AChR-Fc融合蛋白靶向BCR治疗重症肌无力的基础研究
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摘要
重症肌无力(Myasthenia gravis, MG)是由自身抗体介导、T细胞参与、补体依赖的自身免疫性疾病,其致病性自身抗体以及病理作用靶点明确,动物模型易建立,是研究自身免疫性疾病治疗手段的理想模型之一。位于神经肌肉接头突触后膜的乙酰胆碱受体(acetylcholine receptor, AChR)是MG致病性自身抗体攻击的靶点,患者体内的抗AChR抗体与AChR结合后,在补体等因素协同下破坏突触后膜的AChR,临床出现横纹肌收缩无力等症状,严重者累及呼吸肌出现肌无力危象而死亡。目前主要采用胆碱酯酶抑制剂和免疫抑制剂进行治疗,但治疗效果仍然不理想。因此,人们一直在探索一种特异而有效的治疗手段。
近年来,越来越多的证据表明B淋巴细胞在免疫应答中具有更为广泛的作用,而不仅仅是被动接受信号刺激而分泌抗体。B细胞还可以递呈抗原、调节抗原递呈细胞和T细胞的功能、分泌多种细胞因子,并表达以往认为只限于其他细胞类型的多种受体/配体。而当机体免疫系统对自身成分失耐受时,病理性B细胞的作用就更加复杂,也更加重要,尤其在以自身抗体为主要效应机制的体液自身免疫性疾病中。因此,以B细胞为靶细胞进行免疫治疗将会有效控制多种自身免疫性疾病。
鉴于AChRAb是MG的重要致病因子,如果能从源头上清除分泌AChRAb的B细胞,将会有效控制病情。然而,目前尚无特异性清除AChR反应性B细胞的手段。本研究拟将AChRα亚单位胞外段基因与Fc基因连接,表达AChR-Fc融合蛋白,其AChRα亚单位胞外段特异性结合AChR反应性B细胞,同时Fc段结合该B细胞表面抑制性受体FcγRⅡB,抑制B细胞活化、促进其凋亡,或Fc结合于单核-巨噬细胞、补体表面的Fcγ受体,介导单核-巨噬细胞或补体对该致病B细胞的特异性杀伤。我们利用AChRAb杂交瘤细胞分析融合蛋白的结合、抑制作用以及其介导的细胞毒作用,同时用重组表达的AChRα亚单位胞外段(extracellular domain,ECD)蛋白免疫Lewis大鼠制备实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis, EAMG)动物模型,分别通过体外以及在体研究,观察融合蛋白对AChR反应性B细胞的作用。
一、AChR-Fc融合蛋白的表达、纯化和鉴定
以AChRα亚单位基因片段为模板,通过PCR的方法扩增AChRα亚单位胞外段全长基因片段(Hα1-210),将其插入含有CMV启动子、小鼠Kappa链先导序列以及人IgG1从铰链区到CH3的基因片段的真核表达载体pAN1782中,构建重组表达载体pAN-Hα1-210;瞬时转染CHO-K1细胞后取上清,ELISA和Western blot鉴定AChR-Fc融合蛋白的表达;G418筛选转染后细胞,建立稳定表达细胞系;Protein A亲和层析柱纯化表达的融合蛋白,SDS-PAGE进一步分析AChR-Fc融合蛋白的表达和纯度。
二、体外细胞系实验分析AChR-Fc融合蛋白对分泌AChRAb杂交瘤细胞的结合和抑制作用
用含10%胎牛血清(fetal calf serum, FCS)RPMI1640适应性培养分泌AChRAb的杂交瘤细胞系TIB175,利用FITC-anti rat CD32和FITC-anti ratκ+λ轻链抗体,流式细胞术(flow cytometry, FCM)分析表明杂交瘤细胞表面表达BCR和FcγRⅡB;将融合蛋白同杂交瘤细胞共孵育之后,利用Biotin-anti humanγchain的抗体结合FITC-Avidin显示融合蛋白对杂交瘤细胞具有较强的结合能力。
将融合蛋白以不同浓度(100μg/ml, 50μg/ml, 10μg/ml以及0μg/ml)分别与培养的杂交瘤细胞TIB175共孵育,以人IgG作为对照,48小时后活细胞计数结果显示融合蛋白作用组活细胞所占比例呈剂量依赖方式下降;AnnexinV/PI染色,FCM分析结果显示融合蛋白可显著促进杂交瘤细胞的凋亡;Tunel染色、激光扫描共聚焦显微镜(laser scanning confocal microscope, LSCM)观察进一步证实了细胞的凋亡;在孵育结束前16h加入BrdU,孵育结束后用BrdU试剂盒进行检测,FCM分析结果显示融合蛋白治疗组BrdU阳性细胞数明显低于其他治疗组;细胞周期结果也显示融合蛋白可使细胞发生G1期阻滞,从而抑制细胞的增生。此外,融合蛋白与杂交瘤细胞共孵育后加入人外周血单核细胞作为效应细胞、补体作为效应分子,FCM分析表明融合蛋白可通过其Fc段介导效应细胞和补体特异性诱导杂交瘤细胞的凋亡。
三、分析AChR-Fc融合蛋白对EAMG动物模型AChR反应性B细胞的作用
用重组表达的ECD蛋白混合完全弗氏佐剂免疫Lewis大鼠构建EAMG动物模型。分离大鼠脾细胞,体外同融合蛋白孵育后,FCM分析结果显示融合蛋白和脾细胞有较高的亲和力;ELISPOT检测发现融合蛋白作用组AChR反应性B细胞数目呈剂量依赖式下降,将融合蛋白和脾细胞作用48h后,FCM结果显示融合蛋白在体外可诱导CD19+B细胞的凋亡。将融合蛋白以及人IgG尾静脉注射EAMG大鼠,对照组给予PBS,每周3次,共4周进行治疗。每周留取血清标本,ELISA检测发现融合蛋白治疗组血清中抗体滴度明显下降。进一步用ELISPOT分析发现融合蛋白治疗组脾脏中AChR反应性B细胞数清除率明显增高。
本研究成功表达了AChR-Fc融合蛋白,并通过体内外实验初步分析了融合蛋白对AChR反应性B细胞的作用,为进一步研究融合蛋白对MG的治疗作用奠定了基础。本研究首次提出靶向清除自身反应性B细胞的新策略,有望建立特异性治疗自身免疫病的新平台。
Myasthenia gravis is an autoimmune disease mediated by autoantibody, participated by T cells and dependent by complement. It has provided a model for studying other antibody-mediated autoimmune disorders, because the pathogenic target as well as antuantibody is clearly defined and animal model is easy to be constructed. Acetylcholine receptor (AChR) at the neuromuscular junction is the binding target of pathogenic autoantibody. This in combination with complement results in the loss of the AChR and skeletal muscle weakness, which can be life-threatening when respiratory muscle is involved. Current treatments mainly include acetylcholinesterase inhibitors and immunosuppresants, but the radical cure still can’t be achieved. Therefore, investigators are searching a more efficient and specific therapy.
