肺癌肿瘤全溶物脉冲自体树突状细胞体外诱生T细胞抗肿瘤反应的研究  

Generation of T cell-mediated antitumor response in vitro by autologous dendritic cells pulsed with tumor lysates in patients with non-small cell lung cancer

作　　者：尤健[1] 于津浦[2] 任秀宝[2] 王长利[1] 张澎[2] 张熙曾[1] 

机构地区：[1]天津医科大学附属肿瘤医院胸科,300060 [2]天津医科大学附属肿瘤医院免疫研究室,300060

出　　处：《中华肿瘤杂志》2004年第6期333-336,共4页Chinese Journal of Oncology

基　　金：天津市自然科学青年基金资助项目(0 0 3 70 2 911)

摘　　要：目的　探讨非小细胞肺癌 (NSCLC)患者全瘤溶解物 (WTL)冲击的自体树突状细胞 (DCs)瘤苗体外诱生T细胞介导的抗肿瘤反应。方法　在含重组人粒细胞 巨噬细胞 集落刺激因子 (rhGM CSF)和重组人白细胞介素 4 (rhIL 4 )的培养条件下 ,以 10例患者的贴壁外周血单核细胞 (PBMCs)衍生不成熟的树突状细胞 (imDCs) ,分别添加肿瘤坏死因子 (TNFα)、自体WTL或WTL +TNFα诱导成熟 ,即分别为DCs/TNF、DCs/WTL和 DCs/WTL ,用流式细胞仪和混合淋巴细胞反应 (MLR) ,检测细胞表面分子的表达变化及其刺激异基因或同基因的T细胞增殖能力。将 DCs/WTL和自体T细胞共培养1～ 2周 ,以诱导细胞毒性T淋巴细胞 (CTL) ,用计数γ 干扰素 (IFN γ)释放的酶联免疫斑点 (ELISPOT)试验和乳酸脱氢酶 (LDH)释放的细胞毒试验 ,分别检测该CTL中识别自体肿瘤细胞释放特异性IFN γ的T细胞数和溶瘤活性。结果　以 30 μg/ml蛋白的WTL体外冲击自体 10 6imDCs,可导致上调DC表型 ,包括CD1a、CD83和CD86以及HLA DR的分子表达 ,与常规TNFα诱导成熟的DCs表型相似。虽然两者均能显著刺激异基因T细胞的增殖 ,但其刺激自体T淋巴细胞的增殖能力 ,DCs/WTL却显著高于DCs/TNF(P <0 .0 5 )。将体外与 DCs/WTL共培养的自体T细胞作为反应或效应细胞 。Objective To investigate whether dendritic cells pulsed with whole tumor lysates (WTL) could in vitro elicit antitumor T cell responses in patients with non small cell lung cancer (NSCLC). Methods Monocyte derived immature DCs (imDCs) generated in the presence of human recombinant granulocyte macrophage colony stimulating factor and interleukin 4 from peripheral blood mononuclear cell of NSCLC patients, and then were induced to mature by pulsing autologous WTL (DCs/WTL) or by the addition of TNF α(TNF/DCs). FACS and MLR assay were used to monitor their phenotypic changes and capacity to stimulate allogeneic and autologous T cell proliferation. DCs/WTL activated with TNF α(*DCs/WTL) were cocultured in vitro with autologous T cells for eliciting antitumor CTLs. T cell mediated antitumor responses were measured by IFN γ enzyme linked immunospot (ELISPOT) assay for WTL specific IFN γ releasing T cells and by lactate dehydrogenase release (LDH) assay for lysis of autologous tumor cells, respectively. Results When monocytes derived imDCs from the patients with NSCLC ( n =10) were pulsed with autologous WTL for a day at 30 μg total protein of WTL per 10 6 DCs /ml , this led to up regulation of CD1a, CD83 and CD86 as well as HLA DR, and also led to marked stimulation of allogeneic T cell proliferating activity, which was comparable to that of TNF/DCs. However, their capacity of stimulating autologous T cell proliferation in vitro was significantly more potent than those of TNF/DCs ( P < 0.05). The numbers of WTL specific IFN γ releasing T cells in 1/3 cultures after one week exposure to *DCs/WTL was increased significantly compared with those pulsing with TNF/DCs plus IL 2 or IL 2 alone ( P =0.05). T cells derived by priming of non adherent PBMCs with *DCs/WTL after 14 days in vitro stimulation were significantly more responsive to autologous tumor cells compared with LAK ( n =3, P <0.05), but its cytotoxicity against K562 cells was also comparable to LAK cells. Conclusion M

关 键 词：肺癌 肿瘤全溶物 NSCLC 树突状细胞 体外诱生 T细胞 抗肿瘤反应 WTL DCs 

分 类 号：R734.2[医药卫生—肿瘤]