DC-CIK细胞免疫治疗晚期结直肠癌患者的远期疗效及其影响因素分析

目的:探讨树突状细胞(DC)联合细胞因子诱导的杀伤(CIK)细胞免疫治疗晚期结直肠癌(CRC)患者的远期疗效及其影响因素分析。方法:收集我院2011年1月至2014年1月收治的112例晚期结直肠癌患者,依据是否接受DC-CIK细胞免疫治疗将患者分为对照组(n=47)和观察组(n=65),分别给予一般化疗方案治疗和化疗联合DC-CIK细胞免疫方案治疗,比较两组患者治疗前后血清肿瘤标志物癌胚抗原(CEA)、淋巴细胞亚群,治疗有效率(RR)、疾病控制率(DCR)、无进展生存期(PFS)和安全性改变,并分析影响疗效的危险因素。结果:两组治疗后CEA水平均显著低于治疗前(P0.05),治疗后对照组CD3+T细胞、CD4+T细胞、CD8+T细胞和NK细胞均显著减少(P0.05),观察组治疗后CD3+T细胞和CD8+T细胞数量显著高于治疗前和同期对照组(均P0.05);对照组RR、DCR和PFS分别为44.68%、65.96%、6.5个月,观察组治疗对应指标分别为46.15%、86.15%、9个月,观察组DCR和FPS均显著高于对照组(P0.05);多因素分析发现TNM分期达到Ⅳ级(P=0.023)和年龄超过60岁(P=0.006)是影响疗效的独立危险因素。结论:DC-CIK细胞免疫治疗晚期结直肠癌安全可靠,能显著改善患者免疫功能控制肿瘤进展,延长生存期,提高生存质量,值得临床推广。     

DC-CIK免疫治疗联合化疗治疗血液恶性肿瘤的安全性和有效性分析

本研究旨在研究树突状细胞-细胞因子诱导的杀伤细胞(dendritic cell and cytokine-induced killer cell, DC-CIK)免疫治疗联合化疗治疗血液恶性肿瘤的临床疗效。本研究从PubMed、 Web of Science、 The Cochrane Library、 Embase、 CNKI、 WanFang Data、 SinoMed和VIP等数据库中,收集2000年1月至2023年2月发表的符合要求的随机对照研究,应用RevMan 5.4.1软件进行Meta分析。共纳入15篇文献,894例患者,包括686例骨髓瘤患者和208例白血病患者,其中DC-CIK免疫治疗联合化疗治疗组共451例,设置为联合治疗组,而单纯化疗组共443例,设置为化疗(对照)组。分析结果显示,联合治疗组在临床疗效、预后情况、生活质量及各项免疫指标(T淋巴细胞亚群分布和细胞因子分泌水平)等方面均优于化疗组,且不会给患者带来特异性的副作用。研究结果表明,DC-CIK免疫治疗联合化疗能够改善恶性血液肿瘤患者的免疫功能,增强机体的抗肿瘤能力,从而显著提高临床治疗的效果。     

细胞因子诱导杀伤细胞过继免疫治疗恶性血液肿瘤的临床应用进展

细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK cells)是将脐血、外周血和骨髓等不同来源的单个核细胞经适当浓度的IFN-γ、IL-2和CD3单抗等联合诱导后获得的异质细胞群。CIK细胞具有增殖能力强、溶瘤谱广、对耐药细胞株也有杀伤作用,且兼有T细胞强大的杀伤活性和NK细胞(nature killer cells)非主要组织相容性复合体(non-major histocompatibility complex,non-MHC)限制性的特点,使其在肿瘤治疗中具有广阔的临床应用前景。恶性血液肿瘤患者接受CIK细胞过继免疫治疗的生存时间延长,不良反应少,但仍面临许多问题。     

DC-CIK治疗大肠癌的研究进展

大肠癌是消化道常见的恶性肿瘤之一,发病率和死亡率均较高。过继免疫治疗是当今肿瘤治疗的热点,已逐步成为一些肿瘤的首选治疗方法。树突状细胞(DC)是目前已知功能最强大的抗原呈递细胞,具有呈递肿瘤抗原和抵制肿瘤细胞免疫逃逸及刺激T淋巴细胞产生免疫应答的作用。细胞因子诱导的杀伤细胞(CIK)由多种细胞因子诱导而成,具有T淋巴细胞及NK细胞抗肿瘤作用的特点。DC和CIK细胞有效结合可以同时促进DC细胞的增殖和免疫功能及加强CIK细胞的抗肿瘤作用。本文就近年来国内外应用DC-CIK治疗大肠癌的研究进展进行综述。     