In recent years, data have emerged suggesting that B lymphocytes play a broader role in immune responses and are not merely the passive recipients of signals that result in the production of antibody. Along with their traditional roles as precursors of antibody-producing plasma cells, B cells have also been found to present antigen, regulate antigen presenting cells (APCs) and T cell functions, produce cytokines, and express receptor/ligand pairs that previously had been thought to be restricted to other cell types. The role of pathogenic B cells becomes more important and complex when the breakdown of self-tolerance, especially in humoral-mediated autoimmune disease. Therefore, therapies that target B cells may be a valuable therapeutic approach for many autoimmune diseases.
Considering the critical role of AChRAb in MG, it will be possible to cure the disease if we can eliminate the root cause-AChRAb producing B cells. However, there is no strategy of specifically eliminating AChR reactive B cells to date. Our aim was to design a protein that consists of the extracellular domain of AChRαsubunit (Hα1–210) and the CH2 and CH3 domains of the human IgG1 heavy chain. This fusion protein is expected to selectively kill AChR-specific B cells by cross-linking the B cell receptor (BCR) that has an identical specificity with that of the secreted antibody and inhibitory receptor FcγRⅡB on the surface of B cells. Besides that, Fc fragment can bind Fc receptor of mononuclear macrophage and complements, and mediate ADCC and cell lysis function. We detected the binding ability and cytotoxicity of AChR-Fc fusion protein in an AChRAb-reactive hybridoma cell line in vitro. In additon, we construct EAMG by immunized Lewis rat with ECD protein and study the effects of fusion protein to AChR reactive B cells in MG.
1. Expression, purification and characterization of AChR-Fc fusion protein
The extracellular domain of the human AChRα1-subunit (Hα1-210) was amplified by PCR with the plasmid of AChRα1-subunit as template and then inser ted into pAN1782 mammalian expression vector with a cytomegalovirus promoter, a murine immunoglobulinκ-chain leader sequence and the human genomic IgGγ1 constant region from the hinge to the end of CH3. Recombinate expression plasmid was constructed and transient transfected into CHO-K1 cells. ELISA and Western blot analysis demonstrated the expression of AChR-Fc fusion protein in the culture supernatant. The CHO-K1 cells stably expressing AChR-Fc fusion protein were obtained after selecting by G418. Then the AChR-Fc fusion protein was purified by Protein A affinity chromatograph method. SDS-PAGE further confirmed the expression and purity of the purified protein.
2. In vitro binding ability and cytotoxicity assay of AChR-Fc fusion protein on hybridoma cells
TIB175 hybridoma cells were cultivated with complex medium RPMI-1640 supplemented with 10% (v/v) heat-inactivated FCS. Hybridoma cell surface were examined to express BCR and FcγRⅡB by using FITC-conjugated mouse anti-rat CD32 and monoclonal anti-ratκ+λlight chains Ab via flow cytometry analysis. Hybridoma cells were incubated with AChR-Fc fusion protein, FCM analysis showed AChR-Fc fusion protein showed a higher binding capacity to hybridoma cells using FITC-anti humanγchain mAb.
The hybridoma cells were incubated with serial dilutions of AChR-Fc or human IgG at different concentrations: 100μg/ml, 50μg/ml, 10μg/ml and medium alone. After 48 h incubation, cell numbers count demonstrated the rate of vial cells was decreased with a dose-dependent way in fusion protein-treated group; Cells were simultaneously double stained with annexin V-?uorescein isothiocyanate (FITC) and PI. FCM analysis showed AChR-Fc can induce the apoptosis of hybridoma cells. Tunel staining and laser scanning confocal 11 microscope (LSCM) further detected the apoptosis; BrdU was added at 16 h before cell harvesting, and at the end of incubation FCM analysis demonstrated BrdU positive cells in fusion protein-treated group were lower than that of other groups. The result of cell cycle showed fusion protein can inhibit the proliferation of cells by G1 phase blockage. In addition, peripheral blood mononuclear cell (PBMC) as effector cells and complement as effector molecule were added to co-incubation system of fusion protein and hybridoma cells, FCM analysis demonstrated fusion protein facilitated PBMC and complement-induced cell apoptosis through Fc region.
