CD 40激发肿瘤抗原特异性DCs对CIK细胞生物学活性的影响

Effects of CD 40-pulsed tumor specific dendritic cells on biological activity of cytokine-induced killer cells

目的:研究CD40激发凋亡肿瘤细胞致敏的树突状细胞(dendritic cells,DCs)对细胞因子诱导杀伤(cytokine-inducedkiller,CIK)细胞的细胞表型、增殖活性及细胞毒活性的影响。方法:常规方法从健康人外周血单个核细胞中诱导DCs和CIK细胞,采用凋亡肿瘤细胞负载DCs,并用或不用激发型CD40单克隆抗体(CD40mAb)激活DCs成熟;成熟DCs与同源的CIK细胞共育5d,分别获得DC40Ag-CIK细胞及DCAg-CIK细胞;观察细胞增殖活性;流式细胞仪检测细胞表型;酶联免疫吸附实验(enzyme-linked immunosorbent assays,ELISA)法检测细胞培养上清液中IFN-γ的含量;[3H]-TdR掺入法测定细胞杀伤活性。结果:凋亡肿瘤细胞负载激发可使DCs上调表达CD1a、CD80、CD86、CD83、HLR-DR,联合CD40mAb激发可进一步促进DCs成熟;培养至第14天时,DC40Ag-CIK细胞、DCAg-CIK细胞、CIK细胞分别扩增(18.2±1.7)倍、(15.0±1.2)倍、(9.3±1.8)倍;DC40Ag-CIK细胞中的CD3+CD56+比例较DCAg-CIK细胞和CIK细胞明显上调(P<0.05);DC40Ag-CIK细胞、DCAg-CIK细胞对A549杀伤活性强于CIK细胞(P<0.05),并且DC40Ag-CIK细胞强于DCAg-CIK细胞(P<0.05);CIK细胞、DCAg-CIK细胞、DC40Ag-CIK细胞培养上清液中IFN-γ的含量递增,分别为(1494.7±246.3)pg/mL、(2706.3±197.0)pg/mL、(3676.3±335.0)pg/mL。结论:与单独凋亡肿瘤细胞负载的DCs相比,CD40激发的凋亡肿瘤细胞负载的DCs可进一步提高CIK细胞的增殖活性及细胞毒活性。

Objective：To investigate the changes of phenotypes, proliferation activity and cytotoxicity of cytokine-induced killer （CIK） cells after co-cultured with autologous dendritic cells sensitized by CD 40-pulsed apoptotic tumor cells. Methods： DCs and CIK cells were induced from peripheral blood mononuclear cells （PBMC） of healthy subjects by the regular method. The immature DCs were loaded with apoptotic tumor cells pulsed with or without CD 40 mAb. The mature DCs were co-cultured with CIK cells for 5 d to obtain DC40Ag-CIK cells and DCAg-CIK cells. The proliferative activity of the effector cells was observed. Cell phenotypes were analyzed by flow cytometry. The level of interferon IFN-γ in the supematant of cultured cells was measured by enzyme-linked immunosorbent assays （ELISA）. The cytotoxicity was detected by [^3 H] TdR incorporation method. Results ：The expression rates of CD 1a, CD 80, CD 83, CD 86, and HLR-DR were up-regulated in DCs after loaded with apoptotic tumor cells. CD 40 mAb-pulsed apoptotic tumor cells further induced maturation of DCs. On the 14th day the DC40Ag-CIK cells, DCAg-CIK cells, and CIK cells proliferated and expanded （ 18.2 ± 1.7） times, （ 15.0 ± 1.2） times, （9.3 ±1.8） times, respectively. The proportion of CD3^＋CD56^＋ cells was markedly up-regulated in DC40Ag-CIK cells compared with DCAg-CIK cells and CIK cells （P 〈0.05 ）. The cytotoxicity of DC40Ag-CIK cells and DCAg-CIK cells on A 549 cells was enhanced compared with CIK cells （P 〈0.05）. DC40Ag-CIK cells had much stronger cytotoxicity than DCAg-CIK cells （ P 〈 0.05 ）. The level of IFN-γ was （ 1494.7 ± 246.3 ） , （ 2706.3 ± 197.0） , and （ 3676.3 ± 335.0） pg/mL in the supernatant of CIK, DCAg-CIK, and DC40Ag-CIK cell cultures, respectively. Conclusion： DCs loaded with apoptotic tumor cells pulsed by CD 40 could further enhanced the proliferative activity and cytotoxicity of CIK cells compared with those without CD 40 pulsation.