedures in mouse peritoneal cavity and the ascitic tker was over 1:2000 dilution by flow cytometry assay, in which every 1 X 106 cells required 0.25-2.00 microgram purified mAb. Fast-strip method analyses displayed that this mAb belonged to mouse IgG1 (1F1).
To further elucidate the recognition ability of the mAb against 4-1BBL molecule, the phenotype analysis was utilized by flow cytometry assay. The result indicated that the mAb could recognize 4-1BBL molecule expressed on SupT, Jurkat, U266, HepG2, HL60, XG1, XG6, Daudi, Raji, monocytes. These results suggested that the antigen-antibody binding 4-1BBL in this particular circumstance to expressing cells had high specificity and affinity.
The monocytes from PBMCs that naturally expressing tansmember molecule 4-1 BBL were cultured in the plates that precoated with the MAb 1F1. The effect of 1F1 on the monocytes was assessed by cell number and tritiated thymidine radioactivity counting. We demonstrated that, compared with rh4-1BB protein, the MAb 1F1 could more dramatically trigger the proliferation of monocytes from PBMCs , which in the context of GM-CSF+IL-4+TNF-a can be induced into mature dendritic cells(DCs). We also found that 1F1 could promoted proliferation of DCs. These results showed a new stratagy for obtaining a larger quantity of monocytes for inducing DCs in vitro. Very interestingly, 1F1 could replace GM-CSF, and induced monocytes into mature dendritic cells in conjugation
of IL-4 and TNF-a. The mechanism is still in darkness, therefore 1F1 may become a new tool for studying the effect of 4-lBB
引文
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2 Saoulli K, Lee SY, Cannons JL et al. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J Exp Med, 1998; 187(11): 1849-62.
3 Cannons J L, Choi Y, Watts TH. Role of TNF receptor-associated factor 2 and p38 mitogen-activated protein kinase activation during 4-1BB-dependent immune response. J Immunol, 2000;165(11):6193-204.
4 Eine J S, Macosko H D, Justice L et al. An inhibitor of CD28-CD80 interration impairs costimulation of human CD4 T cells. Cell Immunol, 1999;191(1):49-54.
5 Boulougouris G, Mcleod J, Patel Y I. Positive and negative regulation of human T cells activation medition by the CD28/CTLA-4 ligation(CDS0). J Immunol, 1998:3919-3925.
6 Melero I, Bach N, Hellstrom KE et al. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur J Immunol, 1998; 28(3): 1116-21.
7 DeBenedette MA, Shahinian A, Mak TW et al. Costimulation of CD28-T lymphocytes by 4-1BB ligand. J Immunol, 1997;158(2):551-9.
8 Hurtado JC, Kim SH, Pollok KE et al. Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J Immunol, 1995; 155(7):3360-7.
9 Michel, J., S. Pauly, P. Langstein, P. H. Krammer, H. Schwarz. 1999. CD137-induced apoptosis is independent of CD95. Immunology 98:42.
10 Guinn BA, DeBenedette MA, Watts TH et al. 4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine. J Immunol, 1999; 162(8): 5003-10.
11 Maliszewski C.Dendritic cells in models of tumor immunity. Role of Flt3 ligand. Pathol Biol (Paris). 2001; 49(6):481-3.
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13 Kashii Y, Giorda R,Herberman RB et al. Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells. J Immunol, 1999; 163(10):5358-66.
14 Ryan A, Wilcox, Koji Tamada, et al. Signaling Through NK Cell-Associated CD137 Promotes Both Helper Function for CD8~+ Cytolytic T Cells and Responsiveness to IL-2 But Not Cytolytic Activity. J Immunol, 2002; 169: 4230-4236.
15 Wiley SR, et al. Reverse signaling via CD30 ligand.J.immuol. 1996; 157: 3635-3639.
16 Suzuki I, et al. Maximal Proliferation of Cytotoxic T Lymphocytes Requires Reverse Signaling through Fas Ligand. J.Exp. Med, 1998; 187:123-128.
17 Watts AD, Hunt NH, Wanigasekara Y et al. A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in 'reverse signalling'. EMBO J, 1999;18(8):2119-26.
18 Joachim Langstein, Jan Michel, and Herbert Schwarz. CD137 Induces Proliferation and Endomitosis in Monocytes. Blood, Vol. 94 No. 9, 1999; 3161-3168.
19 Ryan A. Wilcox, Andrei I. Chapoval et al. Cutting Edge: Expression of Functional CD137 Receptor by Dendritic Cells. J Immunol, 2002, 168: 4262-4267.