3. The effects of AChR-Fc fusion protein to AChR reactive B cells in EAMG animal model
Recombinant expression ECD protein of human muscle AChR, emulsified in complete Freund’s adjuvant immunized female Lewis rats to induce EAMG animal model. Spleen cells were isolated and incubated with AChR-Fc fusion protein in vitro. ELISPOT assay showed incubation with AChR-Fc fusion protein resulted in a dose-dependent reduction in spleen cells of EAMG. FCM analysis of spleen cells after incubation with fusion protein for 48h showed fusion protein can lead to the apoptosis of CD19+B cells. Rats were treated with AChR-Fc fusion protein or human IgG or PBS by vena caudalis administration for 4 weeks, 3 times per week. Sera were obtained every week and detected by ELISA assay. Treatment with fusion protein resulted in a sharp reduction of level of IgG anti-AChR. ELISPOT analysis showed reduction in the number of AChR-reactive B cells in AChR-Fc treatment Splenocytes from EAMG rats.
Our study expressed successfully AChR-Fc fusion protein and analyzed the effects of the fusion protein to AChR reactive B cells through in vivo as well as in vitro study, which may lay a foundation to further study the efficacy of the fusion protein to MG. Such a new strategy that target elimination autoreactive B cells may provide a platform for the treatment of autoimmune diseases.
近年来,越来越多的证据表明B淋巴细胞在免疫应答中具有更为广泛的作用,而不仅仅是被动接受信号刺激而分泌抗体。B细胞还可以递呈抗原、调节抗原递呈细胞和T细胞的功能、分泌多种细胞因子,并表达以往认为只限于其他细胞类型的多种受体/配体。而当机体免疫系统对自身成分失耐受时,病理性B细胞的作用就更加复杂,也更加重要,尤其在以自身抗体为主要效应机制的体液自身免疫性疾病中。因此,以B细胞为靶细胞进行免疫治疗将会有效控制多种自身免疫性疾病。
鉴于AChRAb是MG的重要致病因子,如果能从源头上清除分泌AChRAb的B细胞,将会有效控制病情。然而,目前尚无特异性清除AChR反应性B细胞的手段。本研究拟将AChRα亚单位胞外段基因与Fc基因连接,表达AChR-Fc融合蛋白,其AChRα亚单位胞外段特异性结合AChR反应性B细胞,同时Fc段结合该B细胞表面抑制性受体FcγRⅡB,抑制B细胞活化、促进其凋亡,或Fc结合于单核-巨噬细胞、补体表面的Fcγ受体,介导单核-巨噬细胞或补体对该致病B细胞的特异性杀伤。我们利用AChRAb杂交瘤细胞分析融合蛋白的结合、抑制作用以及其介导的细胞毒作用,同时用重组表达的AChRα亚单位胞外段(extracellular domain,ECD)蛋白免疫Lewis大鼠制备实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis, EAMG)动物模型,分别通过体外以及在体研究,观察融合蛋白对AChR反应性B细胞的作用。
一、AChR-Fc融合蛋白的表达、纯化和鉴定
以AChRα亚单位基因片段为模板,通过PCR的方法扩增AChRα亚单位胞外段全长基因片段(Hα1-210),将其插入含有CMV启动子、小鼠Kappa链先导序列以及人IgG1从铰链区到CH3的基因片段的真核表达载体pAN1782中,构建重组表达载体pAN-Hα1-210;瞬时转染CHO-K1细胞后取上清,ELISA和Western blot鉴定AChR-Fc融合蛋白的表达;G418筛选转染后细胞,建立稳定表达细胞系;Protein A亲和层析柱纯化表达的融合蛋白,SDS-PAGE进一步分析AChR-Fc融合蛋白的表达和纯度。
二、体外细胞系实验分析AChR-Fc融合蛋白对分泌AChRAb杂交瘤细胞的结合和抑制作用
用含10%胎牛血清(fetal calf serum, FCS)RPMI1640适应性培养分泌AChRAb的杂交瘤细胞系TIB175,利用FITC-anti rat CD32和FITC-anti ratκ+λ轻链抗体,流式细胞术(flow cytometry, FCM)分析表明杂交瘤细胞表面表达BCR和FcγRⅡB;将融合蛋白同杂交瘤细胞共孵育之后,利用Biotin-anti humanγchain的抗体结合FITC-Avidin显示融合蛋白对杂交瘤细胞具有较强的结合能力。
将融合蛋白以不同浓度(100μg/ml, 50μg/ml, 10μg/ml以及0μg/ml)分别与培养的杂交瘤细胞TIB175共孵育,以人IgG作为对照,48小时后活细胞计数结果显示融合蛋白作用组活细胞所占比例呈剂量依赖方式下降;AnnexinV/PI染色,FCM分析结果显示融合蛋白可显著促进杂交瘤细胞的凋亡;Tunel染色、激光扫描共聚焦显微镜(laser scanning confocal microscope, LSCM)观察进一步证实了细胞的凋亡;在孵育结束前16h加入BrdU,孵育结束后用BrdU试剂盒进行检测,FCM分析结果显示融合蛋白治疗组BrdU阳性细胞数明显低于其他治疗组;细胞周期结果也显示融合蛋白可使细胞发生G1期阻滞,从而抑制细胞的增生。此外,融合蛋白与杂交瘤细胞共孵育后加入人外周血单核细胞作为效应细胞、补体作为效应分子,FCM分析表明融合蛋白可通过其Fc段介导效应细胞和补体特异性诱导杂交瘤细胞的凋亡。
三、分析AChR-Fc融合蛋白对EAMG动物模型AChR反应性B细胞的作用
用重组表达的ECD蛋白混合完全弗氏佐剂免疫Lewis大鼠构建EAMG动物模型。分离大鼠脾细胞,体外同融合蛋白孵育后,FCM分析结果显示融合蛋白和脾细胞有较高的亲和力;ELISPOT检测发现融合蛋白作用组AChR反应性B细胞数目呈剂量依赖式下降,将融合蛋白和脾细胞作用48h后,FCM结果显示融合蛋白在体外可诱导CD19+B细胞的凋亡。将融合蛋白以及人IgG尾静脉注射EAMG大鼠,对照组给予PBS,每周3次,共4周进行治疗。每周留取血清标本,ELISA检测发现融合蛋白治疗组血清中抗体滴度明显下降。进一步用ELISPOT分析发现融合蛋白治疗组脾脏中AChR反应性B细胞数清除率明显增高。
本研究成功表达了AChR-Fc融合蛋白,并通过体内外实验初步分析了融合蛋白对AChR反应性B细胞的作用,为进一步研究融合蛋白对MG的治疗作用奠定了基础。本研究首次提出靶向清除自身反应性B细胞的新策略,有望建立特异性治疗自身免疫病的新平台。
Myasthenia gravis is an autoimmune disease mediated by autoantibody, participated by T cells and dependent by complement. It has provided a model for studying other antibody-mediated autoimmune disorders, because the pathogenic target as well as antuantibody is clearly defined and animal model is easy to be constructed. Acetylcholine receptor (AChR) at the neuromuscular junction is the binding target of pathogenic autoantibody. This in combination with complement results in the loss of the AChR and skeletal muscle weakness, which can be life-threatening when respiratory muscle is involved. Current treatments mainly include acetylcholinesterase inhibitors and immunosuppresants, but the radical cure still can’t be achieved. Therefore, investigators are searching a more efficient and specific therapy.
