凋亡肿瘤细胞负载的树突状细胞对抗原特异性CTL的激活

Study on Tumor-Specific Cytotoxicity T Lymphocytes Activated by Dendritic Cells Loaded with Apoptotic Tumor Cells

为探讨凋亡Daudi细胞负载的树突状细胞 (DC )激发诱导肿瘤抗原特异性CTL及其生物学特性 ,采用常规方法从人外周血单个核细胞 (PBMC )诱导DC ,激发型CD40mAb联合 50Gyγ射线辐照诱导Daudi凋亡后负载DC ,然后与自体T细胞共育 ,并联合IL 2激发诱导肿瘤特异性CTL ;采用免疫荧光标记和流式细胞仪测定分析膜分子的表达 ;ELISA检测细胞因子的产生 ;Dextran FITC内吞实验分析DC的抗原摄取能力 ;3H掺入试验和51 Cr释放试验分别测定CTL的增殖和细胞毒效应。结果 :(1 )细胞因子序贯体外诱导 7d的DC ,对Dextran吞噬能力最强 ;(2 )CD40mAb诱导联合γ射线辐照 ,能有效介导Daudi细胞的凋亡 ;(3 )抗原负载联合CD40mAb激发可使DC上调表达CD1a、CD80、CD86和HLA DR ,并能促进IL 1 2的分泌 ;(4)凋亡Daudi负载后的DC在激发型CD40mAb作用下 ,能激发和扩增对Daudi细胞具有高效和特异杀伤作用的细胞毒性T细胞。因而认为凋亡肿瘤细胞负载联合CD40mAb刺激的DC可有效激活和扩增肿瘤特异性CTL。

To investigate of tumor Ag specific CTL activated by apoptotic tumor cells loaded DC to anti malignant B lymphoma,DC was derived from peripheral blood monocyte induced by rhGM CSF and rhIL 4;Daudi cells were incubated with agonist mAb CD40 combined with 60 Co γ 50 Gy irradiation to be induced to apoptosis Then the apoptotic cells were loaded to DC, and tumor loaded DC combined with IL 2 to activated tumor specific CTL Immune florescent and FCM analyzed the expression of membrane molecules;ELISA kit assayed the cytokine levels The ability of DC capturing antigens was measured by Dextran FITC endocytosis,proliferation of CTL was assayed by 3 H thymidine release, and CTL activity was analyzed by a 4 h 51 Cr release assay The results showed that on the 7th day culture,DC had the most powerful ability of endocytosis After incubation with agonist mAb CD40 (5 μg/ml) combined with 50 Gy irradiation,the apoptotic rate of Daudi cells was 27 6%±2 1% CD40 mAb treated the maturation of tumor loaded DC,and DC's phenotypes CD1a,CD80,CD83,CD86 and HLA DR were upregulated The production of IL 12 was higher than any other group Tumor loaded DC induced a strong and specific CTL cytotoxicity to Daudi cells The cytotoxicity rate was 57 6%±5 3% for Daudi cells,Raji 29 5%±3 4%,XG 2 20 1%±4 0% The death percentage of Daudi coculturing with CTL was 24 3%±3 9% 24 h later,and 48 h later>95%,measured by trypan blue dyeing It concludes that apoptotic tumor cell loaded DC stimulated by CD40 mAb can efficiently induce the proliferation and activation of tumor specific CTL against target cells