Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen (CEA) peptide and cultured dendritic cells.
Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen (CEA) peptide and cultured dendritic cells.
复制标题
使用癌胚抗原 (CEA) 肽和培养的树突状细胞生成细胞毒性效应淋巴细胞的新系统。
作者：
K. Ohta;Y. Yamaguchi;K. Shimizu;E. Miyahara;T. Toge
我们已经建立了一个实用的系统，用于产生抗肿瘤效应淋巴细胞，使用肿瘤抗原肽CEA和培养的树突状细胞（DC），也具有其特征的效应细胞。使用IL-4和GM-CSF从HLA-A0201正常或荷瘤供体获得的自体外周血单核细胞（PBMC）的贴壁细胞群诱导DC。培养的DC显示表达I类、II类、CD 80和CD 86分子。用HLA-A0201限制性CEA肽CEA 9 671脉冲的DC刺激PBMC 7天，然后在80 U/ml IL-2（IL-2/CD 3系统）存在下在抗CD 3抗体（1微克/ml）包被的烧瓶中扩增。被称为CEA肽脉冲的树突状细胞激活的杀伤（CEA-PDAK）细胞的效应细胞优先为CD 3 + CD 8+，并且能够杀伤用CEA肽脉冲的T2细胞，但不能单独杀伤T2细胞。CA-PDAK细胞也溶解胃癌细胞系KATO III（HLA-A0201，CEA（+）），但不溶解WiDr（HLA-A2402，CEA（+））细胞。当CEA-PDAK细胞用抗TCR α β抗体处理时或当靶细胞在细胞毒性测定之前用抗I类抗体处理时，细胞毒性被消除。即使在效应期存在大量CEA蛋白的情况下，CEA-PDAK细胞也发挥其细胞毒性活性，这模拟了临床环境。CEA-PDAK细胞在用IL-2/CD 3系统刺激时，与单独用IL-2刺激的细胞相比，在产生的总细胞数中显示出约百倍的扩增，而没有任何特异性裂解的损失。通过RT-PCR-Southern印迹法对CEA-PDAK细胞进行的TCR V β基因分析证明了TCR β 7和12的寡克隆表达，并且后者被证明负责杀伤活性。SSCP分析表明TCR V β 12基因的克隆型，表明通过用CEA肽脉冲的DC刺激携带有限TCR可变区的淋巴细胞的选择性扩增。总之，与CEA抗原反应的效应淋巴细胞可以从具有抗原性CEA肽的PBMC和培养的DC产生。IL-2/CD_3系统对CEA-PDAK细胞激活效应细胞的作用是有效和实用的。使用该系统的连续免疫疗法可能有希望用于治疗CEA表达肿瘤。
We have established a practical system for generating antitumor effector lymphocytes using the tumor antigen peptide CEA and cultured dendritic cells (DCs), and have also characterized effector cells. DCs were induced from the adherent cell population of autologous peripheral blood mononuclear cells (PBMCs) obtained from HLA-A0201 normal or tumor-bearing donors using IL-4 and GM-CSF. The cultured DCs were shown to express class I, class II, CD80 and CD86 molecules. The PBMCs were stimulated for 7 days with the DCs pulsed with the HLA-A0201-restricted CEA peptide CEA9 671 and then expanded in an anti-CD3 antibody (1 microgram/ml)-coated flask in the presence of a 80 U/ml IL-2 (IL-2/CD3 system). The effector cells, which were designated as CEA peptide-pulsed dendritic cell-activated killer (CEA-PDAK) cells, were preferentially CD3+CD8+, and capable of killing T2 cells pulsed with CEA peptide but not T2 cells alone. The CA-PDAK cells also lysed the gastric cancer cell line KATO III (HLA-A0201, CEA (+)), but not the WiDr (HLA-A2402, CEA(+)) cells. The cytotoxicity was abrogated when the CEA-PDAK cells were treated with anti-TCR alpha beta antibody or when the target cells were treated with the anti-class I antibody prior to the cytotoxicity assay. The CEA-PDAK cells exerted their cytotoxic activity even in the presence of a high amount of CEA protein at the effector phase, which mimicked the clinical setting. The CEA-PDAK cells showed approximately a hundred-fold expansion in total cell numbers yielded without any loss of the specific lysis, when stimulated with the IL-2/CD3 system compared to those stimulated with IL-2 alone. The TCR V beta gene analysis for the CEA-PDAK cells, conducted by means of RT-PCR-Southern blotting, demonstrated oligoclonal expression of TCR beta 7 and 12, and the latter was shown to be responsible for the killing activity. SSCP analysis indicated the clonotype of the TCR V beta 12 gene, indicating a selective expansion of lymphocytes bearing a limited TCR variable region by the stimulation with CEA peptide-pulsed DCs. Taken together, the effector lymphocytes reactive with the CEA antigen can be generated from PBMCs with the antigenic CEA peptide and cultured DCs. The IL-2/CD3 system is effective and practical in activating the effector cells for the clinical use of CEA-PDAK cells. Adoptive immunotherapy using this system may be promising for treating CEA-expressing tumors.