In recent years, data have emerged suggesting that B lymphocytes play a broader role in immune responses and are not merely the passive recipients of signals that result in the production of antibody. Along with their traditional roles as precursors of antibody-producing plasma cells, B cells have also been found to present antigen, regulate antigen presenting cells (APCs) and T cell functions, produce cytokines, and express receptor/ligand pairs that previously had been thought to be restricted to other cell types. The role of pathogenic B cells becomes more important and complex when the breakdown of self-tolerance, especially in humoral-mediated autoimmune disease. Therefore, therapies that target B cells may be a valuable therapeutic approach for many autoimmune diseases.
Considering the critical role of AChRAb in MG, it will be possible to cure the disease if we can eliminate the root cause-AChRAb producing B cells. However, there is no strategy of specifically eliminating AChR reactive B cells to date. Our aim was to design a protein that consists of the extracellular domain of AChRαsubunit (Hα1–210) and the CH2 and CH3 domains of the human IgG1 heavy chain. This fusion protein is expected to selectively kill AChR-specific B cells by cross-linking the B cell receptor (BCR) that has an identical specificity with that of the secreted antibody and inhibitory receptor FcγRⅡB on the surface of B cells. Besides that, Fc fragment can bind Fc receptor of mononuclear macrophage and complements, and mediate ADCC and cell lysis function. We detected the binding ability and cytotoxicity of AChR-Fc fusion protein in an AChRAb-reactive hybridoma cell line in vitro. In additon, we construct EAMG by immunized Lewis rat with ECD protein and study the effects of fusion protein to AChR reactive B cells in MG.
1. Expression, purification and characterization of AChR-Fc fusion protein
The extracellular domain of the human AChRα1-subunit (Hα1-210) was amplified by PCR with the plasmid of AChRα1-subunit as template and then inser ted into pAN1782 mammalian expression vector with a cytomegalovirus promoter, a murine immunoglobulinκ-chain leader sequence and the human genomic IgGγ1 constant region from the hinge to the end of CH3. Recombinate expression plasmid was constructed and transient transfected into CHO-K1 cells. ELISA and Western blot analysis demonstrated the expression of AChR-Fc fusion protein in the culture supernatant. The CHO-K1 cells stably expressing AChR-Fc fusion protein were obtained after selecting by G418. Then the AChR-Fc fusion protein was purified by Protein A affinity chromatograph method. SDS-PAGE further confirmed the expression and purity of the purified protein.
2. In vitro binding ability and cytotoxicity assay of AChR-Fc fusion protein on hybridoma cells
TIB175 hybridoma cells were cultivated with complex medium RPMI-1640 supplemented with 10% (v/v) heat-inactivated FCS. Hybridoma cell surface were examined to express BCR and FcγRⅡB by using FITC-conjugated mouse anti-rat CD32 and monoclonal anti-ratκ+λlight chains Ab via flow cytometry analysis. Hybridoma cells were incubated with AChR-Fc fusion protein, FCM analysis showed AChR-Fc fusion protein showed a higher binding capacity to hybridoma cells using FITC-anti humanγchain mAb.
The hybridoma cells were incubated with serial dilutions of AChR-Fc or human IgG at different concentrations: 100μg/ml, 50μg/ml, 10μg/ml and medium alone. After 48 h incubation, cell numbers count demonstrated the rate of vial cells was decreased with a dose-dependent way in fusion protein-treated group; Cells were simultaneously double stained with annexin V-?uorescein isothiocyanate (FITC) and PI. FCM analysis showed AChR-Fc can induce the apoptosis of hybridoma cells. Tunel staining and laser scanning confocal 11 microscope (LSCM) further detected the apoptosis; BrdU was added at 16 h before cell harvesting, and at the end of incubation FCM analysis demonstrated BrdU positive cells in fusion protein-treated group were lower than that of other groups. The result of cell cycle showed fusion protein can inhibit the proliferation of cells by G1 phase blockage. In addition, peripheral blood mononuclear cell (PBMC) as effector cells and complement as effector molecule were added to co-incubation system of fusion protein and hybridoma cells, FCM analysis demonstrated fusion protein facilitated PBMC and complement-induced cell apoptosis through Fc region.
3. The effects of AChR-Fc fusion protein to AChR reactive B cells in EAMG animal model
Recombinant expression ECD protein of human muscle AChR, emulsified in complete Freund’s adjuvant immunized female Lewis rats to induce EAMG animal model. Spleen cells were isolated and incubated with AChR-Fc fusion protein in vitro. ELISPOT assay showed incubation with AChR-Fc fusion protein resulted in a dose-dependent reduction in spleen cells of EAMG. FCM analysis of spleen cells after incubation with fusion protein for 48h showed fusion protein can lead to the apoptosis of CD19+B cells. Rats were treated with AChR-Fc fusion protein or human IgG or PBS by vena caudalis administration for 4 weeks, 3 times per week. Sera were obtained every week and detected by ELISA assay. Treatment with fusion protein resulted in a sharp reduction of level of IgG anti-AChR. ELISPOT analysis showed reduction in the number of AChR-reactive B cells in AChR-Fc treatment Splenocytes from EAMG rats.