树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用

由树突状细胞(DC)与细胞因子诱导的同源杀伤细胞(CIK)的共培养诱生的细胞群(DCCIK)对肿瘤细胞的细胞毒活性的研究。DCCIK细胞体外杀伤肿瘤靶细胞A549(MTT法),效靶比为10∶1、5∶1时杀伤率分别为61%、52%。DCCIK细胞诱导培养3周后,效靶比为10∶1、5∶1时杀伤率分别为64%和56%。数据亦表明DCCIK细胞对靶细胞的杀伤优于CIK细胞。动物体内实验分荷瘤A549、BEL7404和A375三组,每组分(A)DCCIK 化疗、(B)单用化疗。治疗20天、35天后测量各组肿瘤消失率。结果显示:DCCIK 化疗的抑瘤效果明显好于单纯化疗。提示DCCIK细胞有临床应用前景。     

胃癌的过继性免疫治疗研究进展

胃癌是目前世界上发病率及致死率较高的恶性肿瘤之一,在东亚地区尤其显著。针对胃癌的治疗手段仍是传统的手术联合化疗、放疗为主,尽管靶向药物治疗提供了新的选择,但其对晚期胃癌的疗效仍然有限。胃癌的免疫治疗作为独特的治疗手段,在近十多年发展较为活跃,特别是过继性免疫治疗手段不断有创新。过继性免疫治疗主要依赖回输具有抗肿瘤活性的细胞,目前回输的细胞由具有非特异性抗肿瘤作用向具有特异性抗肿瘤作用演变,特别是嵌合性抗原T细胞治疗的出现,为进展期胃癌患者提供了有一种潜在的选择。本文对胃癌过继性免疫治疗中采用的不同免疫活性细胞的作用机制、临床应用等进行总结,并针对其不足提出利用基因工程技术增强治疗靶向性、降低免疫逃逸的研究方向。     

抗原负载DCs诱导的CIK细胞对B16黑色素瘤抑制作用的形态学观察

目的:探讨抗原负载树突状细胞(dentritic cells,DCs)诱导的CIK(cytokine induced killer)细胞对B16黑色素瘤的抑瘤作用。方法:分离、培养DC和CIK细胞,取部分DC进行肿瘤抗原负载,将其与CIK细胞按1:10的比例共培养3d,即为抗原负载的DC-CIK。建立B16黑色素瘤小鼠模型,分别于瘤周围皮下注射经Brdu标记的CIK、DC-CIK、抗原负载DC-CIK。按注射细胞进行分组,测量注射前后各组小鼠的瘤体积,计算抑瘤率,比较其抑瘤作用。应用免疫组化方法和透射电镜观察抗原负载DC-CIK细胞在皮肤中的分布及杀伤肿瘤细胞的形态学表现。结果:抗原负载DC诱导的CIK(细胞组抑瘤率(86.57%)高于CIK细胞组(33.34%,P<0.05)和DC-CIK细胞组(61.08%,P<0.05);光镜下抗原负载DC-CIK细胞主要分布在皮下组织,癌组织周围,特别是癌巢周边。透射电镜下抗原负载DC-CIK细胞体积大,核有切迹,细胞质内细胞器丰富,粗面内质网扩张。细胞表面有突起,与肿瘤细胞密切接触。大量肿瘤细胞凋亡、坏死。结论:CIK细胞经抗原负载DC诱导后抑瘤作用明显强于单纯CIK细胞和DC-CIK细胞。     

DCIK细胞用于肺癌临床免疫治疗

观察细胞因子诱导的杀伤细胞(CIK 细胞)和同源树突状细胞(DC)共培养后,共培养细胞树突状细胞调节的细胞因子诱导的杀伤细胞(DCIK 细胞)体外细胞毒活性,并观察DCIK细胞治疗肺癌的近期临床疗效、免疫学活性及副反应.收录 12例确诊肺癌经标准治疗方案治疗的患者,取外周血分离单个核细胞(PBMC),体外诱导出DC和CIK细胞共培养后,观察DCIK细胞表型,用MTT法测体外细胞毒活性;当效靶比为20∶1、10∶1时,DCIK细胞体外细胞毒活性杀伤率分别为55%、46.2%.所有患者均接受一定剂量的 DCIK细胞过继免疫治疗,观察其近期临床疗效、免疫反应、不良反应.12例患者中完全缓解 1例,部分缓解4例,病情稳定1例,近期有效率为41.6%,疾病控制率为50%,病情进展共6例,其中死亡2例.与DCIK细胞回输前相比,患者CD4 、CD8 、CD56 均有明显的升高(P＜0.05),这表示可以诱导患者产生特异性的免疫反应.除两例患者出现一过性的发热外,其余患者基本无不良反应.DCIK细胞在肺癌免疫治疗中能诱导机体产生特异性的免疫反应,亦是新的杀伤肿瘤细胞的效应细胞,有较好的临床疗效.     

