IL-15上调NKG2D表达对CIK细胞杀伤活性的增强效应

目的观察IL-15对细胞因子诱导的杀伤细胞( Cytokine-induced killer cells,CIK)NKG2D受体表达及其对食管癌EC9706细胞杀伤活性的影响。方法体外分离外周血单个核细胞,分为两组。对照组：干扰素-γ、白细胞介素-2、CD3单抗诱导培养CIK细胞。IL-15组：加用IL-15培养。流式细胞仪检测细胞免疫表型及CD3+细胞、CD56+细胞表面NKG2D的表达,LDH法测定第14天细胞在效靶比20∶1、30∶1时对EC9706细胞的杀伤活性;效靶比30∶1时,观察NKG2D单抗封闭细胞表面NKG2D分子后对两组细胞杀伤活性的影响。结果随着培养时间的延长,CIK群体细胞及CD56+细胞表面NKG2D表达逐渐增强,IL-15组与对照组相比差异有统计学意义(P＜0.05);效靶比20∶1、30∶1时,IL-15组细胞对EC9706细胞的杀伤活性均较对照组明显增强,差异均有统计学意义(P＜0.05);效靶比30∶1时NKG2D单抗封闭CIK细胞表面NKG2D分子后,对照组细胞、IL-15组细胞对EC9706细胞的杀伤活性均较阻断前明显下降,差异均有统计学意义 (P＜0.05)。结论IL-15上调CIK细胞表面NKG2D分子表达,增强CIK细胞对EC9706细胞的杀伤活性,CIK细胞通过NKG2D发挥作用。     

Optimized protocols for generation of cord blood-derived cytokine-induced killer/natural killer cells

The efficacy of various combinations of stem cell factor (SCF), FLT3 ligand, interleukin (IL)-2, IL-7 and IL-15 to induce and expand cord blood-derived cytokine-induced killer (CIK) cells was investigated. There were three treatment groups: group A: SCF combined with IL-2, IL-7 and IL-15; group B: SCF, FLT3 ligand combined with IL-2, IL-7 and IL-15, and group C: IL-2, IL-7 and IL-15, the control group. Proliferation rates of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) natural killer (NK) cells were highest in group B; expansion of CIK cells increased 796.1 ± 278.5-fold, and that of NK cells increased 36.6 ± 3.5-fold. All expanded cord blood-derived CIK/NK cells showed cytotoxic activity against the K562 cell line. Interestingly, the cytotoxicity of group A was highest and significantly higher than that of other groups. These protocols might provide an alternative choice for CIK/NK cell expansion.     

抗PD-1单克隆抗体联合CIK细胞对肺癌细胞株杀伤作用的研究

目的:研究程序性死亡受体1（program cell death-1,PD-1）单克隆抗体联合细胞因子诱导的杀伤细胞（cytokine-induced killer cells,CIK cells）对肺癌细胞株的杀伤作用及相关机制,探究该治疗方法在肺癌治疗中的可行性。方法:患者外周血单个核细胞（peripheral blood mononuclear cell,PBMC）在体外采用多种细胞因子诱导为CIK,并用流式细胞术(flow cytometry,FCM)分析CIK细胞的表型。培养A520 及H1975肺癌细胞,利用FCM检测其表面HLA-ABC,HLA-A2,HLA-DR及PD-L1的表达情况。将CIK细胞和抗PD-1单抗Nivolumab单独或者联合作用于A520 及H1975肺癌细胞。用ELISPOT法测定不同组γ干扰素（interferon gamma,IFN-γ）的释放,用CCK8法检测CIK细胞对肺癌细胞株的杀伤率。结果:CIK细胞对于肺癌细胞株的杀伤随着效靶比（effect target ratio,E∶T）的增加而加强;对于PD-L1高表达的肺癌细胞,Nivolumab与CIK细胞联合使用对肺癌细胞株的杀伤优于单一的CIK细胞及Nivolumab。结论:对于PD-L1高表达的A520肺癌细胞,PD-1单抗Nivolumab能够提升CIK细胞的杀伤作用,而对于PD-L1表达水平低的H1975细胞,Nivolumab不能提升CIK细胞的杀伤作用。     