Our study expressed successfully AChR-Fc fusion protein and analyzed the effects of the fusion protein to AChR reactive B cells through in vivo as well as in vitro study, which may lay a foundation to further study the efficacy of the fusion protein to MG. Such a new strategy that target elimination autoreactive B cells may provide a platform for the treatment of autoimmune diseases.
引文
1. Lipsky, P.E., Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat Immunol, 2001. 2(9): p. 764-6.
2. Mease, P.J., B cell-targeted therapy in autoimmune disease: rationale, mechanisms, and clinical application. J Rheumatol, 2008. 35(7): p. 1245-55.
3. Stubgen, J.P., B cell-targeted therapy with rituximab and autoimmune neuromuscular disorders. J Neuroimmunol, 2008. 204(1-2): p. 1-12.
4. Kao, I. and D.B. Drachman, Thymic muscle cells bear acetylcholine receptors: possible relation to myasthenia gravis. Science, 1977. 195(4273): p. 74-5.
5. Spuler, S., et al., Myogenesis in thymic transplants in the severe combined immunodeficient mouse model of myasthenia gravis. Differentiation of thymic myoid cells into striated muscle cells. Am J Pathol, 1994. 145(4): p. 766-70.
6. Matsumoto, M.Y., et al., Thymic myoid cells as a myasthenogenic antigen and antigen-presenting cells. J Neuroimmunol, 2004. 150(1-2): p. 80-7.
7. Hohlfeld, R. and H. Wekerle, Reflections on the "intrathymic pathogenesis" of myasthenia gravis. J Neuroimmunol, 2008. 201-202: p. 21-7.
8.张勇,李柱一,宿长军等,树突状细胞及相关细胞因子在重症肌无力患者胸腺中的变化.中国神经免疫学和神经病学杂志, 2005. 12(2): p. 70-72.
9. Garcia, Y.R., J.C. Pothitakis, and K.A. Krolick, Myocyte production of nitric oxide in response to AChR-reactive antibodies in two inbred rat strains may influence disease outcome in experimental myasthenia gravis. Clin Immunol, 2003. 106(2): p. 116-26.
10. Roxanis, I., et al., Thymic myoid cells and germinal center formation in myasthenia gravis; possible roles in pathogenesis. J Neuroimmunol, 2002. 125(1-2): p. 185-97.
11. Rapoport, B., S. Portolano, and S.M. McLachlan, Combinatorial libraries: new insights into human organ-specific autoantibodies. Immunol Today, 1995. 16(1): p. 43-9.
12. Hohlfeld, R., et al., Autoimmune human T lymphocytes specific for acetylcholine receptor. Nature, 1984. 310(5974): p. 244-6.
13. Wang, Z.Y., et al., T cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis. J Neuroimmunol, 2000. 108(1-2): p. 29-39.
14. Araga, S., et al., A peptide vaccine that prevents experimental autoimmune myasthenia gravis by specifically blocking T cell help. Faseb J, 2000. 14(1): p. 185-96.
15. Melms, A., et al., Thymus in myasthenia gravis. Isolation of T-lymphocyte lines specific for the nicotinic acetylcholine receptor from thymuses of myasthenic patients. J Clin Invest, 1988. 81(3): p. 902-8.
16. Wang, H.B., et al., Role for interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis. Clin Immunol, 2000. 95(2): p. 156-62.
17. Sun, Y., et al., Increase of circulating CD4+CD25+ T cells in myasthenia gravis patients with stability and thymectomy. Clin Immunol, 2004. 112(3): p. 284-9.
18.金伯泉主编,细胞和分子免疫学. 2001,北京:科学出版社.
19. Lin, F., et al., Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies. Immunology, 2001. 104(2):p. 215-25.
20. Tuzun, E., et al., Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis. J Immunol, 2003. 171(7): p. 3847-54.
21. Kaminski, H.J., et al., Susceptibility of ocular tissues to autoimmune diseases. Ann N Y Acad Sci, 2003. 998: p. 362-74.
22. Kaminski, H.J., et al., Complement regulators in extraocular muscle and experimental autoimmune myasthenia gravis. Exp Neurol, 2004. 189(2): p. 333-42.
23.刘睿,李柱一,王桂平等,大鼠E AMG模型眼外肌下调差异基因的初步筛选中国临床神经科学, 2004. 12(3): p. 223-225.
24. Lin, F., et al., Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection. J Clin Invest, 2002. 110(9): p. 1269-74.
25. Kaminski, H.J., et al., Deficiency of decay accelerating factor and CD59 leads to crisis in experimental myasthenia. Exp Neurol, 2006. 202(2): p. 287-93.
26. Kadowaki, N., Immunotherapy exploiting the versatility of dendritic cells. Ther Apher Dial, 2003. 7(3): p. 312-7.
27. Nagane, Y., et al., Dendritic cells in hyperplastic thymuses from patients with myasthenia gravis. Muscle Nerve, 2003. 27(5): p. 582-9.
28. Yarilin, D., et al., Dendritic cells exposed in vitro to TGF-beta1 ameliorate experimental autoimmune myasthenia gravis. Clin Exp Immunol, 2002. 127(2): p. 214-9.
29. Duan, R.S., et al., Protective potential of experimental autoimmune myasthenia gravis in Lewis rats by IL-10-modified dendritic cells.Neurobiol Dis, 2004. 16(2): p. 461-7.