嵌合抗原受体疗法在肿瘤免疫治疗中的研究进展

嵌合抗原受体(chimeric antigen receptor, CAR)是通过基因工程技术构建的融合蛋白,经其修饰的免疫细胞可以特异性靶向和杀伤表达特定抗原的肿瘤细胞。到目前为止,大多数关于CAR的研究主要集中在T细胞上。CAR-T细胞疗法在治疗恶性血液疾病方面取得了突破性进展,目前已有7款药物获批上市,研究者在不断深入研究CAR-T细胞疗法的同时也开始寻找新的免疫效应细胞, CAR自然杀伤细胞(CAR natural killer cell, CAR-NK)疗法、CAR自然杀伤T细胞(CAR NK T cell,CAR-NKT)疗法以及CAR巨噬细胞(CAR macrophage, CAR-MP)疗法逐渐成为免疫治疗的新策略。尽管围绕CAR的研究策略不断增加,该领域也依然面临一些挑战,例如有效地靶向实体瘤、减少治疗时的毒副作用。该文对以CAR为基础修饰不同效应细胞的细胞疗法的研究进展进行了综述。     

DC-CIK对K562/A多药耐药基因mdr1表达的影响

目的：体外观察树突状细胞（dendritic cell,DC）联合细胞因子诱导的杀伤细胞（cytokine inducedkiller,CIK）对K562/A细胞株多药耐药基因mdr1表达的影响。方法：采集健康人的外周血,分离出单个核细胞（peripheral blood mononuclear cell,PBMC）,在体外加入多种细胞因子经诱导生成DC及CIK细胞,以流式细胞仪检测其表面标志,将DC细胞内加入K562/A细胞裂解物致敏后,再与CIK细胞混合培养48小时。将致敏后的DC-CIK细胞与K562/A及K562分组培养后以荧光定量PCR检测其mdr1基因表达的情况,PBMC作为对照组。结果：RT-PCR中可见K562/A＋DC-CIK组中mdr1 mRNA表达较K562/A明显降低,经荧光定量PCR观察到K562/A内mdr1 mRNA表达为K562的10.27倍、K562/A/PBMC略低于未处理的K562/A（P〉0.05）,K562/A/DC-CIK细胞中mdr1 mRNA含量较K562/A、K562/A/PBMC少（P〈0.05）。DC-CIK细胞与细胞株混合培养后,mdr1基因表达较混合培养前明显降低。结论：实验数据显示DC-CIK可使耐药细胞株内mdr1基因表达下调。但K562与DC-CIK混合培养后该基因降低不明显,提示该基因在细胞中存在着基础表达,意义在于维持细胞内稳态。目前针对逆转白血病耐药的研究较少,需要多进行相关研究以拓宽细胞免疫治疗在逆转耐药领域的应用。DC-CIK是具有发展潜力的抗肿瘤方法。本实验将为下一阶段研究逆转耐药的机制提供依据,DC-CIK细胞免疫疗法有望成为逆转肿瘤耐药的新方法。     

Selective Expansion of Chimeric Antigen Receptor-targeted T-cells with Potent Effector Function using Interleukin-4

Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR⁺ T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4αβ) was engineered by fusion of the IL-4 receptor α (IL-4Rα) ectodomain to the β_c subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4αβ resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4αβ with γ_c was implicated in signal delivery. Next, human T-cells were engineered to co-express 4αβ with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.     

人外周血树突状细胞体外诱导扩增的研究

目的:探讨可用于临床治疗功能成熟的DC体外扩增的优化培养方案。方法:胎牛血清培养基联合细胞因子rhGM-CSF(100ng/mL)和rhIL-4(50 ng/mL)扩增人外周血分离的单个核细胞,细胞培养分别按5×106/mL、6×106/mL和7×106/mL的密度,加入6孔培养板。第6d加入rhTNF-a(100 ng/mL)联合培养,分别于第6 d,第9 d和12 d收获细胞。从形态学、细胞表面标志方面进行鉴定。结果:显微镜观察,经过9 d诱导后,培养细胞具有典型树突细胞外形。流式细胞仪分析,6×106/mL密度的细胞培养组培养到第9天最宜。结论:细胞具有典型的DC的形态特征,细胞表型及功能实验证实其DC的特性,说明建立的血清培养基联合细胞因子rhGM-CSF、rhIL-4和rhTNF-a体外诱导DC的方法是切实可行的。     