Role of NKG2D in cytokine-induced killer cells against multiple myeloma cells

The cytokine-induced killer cells (CIK) have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanisms that CIK cell recognizing MM cells remain unknown. Recent studies indicated that the interaction between NKG2D receptor and NKG2D ligands plays an important role in inducing cytotoxicity against various target cells by natural killer cells (NK). We suspect whether NKG2D receptor and NKG2D ligands interaction is also responsible for the killing of MM cells by CIK as the same way did NK cells. We expanded CIK cells from healthy controls with interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2), and checked expression of NK cell receptors on CIK cells by flow cytometry. About 86% bulk CIK cells expressed NKG2D receptor but not other NK receptors, such as CD158a, CD158b and NCRs. We analyzed NKG2D ligands expression in MM patients by flow cytometry, primary plasma cells from 8 out of 13 (62%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. Interestingly, when stimulated with MM cell line U266 that expressed some levels of MICA/B, only NKG2D expressing CIK cells released IFN-γ. CIK cells showed cytotoxicity against NKG2D ligands expressing U266 and primary MM cells, and the cytotoxicity was partially blocked by treating CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligand interaction may be one of the mechanisms by which CIK cells kill MM cells.     

CIK的体外增殖及体内外杀瘤活性的实验研究

目的:从人骨髓造血前体细胞体外培养扩增树突状细胞(dendritic cells,DCs),测定其表型及T细胞刺激活性.方法:采用Mini-MACS分离技术,从正常人骨髓、脐血分离CD34~ 造血干细胞,体外以重组hGM-CSF,hTNF-α,hIL-3诱导培养2周,流式细胞术检测扩增细胞的表面表型及细胞内IL-12的表达,体外同种混合淋巴细胞反应检测扩增DCs的T细胞刺激活性.结果:从正常人骨髓、脐血分离得到高纯度(>90%)的CD34~ 造血干细胞,经重组hGM-CSF,hTNF-α的共同诱导培养,扩增得到大量DCs,加人hIL-3可以进一步增加DCs产量;FACS检测表明,扩增的DCs表达HLA-DR,CD40,CD54,CD80,CD86分子,细胞内有hIL-12的P35,P40亚基的表达;与外周血单核细胞培养生成的DCs相比,由CD34~ 干细胞扩增的DCs具有更强的激发同种T细胞增殖的能力.结论:人CD34~ 干细胞体外经诱导培养,可以生成大量功能成熟的DCs,从而为进一步开展DCs的基础及临床研究打下了基础.     

人肺癌细胞NHE—1基因片段的克隆及其反义表达载体的构建

目的:动态观察CIK(cytokine induced killer)细胞的体外增殖,体外的细胞毒活性,及通过动物实验研究其体内的抗肿瘤作用.方法:通过提取健康供血者的PBMC,第0天加入γ-IFN,第1天加入IL-2、抗-CD3单抗和IL-1培养CIK细胞;在流式细胞仪上做动态培养物的表型分析;与LAK细胞作对比,分别用MIT法测定其体外细胞毒活性及对S180荷瘤鼠的体内抗肿瘤作用.结果:CIK细胞在培养2周后获得大量增殖,表型分析表明,CIK细胞属异质性细胞群,在培养的过程中,群体的CD3~ CD56~ 细胞大量扩增达1000多倍,是CIK细胞的主要效应细胞;实验证明,CIK细胞的体外细胞毒活性及对S180荷瘤鼠的体内抗肿瘤作用均强于LAK细胞;其较强的体内抗癌活性可能与荷瘤鼠主体内T细胞活化有关.结论:CIK细胞是一种强于LAK细胞的、新型、高效、具有广谱杀瘤活力的免疫活性细胞.     

Cytotoxicity of Cytokine-Induced Killer Cells Coated with Bispecific Antibody Against Acute Myeloid Leukemia Cells

Various types of cytokines have been used in in vitro experiments to generate cytokine-induced killer (CIK) cells that are reactive to patient acute myeloid leukemia (AML) cells. Of these CIK cells, interleukin-2 (IL-2)-activated peripheral blood mononuclear cells, i.e., lymphokine-activated killer (LAK) cells, with the initial addition of the anti-CD3 monoclonal antibody (T3 LAK cells), are the most potent cytotoxic lymphocytes, and have marked proliferative capacity. The cytotoxicity of such T3 LAK cells against CD13⁺ AML cells is further enhanced by the addition of anti-CD3 x anti-CD 13 bispecific antibody (BsAb) during the cytotoxicity assay. The combined use of T3 LAK cells and the BsAb can be used for ex vivo purging of CD13⁺ AML cells in autologous bone marrow transplantation. Other cytokines, such as IL-7 or IL-7 in combination with IL-2, or newly identified cytokines, will also be tested in attempts to obtain more specific and more potent effector cells. Studies of methods to increase the susceptibility of AML cells to CIK are also required.     