30. Link, H. and B.G. Xiao, Rat models as tool to develop new immunotherapies. Immunol Rev, 2001. 184: p. 117-28.
31. Yi, H.J., et al., Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor. Mol Immunol, 2008. 46(1): p. 192-201.
32. Richman, D.P. and M.A. Agius, Treatment of autoimmune myasthenia gravis. Neurology, 2003. 61(12): p. 1652-61.
33. Vincent, A., et al., Seronegative myasthenia gravis. Evidence for plasma factor(s) interfering with acetylcholine receptor function. Ann N Y Acad Sci, 1993. 681: p. 529-38.
34. Hoch, W., et al., Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Nat Med, 2001. 7(3): p. 365-8.
35. McConville, J., et al., Detection and characterization of MuSK antibodies in seronegative myasthenia gravis. Ann Neurol, 2004. 55(4): p. 580-4.
36. Lederman, S., et al., The central role of the CD40-ligand and CD40 pathway in T-lymphocyte-mediated differentiation of B lymphocytes. Curr Opin Hematol, 1996. 3(1): p. 77-86.
37. Diehl, L., et al., The role of CD40 in peripheral T cell tolerance and immunity. J Mol Med, 2000. 78(7): p. 363-71.
38. Reiser, H. and M.J. Stadecker, Costimulatory B7 molecules in the pathogenesis of infectious and autoimmune diseases. N Engl J Med, 1996. 335(18): p. 1369-77.
39. Mihara, M., et al., CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J Clin Invest, 2000. 106(1): p.91-101.
40. Davidson, A., et al., Co-stimulatory blockade in the treatment of murine systemic lupus erythematosus (SLE). Ann N Y Acad Sci, 2003. 987: p. 188-98.
41. Prud'homme, G.J., Y. Chang, and X. Li, Immunoinhibitory DNA vaccine protects against autoimmune diabetes through cDNA encoding a selective CTLA-4 (CD152) ligand. Hum Gene Ther, 2002. 13(3): p. 395-406.
42. Wang, X., et al., Effects of anti-CD154 treatment on B cells in murine systemic lupus erythematosus. Arthritis Rheum, 2003. 48(2): p. 495-506.
43. Durie, F.H., et al., Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science, 1993. 261(5126): p. 1328-30.
44. Gerritse, K., et al., CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci U S A, 1996. 93(6): p. 2499-504.
45. Durie, F.H., et al., Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft-vs-host disease. J Clin Invest, 1994. 94(3): p. 1333-8.
46. Huang, W., et al., The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum, 2002. 46(6): p. 1554-62.
47. Kalled, S.L., A.H. Cutler, and L.C. Burkly, Apoptosis and altered dendritic cell homeostasis in lupus nephritis are limited by anti-CD154 treatment. J Immunol, 2001. 167(3): p. 1740-7.
48. Boon, L., et al., Preclinical assessment of anti-CD40 Mab 5D12 in cynomolgus monkeys. Toxicology, 2002. 174(1): p. 53-65.
49. Reff, M.E., et al., Depletion of B cells in vivo by a chimeric mouse humanmonoclonal antibody to CD20. Blood, 1994. 83(2): p. 435-45.
50. Bournia, V.K., et al., Recent advances in the treatment of systemic sclerosis. Clin Rev Allergy Immunol, 2009. 36(2-3): p. 176-200.
51. Oligino, T.J. and S.A. Dalrymple, Targeting B cells for the treatment of rheumatoid arthritis. Arthritis Res Ther, 2003. 5 Suppl 4: p. S7-11.
52. Sabahi, R. and J.H. Anolik, B-cell-targeted therapy for systemic lupus erythematosus. Drugs, 2006. 66(15): p. 1933-48.
53. Kurosaki, T., Paradox of B cell-targeted therapies. J Clin Invest, 2008. 118(10): p. 3260-3.
54. Proby, C.M., et al., Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris. Br J Dermatol, 2000. 142(2): p. 321-30.
55. Reiners, K.S., et al., Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen. Immunology, 2004. 112(2): p. 228-36.
56. Nimmerjahn, F. and J.V. Ravetch, Fcgamma receptors as regulators of immune responses. Nat Rev Immunol, 2008. 8(1): p. 34-47.
57. Nimmerjahn, F., Activating and inhibitory FcgammaRs in autoimmune disorders. Springer Semin Immunopathol, 2006. 28(4): p. 305-19.
58. Fukuyama, H., F. Nimmerjahn, and J.V. Ravetch, The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat Immunol, 2005. 6(1): p. 99-106.
59. Tchorbanov, A.I., et al., Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice. Eur J Immunol, 2007. 37(12): p. 3587-96.
60. Mihaylova, N., et al., Selective silencing of disease-associatedB-lymphocytes by chimeric molecules targeting their Fc gamma IIb receptor. Int Immunol, 2008. 20(2): p. 165-75.
61. Weinstein, E., et al., B-cell biology. Rheum Dis Clin North Am, 2004. 30(1): p. 159-74.
62. Constant, S.L., B lymphocytes as antigen-presenting cells for CD4+ T cell priming in vivo. J Immunol, 1999. 162(10): p. 5695-703.
63. Carson, D.A., P.P. Chen, and T.J. Kipps, New roles for rheumatoid factor. J Clin Invest, 1991. 87(2): p. 379-83.
64. Edwards, J.C., et al., Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med, 2004. 350(25): p. 2572-81.
65. Garcia-Carrasco, M., et al., Use of rituximab in patients with systemic lupus erythematosus: an update. Autoimmun Rev, 2009. 8(4): p. 343-8.
66. Matsushita, T., et al., Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest, 2008. 118(10): p. 3420-30.
67. Mauri, C. and M.R. Ehrenstein, The 'short' history of regulatory B cells. Trends Immunol, 2008. 29(1): p. 34-40.
68. Li, J., et al., Inhibitory IgG receptor FcgammaRIIB fails to inhibit experimental autoimmune myasthenia gravis pathogenesis. J Neuroimmunol, 2008. 194(1-2): p. 44-53.
