激发型CD40单克隆抗体联合特异性细胞毒T淋巴细胞对B细胞淋巴瘤的免疫治疗作用

Therapeutic effect of agonistic CD40 monoclonai antibody combined with CTL on hu-SCID mouse B iymphoma model

目的 研究激发型CD40单克隆抗体5C11联合特异性细胞毒T淋巴细胞（CTL）对B细胞淋巴瘤的免疫治疗作用。方法 人B细胞淋巴瘤Daudi细胞株经5C11刺激24或48h，Annexin V／PI结合实验测定凋亡率，流式细胞术检测Fas表达率。外周血单个核细胞来源的树突状细胞（DC），经凋亡Daudi细胞负载、5C11诱导成熟后，与自体T细胞混合培养，激发特异性CTL，JAM法检测5C11、CTL或5C11联合CTL对Daudi细胞的杀伤效应。皮下注射Daudi细胞，建立人源化SCID小鼠B细胞淋巴瘤模型；接种肿瘤细胞1周、3周后，分别经腹腔注射5C11、CTL、5C11＋CTL进行治疗，通过肿瘤生长曲线、荷瘤小鼠生存期评价疗效。结果 5C11作用后，Daudi细胞表面Fas的表达率显著上调，但细胞凋亡率没有明显改变。特异性CTL能有效杀伤靶细胞，且与5C11联合应用后，Daudi细胞DNA片段形成率显著增高，8h为71．9％±4．0％，12h达82．6％±4．4％。经5C11、CTL、5C11联合CTL治疗后，荷瘤小鼠体内肿瘤生长速度均显著减缓，而且在低肿瘤负荷小鼠中，CTL、5C11联合CTL治疗分别取得了30．0％和70．0％的完全缓解率，与对照组相比，各治疗组小鼠生存期显著延长。结论 激发型CD40单抗5C11可显著上调细胞表面Fas的表达，增强Daudi细胞对诱导凋亡的敏感性，从而促进肿瘤特异性CTL的体外杀伤和体内治疗效应。

Objective To study the therapeutic effect of agonistic CD40 monoclonal antibody combined with tumor specific cytotoxic T lymphocyte （CTL） on B lymphoma. Methods Human B lymphoma cell line, Daudi cells, were cultured with CD40 mAb （5C11 ） for 24 and 48 hours, respectively. Annexin V/PI-binding assay was employed to analyze apoptosis, and FCM to analyze Fas （CD95） expression. Human peripheral monocyte-derived DC were loaded with apoptotic Daudi cells and stimulated by 5C11 for further maturation. Tumor specific CTL were generated in vitro by co-culture of mature DC with autologous T lymphocytes. DNA fragmentations of Daudi cells treated with 5C11, CTL or 5C11 combined with CTL were determined by JAM assay. To establish the B lymphoma model, Daudi cells were subcutaneously injected into humanized SCID mice （hu-SCID）. 1 or 3 weeks after tumor transfer, tumorbearing mice were respectively treated with 5C11, CTL, 5C11 combined with CTL by intraperitoneal injection. Tumor volume in differently treated mice was measured every week after therapy, and the survival of tumor-bearing mice was recorded. Results 5C11 significantly up-regulated FAS expression in Daudi cells, but had no significant effect on apoptosis rate of Daudi cells. Tumor-specific CTL could effectively kill Daudi cells. Fragmentation of Daudi cells co-cultured with CTL was remarkably enhanced by combination with 5C11. Tumor growth in hu-SCID mice was apparently delayed by treatment with 5C11, CTL, or 5C11 combined with CTL. Moreover, minimal tumor burden mice got 30. 0% or 70. 0% complete remission （CR）, respectively, when received CTL treatment or combination treatment of 5C11 with CTL, and the lifespan of tumor bearing mice was also prolonged significantly. Conclusion 5C11 may enhance the sensitivity of Daudi cells to apoptosis by up-regulation of Fas expression and promote cytotoxicity of CTL in vitro and therapeutic effect in vivo.