酸性环境抑制CIK细胞对肝癌HepG2细胞的杀伤活性

目的 研究酸性环境对CIK（cytokine-induced killer, CIK）细胞杀伤肝癌HepG2细胞的影响。方法 利用IFN-γ、IL-2及CD3抗体诱导外周血单个核细胞获得CIK细胞。在pH6.5及pH7.4条件下将CIK细胞和荧光素酶标记的HepG2细胞（HepG2-luc）按不同的效靶比混合培养,用小动物活体成像系统检测HepG2-luc荧光强度并计算杀伤活性,用MTT法检测并计算杀伤活性。在pH6.5及pH7.4条件下,在含CIK条件培养液0、50%和100%的情况下培养HepG2细胞,流式细胞仪检测凋亡坏死的细胞比例。结果 pH7.4时CIK细胞对HepG2细胞的杀伤率明显高于pH6.5时。CIK条件培养液作用下,pH7.4时HepG2细胞的凋亡坏死比例明显高于pH6.5。结论 酸性环境明显抑制了CIK细胞对肝癌细胞HepG2的杀伤活性。     

Enhanced killing of primary ovarian cancer by retargeting autologous cytokine-induced killer cells with bispecific antibodies: a preclinical study.

Cytokine-induced killer (CIK) cells are ex vivo activated and expanded CD8+ natural killer T cells that have been shown to have antitumor activity. This is the first study exploring cell killing of primary ovarian carcinoma cells with and without bispecific antibodies. Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. Bispecific antibodies against cancer antigen-125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On fluorescence-activated cell sorting analysis, the expansion of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by bispecific antibodies, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 +/- 0.3% to 89.4 +/- 2.1% at an E:T ratio of 100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001 and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.     

CIK细胞对晚期非小细胞肺癌预后的影响

目的观察细胞因子诱导的杀伤细胞（CIK细胞）体外培养后,对晚期非小细胞肺癌的疗效。方法收集经确诊的、并发生转移、且采用标准治疗方案的非小细胞肺癌患者20例作为观察组,取外周血分离单个核细胞（PBMC）,体外细胞因子诱导培养CIK细胞,流式细胞仪检测细胞表型。20例患者均接受CIK细胞免疫治疗,观察其免疫活性及生存期。并以20例采用标准治疗方案而未经CIK治疗的晚期非小细胞肺癌患者作为对照组。结果观察组培养后的细胞表型CD3、CD3＋CD56＋明显升高,生存期为（16.15±3.8）个月,对照组生存期为（9.12±3.2）个月。结论经CIK细胞治疗后晚期非小细胞肺癌患者的生存期明显延长。     

体外扩增CIK细胞杀伤胃癌细胞SGC-7901

目的：观察CIK细胞增殖情况,了解扩增后的最佳应用时机,并观察体外对SGC-7901的杀伤作用。方法：健康人外周血单核细胞（PBMC）在体外条件下经过多种细胞因子的共同刺激诱导成CIK细胞,计数培养不同时间的CIK细胞,用流式细胞术检测CIK细胞的表型特征。用MTT法检测杀伤活性,对比CIK细胞与5-FU对SGC-7901的杀伤作用,并观察了两者联合应用的效果。结果：CIK细胞随体外培养时间的延长,数量及杀伤活性均增加。体外培养20d增殖124倍,CD3^＋、CD56^＋双阳性细胞的比例达66．1％,其后两者数量增长缓慢;体外实验显示CIK细胞对胃癌SGC-7901细胞株有明显的杀伤作用,最高杀伤效率为73．13％,其杀伤作用优于5-FU,P〈0．05。二者联合应用杀伤作用降低。结论：CIK细胞具有较强的抗胃癌细胞活性;体外培养15～20d时应用较为合适;联合应用时5-FU可降低CIK细胞的杀伤效率。     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