69. Zocher, M., et al., Specific depletion of autoreactive B lymphocytes by a recombinant fusion protein in vitro and in vivo. Int Immunol, 2003. 15(7): p. 789-96.
2. Mease, P.J., B cell-targeted therapy in autoimmune disease: rationale, mechanisms, and clinical application. J Rheumatol, 2008. 35(7): p. 1245-55.
3. Stubgen, J.P., B cell-targeted therapy with rituximab and autoimmune neuromuscular disorders. J Neuroimmunol, 2008. 204(1-2): p. 1-12.
4. Kao, I. and D.B. Drachman, Thymic muscle cells bear acetylcholine receptors: possible relation to myasthenia gravis. Science, 1977. 195(4273): p. 74-5.
5. Spuler, S., et al., Myogenesis in thymic transplants in the severe combined immunodeficient mouse model of myasthenia gravis. Differentiation of thymic myoid cells into striated muscle cells. Am J Pathol, 1994. 145(4): p. 766-70.
6. Matsumoto, M.Y., et al., Thymic myoid cells as a myasthenogenic antigen and antigen-presenting cells. J Neuroimmunol, 2004. 150(1-2): p. 80-7.
7. Hohlfeld, R. and H. Wekerle, Reflections on the "intrathymic pathogenesis" of myasthenia gravis. J Neuroimmunol, 2008. 201-202: p. 21-7.
8.张勇,李柱一,宿长军等,树突状细胞及相关细胞因子在重症肌无力患者胸腺中的变化.中国神经免疫学和神经病学杂志, 2005. 12(2): p. 70-72.
9. Garcia, Y.R., J.C. Pothitakis, and K.A. Krolick, Myocyte production of nitric oxide in response to AChR-reactive antibodies in two inbred rat strains may influence disease outcome in experimental myasthenia gravis. Clin Immunol, 2003. 106(2): p. 116-26.
10. Roxanis, I., et al., Thymic myoid cells and germinal center formation in myasthenia gravis; possible roles in pathogenesis. J Neuroimmunol, 2002. 125(1-2): p. 185-97.
11. Rapoport, B., S. Portolano, and S.M. McLachlan, Combinatorial libraries: new insights into human organ-specific autoantibodies. Immunol Today, 1995. 16(1): p. 43-9.
12. Hohlfeld, R., et al., Autoimmune human T lymphocytes specific for acetylcholine receptor. Nature, 1984. 310(5974): p. 244-6.
13. Wang, Z.Y., et al., T cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis. J Neuroimmunol, 2000. 108(1-2): p. 29-39.
14. Araga, S., et al., A peptide vaccine that prevents experimental autoimmune myasthenia gravis by specifically blocking T cell help. Faseb J, 2000. 14(1): p. 185-96.
15. Melms, A., et al., Thymus in myasthenia gravis. Isolation of T-lymphocyte lines specific for the nicotinic acetylcholine receptor from thymuses of myasthenic patients. J Clin Invest, 1988. 81(3): p. 902-8.
16. Wang, H.B., et al., Role for interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis. Clin Immunol, 2000. 95(2): p. 156-62.
17. Sun, Y., et al., Increase of circulating CD4+CD25+ T cells in myasthenia gravis patients with stability and thymectomy. Clin Immunol, 2004. 112(3): p. 284-9.
18.金伯泉主编,细胞和分子免疫学. 2001,北京:科学出版社.
19. Lin, F., et al., Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies. Immunology, 2001. 104(2):p. 215-25.
20. Tuzun, E., et al., Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis. J Immunol, 2003. 171(7): p. 3847-54.
21. Kaminski, H.J., et al., Susceptibility of ocular tissues to autoimmune diseases. Ann N Y Acad Sci, 2003. 998: p. 362-74.
22. Kaminski, H.J., et al., Complement regulators in extraocular muscle and experimental autoimmune myasthenia gravis. Exp Neurol, 2004. 189(2): p. 333-42.
23.刘睿,李柱一,王桂平等,大鼠E AMG模型眼外肌下调差异基因的初步筛选中国临床神经科学, 2004. 12(3): p. 223-225.
24. Lin, F., et al., Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection. J Clin Invest, 2002. 110(9): p. 1269-74.
25. Kaminski, H.J., et al., Deficiency of decay accelerating factor and CD59 leads to crisis in experimental myasthenia. Exp Neurol, 2006. 202(2): p. 287-93.
26. Kadowaki, N., Immunotherapy exploiting the versatility of dendritic cells. Ther Apher Dial, 2003. 7(3): p. 312-7.
27. Nagane, Y., et al., Dendritic cells in hyperplastic thymuses from patients with myasthenia gravis. Muscle Nerve, 2003. 27(5): p. 582-9.
28. Yarilin, D., et al., Dendritic cells exposed in vitro to TGF-beta1 ameliorate experimental autoimmune myasthenia gravis. Clin Exp Immunol, 2002. 127(2): p. 214-9.
29. Duan, R.S., et al., Protective potential of experimental autoimmune myasthenia gravis in Lewis rats by IL-10-modified dendritic cells.Neurobiol Dis, 2004. 16(2): p. 461-7.
30. Link, H. and B.G. Xiao, Rat models as tool to develop new immunotherapies. Immunol Rev, 2001. 184: p. 117-28.
31. Yi, H.J., et al., Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor. Mol Immunol, 2008. 46(1): p. 192-201.
32. Richman, D.P. and M.A. Agius, Treatment of autoimmune myasthenia gravis. Neurology, 2003. 61(12): p. 1652-61.
33. Vincent, A., et al., Seronegative myasthenia gravis. Evidence for plasma factor(s) interfering with acetylcholine receptor function. Ann N Y Acad Sci, 1993. 681: p. 529-38.