20 Broll K, Richter G, Pauly S et al. CD137 expression in tumor vessel walls. High correlation with malignant tumors. Am J Clin Pathol, 2001; 115(4):543-9.
21 Salih HR, Kosowski SG, Haluska VF et al. Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J Immunol, 2000;165(5):2903-10.
22 Salih HR, Schmetzer HM, Burke C et al. Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J Immunol, 2001; 167(7):4059-66.
23 Yndestad A, Kristian Damas J et al. Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure.
24 Mark A. DeBenedette, Tao Wen et al. Analysis of 4-1BB Ligand (4-1BBL)-Deficient Mice and of Mice Lacking Both 4-1BBL and CD28 Reveals a Role for 4-1BBL in Skin Allograft Rejection and in the Cytotoxic T Cell Response to Influenza Virus. J Immunol, 1999, 163: 4833-4841.
25 Seko Y, Takahashi N, Oshima H et al. Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3. J Pathol 2001, Dec; 195(5):593-603.
26 Bruce R. Blazar, Byoung S et al. Ligation of 4-1BB (CDw137) Regulates Graft-Versus-Host Disease, Graft-Versus-Leukemia, and Graft Rejection in Allogeneic Bone Marrow Transplant Recipients. J Immunol, 2001, 166:3174-3183.
27 Bretscher P, Cohn M. A theory of self-nonself discrimination. Science, 1970; 169(950): 1042-9.
2 Saoulli K, Lee SY, Cannons JL et al. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J Exp Med, 1998; 187(11): 1849-62.
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8 Hurtado JC, Kim SH, Pollok KE et al. Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J Immunol, 1995; 155(7):3360-7.
9 Michel, J., S. Pauly, P. Langstein, P. H. Krammer, H. Schwarz. 1999. CD137-induced apoptosis is independent of CD95. Immunology 98:42.
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11 Maliszewski C.Dendritic cells in models of tumor immunity. Role of Flt3 ligand. Pathol Biol (Paris). 2001; 49(6):481-3.
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13 Kashii Y, Giorda R,Herberman RB et al. Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells. J Immunol, 1999; 163(10):5358-66.
14 Ryan A, Wilcox, Koji Tamada, et al. Signaling Through NK Cell-Associated CD137 Promotes Both Helper Function for CD8~+ Cytolytic T Cells and Responsiveness to IL-2 But Not Cytolytic Activity. J Immunol, 2002; 169: 4230-4236.
15 Wiley SR, et al. Reverse signaling via CD30 ligand.J.immuol. 1996; 157: 3635-3639.
16 Suzuki I, et al. Maximal Proliferation of Cytotoxic T Lymphocytes Requires Reverse Signaling through Fas Ligand. J.Exp. Med, 1998; 187:123-128.
17 Watts AD, Hunt NH, Wanigasekara Y et al. A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in 'reverse signalling'. EMBO J, 1999;18(8):2119-26.
18 Joachim Langstein, Jan Michel, and Herbert Schwarz. CD137 Induces Proliferation and Endomitosis in Monocytes. Blood, Vol. 94 No. 9, 1999; 3161-3168.
19 Ryan A. Wilcox, Andrei I. Chapoval et al. Cutting Edge: Expression of Functional CD137 Receptor by Dendritic Cells. J Immunol, 2002, 168: 4262-4267.
20 Broll K, Richter G, Pauly S et al. CD137 expression in tumor vessel walls. High correlation with malignant tumors. Am J Clin Pathol, 2001; 115(4):543-9.
21 Salih HR, Kosowski SG, Haluska VF et al. Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells. J Immunol, 2000;165(5):2903-10.
22 Salih HR, Schmetzer HM, Burke C et al. Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies. J Immunol, 2001; 167(7):4059-66.
23 Yndestad A, Kristian Damas J et al. Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure.
24 Mark A. DeBenedette, Tao Wen et al. Analysis of 4-1BB Ligand (4-1BBL)-Deficient Mice and of Mice Lacking Both 4-1BBL and CD28 Reveals a Role for 4-1BBL in Skin Allograft Rejection and in the Cytotoxic T Cell Response to Influenza Virus. J Immunol, 1999, 163: 4833-4841.
25 Seko Y, Takahashi N, Oshima H et al. Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3. J Pathol 2001, Dec; 195(5):593-603.
26 Bruce R. Blazar, Byoung S et al. Ligation of 4-1BB (CDw137) Regulates Graft-Versus-Host Disease, Graft-Versus-Leukemia, and Graft Rejection in Allogeneic Bone Marrow Transplant Recipients. J Immunol, 2001, 166:3174-3183.
27 Bretscher P, Cohn M. A theory of self-nonself discrimination. Science, 1970; 169(950): 1042-9.