CIK细胞对乳腺癌细胞株抗增殖及诱导凋亡的作用

目的研究多种细胞因子诱导的杀伤细胞(Cytokine-inducedkillcells,CIK)对ZK-75-1乳腺癌细胞株的抗增殖和诱导凋亡作用,并探讨其作用机制。方法应用MTT法检测CIK细胞对ZK-75-1乳腺癌细胞株的抗增殖作用的影响及细胞毒活性;通过倒置显微镜、HE染色、透射电镜观察凋亡细胞的形态学改变。结果MTT比色法表明随着CIK细胞与乳腺癌细胞效靶比增加及作用时间延长,抑制率明显增强(P<0.01)。CIK细胞作用于乳腺癌细胞24h后,形态学观察乳腺癌细胞发生凋亡或坏死。结论CIK细胞是一种新型高效具有较强杀伤体外乳腺癌细胞的免疫活性细胞。     

顺铂预处理对CIK杀伤肺癌细胞的影响

目的探讨顺铂（DDP）预处理对细胞因子诱导的杀伤细胞（CIK）杀伤活性的影响。方法采用CFSE/PI 双标法检测DDP预处理不同浓度、不同时间及预处理前后不同效靶比,CIK对肺癌细胞的杀伤活性。结果经IFN-γ,CD3单抗,IL-2诱导后20 d,获得典型的CIK细胞做为效应细胞。随着DDP预处理浓度的增加,CIK细胞的杀伤活性随之增强,随着DDP预处理时间的延长,CIK细胞的杀伤活性亦随之增强。在DDP预处理浓度25 ng/ml,预处理时间18 h的条件下,DDP对靶细胞并无明显影响,但联合CIK后,随着效靶比的增高,CIK细胞杀伤肿瘤细胞的活性明显增强。结论顺铂预处理肺癌细胞后,提高了CIK细胞的杀伤活性,对CIK细胞的临床应用具有借鉴意义。     

Comparative analysis of interleukin 15 and interleukin 2 for induction of killer activity and of type 2 cytokine production by mononuclear cells from lung cancer patients.

Interleukin (IL) 15 is a novel cytokine with IL-2-like activity. In this study, we examined the effect of IL-15 on induction of non major histocompatibility complex (MHC)-restricted killer activity and of type 2 cytokine production by peripheral blood and pleural mononuclear cells (MNCs), from 34 lung cancer patients and 20 control subjects. IL-15 induced significant killer activity in blood MNCs from lung cancer patients as well as control subjects against a small-cell lung cancer cell line (SBC-3). Effective killer induction by IL-15 was observed even in blood MNCs and pleural MNCs from the site of tumour growth in advanced lung cancer patients. IL-12 had an additive effect with a suboptimal dose of IL-15 in induction of killer activity. In the case of MNCs from lung cancer patients, IL-10 production was more prominent when cells were incubated with IL-2 than with IL-15. IL-5 production was observed in MNCs from lung cancer patients stimulated with IL-2, but not with IL-15. These observations suggest that IL-15, by virtue of its lesser induction of type 2 cytokine, may be a better candidate than IL-2 for lung cancer immunotherapy.     

化疗药物对肿瘤细胞免疫原性的调变作用

目的 观察化疗药物对肿瘤细胞免疫原性的调变作用,探讨介导预处理化疗增效作用的机制.方法 MTT法筛选化疗药物5-氟尿嘧啶(5- Fluorouracil,5- FU)、顺铂(Cisplatin,DDP)、紫杉醇(Paclitaxel,PTX)、多西紫杉醇( Docetaxel,DTX)的体外高、中、低毒性浓度,将这些浓...     

厄洛替尼增强肺腺癌A549细胞对CIK细胞杀伤的敏感性

  目的  研究表皮生长因子受体酪氨酸激酶抑制剂厄洛替尼对人肺腺癌A549 细胞NKG2D配体表达及CIK细胞杀伤活性的影响及其分子机制。  方法  流式细胞仪检测厄洛替尼、EGFR下游分子LY294002（PI3K抑制剂）、SB203580（MAPK抑制剂）、STAT21（STAT3抑制剂）作用A549细胞24 h后A549细胞NKG2D配体的表达。乳酸脱氢酶释放法测定不同效靶比时,CIK细胞对10 μmol/L厄洛替尼作用前、后A549细胞的杀伤活性。  结果  厄洛替尼下调A549细胞MICA表达,上调MICB、ULBP1表达,EGFR下游分子 MAPK、STAT3 抑制剂不影响 A549 细胞 NKG2D 配体的表达,PI3-K 抑制剂下调 A549 细胞 MICA 表达,厄洛替尼增强A549细胞对CIK细胞杀伤的敏感性。  结论  EGFR TKI 抗肺癌作用与其增强肺癌细胞对免疫细胞杀伤的敏感性有关。      