34. Hoch, W., et al., Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Nat Med, 2001. 7(3): p. 365-8.
35. McConville, J., et al., Detection and characterization of MuSK antibodies in seronegative myasthenia gravis. Ann Neurol, 2004. 55(4): p. 580-4.
36. Lederman, S., et al., The central role of the CD40-ligand and CD40 pathway in T-lymphocyte-mediated differentiation of B lymphocytes. Curr Opin Hematol, 1996. 3(1): p. 77-86.
37. Diehl, L., et al., The role of CD40 in peripheral T cell tolerance and immunity. J Mol Med, 2000. 78(7): p. 363-71.
38. Reiser, H. and M.J. Stadecker, Costimulatory B7 molecules in the pathogenesis of infectious and autoimmune diseases. N Engl J Med, 1996. 335(18): p. 1369-77.
39. Mihara, M., et al., CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J Clin Invest, 2000. 106(1): p.91-101.
40. Davidson, A., et al., Co-stimulatory blockade in the treatment of murine systemic lupus erythematosus (SLE). Ann N Y Acad Sci, 2003. 987: p. 188-98.
41. Prud'homme, G.J., Y. Chang, and X. Li, Immunoinhibitory DNA vaccine protects against autoimmune diabetes through cDNA encoding a selective CTLA-4 (CD152) ligand. Hum Gene Ther, 2002. 13(3): p. 395-406.
42. Wang, X., et al., Effects of anti-CD154 treatment on B cells in murine systemic lupus erythematosus. Arthritis Rheum, 2003. 48(2): p. 495-506.
43. Durie, F.H., et al., Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science, 1993. 261(5126): p. 1328-30.
44. Gerritse, K., et al., CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci U S A, 1996. 93(6): p. 2499-504.
45. Durie, F.H., et al., Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft-vs-host disease. J Clin Invest, 1994. 94(3): p. 1333-8.
46. Huang, W., et al., The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum, 2002. 46(6): p. 1554-62.
47. Kalled, S.L., A.H. Cutler, and L.C. Burkly, Apoptosis and altered dendritic cell homeostasis in lupus nephritis are limited by anti-CD154 treatment. J Immunol, 2001. 167(3): p. 1740-7.
48. Boon, L., et al., Preclinical assessment of anti-CD40 Mab 5D12 in cynomolgus monkeys. Toxicology, 2002. 174(1): p. 53-65.
49. Reff, M.E., et al., Depletion of B cells in vivo by a chimeric mouse humanmonoclonal antibody to CD20. Blood, 1994. 83(2): p. 435-45.
50. Bournia, V.K., et al., Recent advances in the treatment of systemic sclerosis. Clin Rev Allergy Immunol, 2009. 36(2-3): p. 176-200.
51. Oligino, T.J. and S.A. Dalrymple, Targeting B cells for the treatment of rheumatoid arthritis. Arthritis Res Ther, 2003. 5 Suppl 4: p. S7-11.
52. Sabahi, R. and J.H. Anolik, B-cell-targeted therapy for systemic lupus erythematosus. Drugs, 2006. 66(15): p. 1933-48.
53. Kurosaki, T., Paradox of B cell-targeted therapies. J Clin Invest, 2008. 118(10): p. 3260-3.
54. Proby, C.M., et al., Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris. Br J Dermatol, 2000. 142(2): p. 321-30.
55. Reiners, K.S., et al., Selective killing of B-cell hybridomas targeting proteinase 3, Wegener's autoantigen. Immunology, 2004. 112(2): p. 228-36.
56. Nimmerjahn, F. and J.V. Ravetch, Fcgamma receptors as regulators of immune responses. Nat Rev Immunol, 2008. 8(1): p. 34-47.
57. Nimmerjahn, F., Activating and inhibitory FcgammaRs in autoimmune disorders. Springer Semin Immunopathol, 2006. 28(4): p. 305-19.
58. Fukuyama, H., F. Nimmerjahn, and J.V. Ravetch, The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat Immunol, 2005. 6(1): p. 99-106.
59. Tchorbanov, A.I., et al., Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice. Eur J Immunol, 2007. 37(12): p. 3587-96.
60. Mihaylova, N., et al., Selective silencing of disease-associatedB-lymphocytes by chimeric molecules targeting their Fc gamma IIb receptor. Int Immunol, 2008. 20(2): p. 165-75.
61. Weinstein, E., et al., B-cell biology. Rheum Dis Clin North Am, 2004. 30(1): p. 159-74.
62. Constant, S.L., B lymphocytes as antigen-presenting cells for CD4+ T cell priming in vivo. J Immunol, 1999. 162(10): p. 5695-703.
63. Carson, D.A., P.P. Chen, and T.J. Kipps, New roles for rheumatoid factor. J Clin Invest, 1991. 87(2): p. 379-83.
64. Edwards, J.C., et al., Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med, 2004. 350(25): p. 2572-81.
65. Garcia-Carrasco, M., et al., Use of rituximab in patients with systemic lupus erythematosus: an update. Autoimmun Rev, 2009. 8(4): p. 343-8.
66. Matsushita, T., et al., Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest, 2008. 118(10): p. 3420-30.
67. Mauri, C. and M.R. Ehrenstein, The 'short' history of regulatory B cells. Trends Immunol, 2008. 29(1): p. 34-40.
68. Li, J., et al., Inhibitory IgG receptor FcgammaRIIB fails to inhibit experimental autoimmune myasthenia gravis pathogenesis. J Neuroimmunol, 2008. 194(1-2): p. 44-53.
69. Zocher, M., et al., Specific depletion of autoreactive B lymphocytes by a recombinant fusion protein in vitro and in vivo. Int Immunol, 2003. 15(7): p. 789-96.