Interleukin-12 augments cytolytic activity of peripheral blood mononuclear cells against autologous lung cancer cells in combination with IL-2

The majority of patients with advanced lung cancer die within a few years. Accordingly, new therapeutic modalities need to be developed. Interleukin (IL)-12 was previously known as natural killer (NK) cell stimulatory factor or cytotoxic lymphocyte maturation factor. By virtue of its effects on T cells and NK cells, IL-12 seems to be one of the key cytokines that regulates cell-mediated anti tumor immune responses. Recently, there has been a substantial interest in the potential applications of IL-12 in the treatment of lung cancer. However, there have been no reports about the effect of IL-12 on peripheral blood mononuclear cells (PBMCs) obtained from lung cancer patients in an autologous setting. In this study, we examined the cytotoxicity of PBMC activated by IL-2, IL-12 or both against K562 or autologous lung cancer cells. In contrast to the effect of IL-2 on NK activity, IL-12 alone augmented NK activity against K562 cells, but not against autologous lung cancer cells. IL-12 augmented the IL-2 mediated cytotoxicity of PBMC against both K562 and autologous lung cancer cells. In the absence of IL-2, IL-12 alone cannot induce an autologous anti-tumor effect in vivo. In summary, our results clearly demonstrated that IL-12 can augment the cytolytic activity of PBMC against K562 and autologous lung cancer cells when combined with IL-2, although, IL-12 alone was unable to induce a marked increase in the cytotoxicity against autologous lung cancer cells. These results suggest that an administration of IL-12 in combination with IL-2 may be a useful therapeutic option for solid tumors.     

肿瘤负荷对CIK/IL-2临床疗效的影响

目的:自从1993年Schmidt Wolf建立细胞因子诱导的杀伤细胞(CIK)制备方法以来,CIK/IL-2在净化骨髓和治疗血液病方面取得较多经验,但缺乏CIK/IL-2治疗实体瘤的临床资料.本文总结应用CIK/IL-2治疗41例实体瘤的临床经验,分析肿瘤负荷对该疗法临床疗效的影响.方法:选择无临床病灶的高危复发患者和存在明显临床病灶的晚期实体瘤患者,分离外周血单个核细胞,制备CIK.CIK联合IL-2静脉输注患者.结果:高危复发组中,姑息性治疗9例,根治性治疗14例,中位随访时间13个月.接受CIK/IL-2治疗后姑息性治疗者复发6例,根治性治疗者复发3例,复发率分别为66.7%和21.4%(P＜0.05).此外,CIK/IL-2治疗常规治疗失败的晚期实体瘤患者18例,4例稳定.结论:CIK/IL-2的临床疗效受肿瘤负荷制约,接受CIK/IL-2辅助治疗前应联合其它治疗方法尽量降低肿瘤负荷.     

负载人结肠癌Lovo细胞总RNA抗原树突状细胞对细胞因子诱导的杀伤细胞在体外特异性杀伤的影响

目的 探讨人结肠癌Lovo细胞总RNA抗原致敏的树突状细胞(DC)对细胞因子诱导的杀伤细胞(CIK)在体外特异性杀伤的影响.方法 利用Ficoll密度梯度离心法提取脐血单核细胞,分别诱导CIK和DC细胞,并用流式细胞仪检测其免疫表型.采用Trizol提取结肠癌Lovo细胞总RNA作为肿瘤细胞抗原,转染脐血来源的DC.实验分为3组:转染Lovo DC共培养CIK组、未转染DC共培养CIK组和单纯CIK组.靶细胞为Lovo细胞,在效靶比为50∶1和20∶1的条件下以噻唑蓝法分别检测CIK的体外杀伤活性.结果 在效靶比为20∶1时,负载Lovo RNA抗原的DC能诱导出CIK对Lovo细胞最强的细胞毒杀伤力为(76.49±4.21)%,DC+CIK组次之为(53.84±2.15)%,CIK组细胞毒性最低为(32.20±3.07)%,且两组间差异有统计学意义(P＜0.05).结论 肿瘤细胞总RNA提取方法简单,易于临床实施,其作为抗原致敏DC能强化CIK的特异性杀伤,将有很好的临床应用前景.