Effect of BCG stimulus on proinflammatory cytokine production in laryngeal cancer

Background  Evaluate the production of TNF and IL-6 in the supernatant of peripheral blood mononuclear cell (PBMC) cultures of patients with supraglottic laryngeal cancer before and after surgical treatment. Materials and methods  Adherent cell cultures were stimulated with LPS and BCG. Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cytokine concentration was determined by ELISA in supernatants of mononuclear cell cultures. Results  In non-stimulated cultures, lower TNF cytokine levels were detected during the late postoperative (LP) period compared to control (P = 0.02). LP TNF and IL-6 levels were high in cultures stimulated with LPS compared with the preoperative period (PREOP) (P = 0.007; P = 0.008, respectively). Stimulation with BCG led to increased levels of TNF and IL-6 during the LP period compared to control (P = 0.001; P = 0.04, respectively). Conclusion  BCG is able to modulate the immune response of patients with advanced supraglottic laryngeal cancer in vitro, increasing the secretion of TNF and IL-6 by macrophages during the postoperative period.     

DCs-CIK细胞免疫治疗联合化疗治疗晚期非小细胞肺癌的疗效观察

目的:探讨树突状细胞(DCs)和细胞因子诱导的杀伤(CIK)细胞免疫治疗联合化疗对晚期非小细胞肺癌患者的治疗效果。方法:将我院2012年2月到2014年2月就诊的72例晚期非小细胞肺癌患者随机分为对照组(n=36,单纯化疗组)和实验组(n=36,DCs-CIK细胞免疫联合化疗组)。比较两组患者治疗后的疗效、治疗前后免疫功能,并运用Kamofsky(KPS)评分来评估两组患者治疗后生活质量的改善情况。结果:实验组的疾病控制率(DCR)77.78%显著高于对照组的52.78%(P0.05)。治疗后实验组患者外周血CD3+、CD8+及NK细胞所占的比值较治疗前均上升显著(P0.05);治疗后对照组患者外周血CD3+、CD8+及NK细胞所占的比值较治疗前下降显著(P0.05)。治疗后实验组KPS评分提高率明显高于对照组(P0.05)。结论:DCs-CIK细胞免疫联合化疗能够提高晚期非小细胞肺癌患者的DCR,且显著改善患者的免疫功能和生活质量。     

Generation of dendritic cell–tumor cell hybrids by electrofusion for clinical vaccine application

Vaccination with hybrids comprising fused dendritic cells (DCs) and tumor cells is a novel cancer immunotherapy approach designed to combine tumor antigenicity with the antigen-presenting and immune-stimulatory capacities of DCs. For clinical purposes, we have incorporated a large-scale process for the generation of clinical-grade DCs together with novel electrofusion technology. The electrofusion system provides for ease and standardization of method, efficient DC–tumor cell hybrid formation, and large-quantity production of hybrids in a high-volume (6-ml) electrofusion chamber. In addition, we have evaluated DC electrofusion with a variety of allogeneic human tumor cell lines with the rationale that these tumor cell partners would prove a ready, suitable source for the generation of DC–tumor cell hybrid vaccines. The DC production process can generate 6×10⁸ to 2×10⁹ DCs from a single leukapheresis product (~180 ml). As determined by FACS analysis, electrofusion of 6×10⁷ total cells (1:1 ratio of DC and tumor cells) resulted in a consistent average of 8–10% DC–tumor cell hybrids, irrespective of the tumor type used. Hybrids were retained in the population for 48 h postfusion and following freezing and thawing. Upon pre-irradiation of the tumor cell partner for vaccine purposes, the overall fusion efficiency was not altered at doses up to 200 Gy. Evaluation of DC–tumor cell hybrid populations for their ability to stimulate T-cell responses demonstrated that electrofused populations are superior to mixed populations of DCs and tumor cells in generating a primary T-cell response, as indicated by IFN- release. Moreover, hybrids comprising HLA-A^*0201 DCs and allogeneic melanoma tumor cells (Colo 829 cell line) stimulated IFN- secretion by antigen-specific CD8⁺ T cells, which are restricted for recognition of a melanoma gp100 peptide antigen (gp100_209–217) within the context of the DC HLA haplotype. Maturation of the DC-Colo 829 cell hybrid population served to further improve this T-cell gp100-specific response. Overall, our results are promising for the large-scale generation of electrofused hybrids comprising DCs and allogeneic tumor cells, that may prove useful in human vaccine trials.     

小鼠肝脏树突状细胞前体分离培养及影响其成熟因素的初步探讨

本次首次建立了从小鼠肝脏分离、培养、扩增肝树突状细胞（ｈｅｐａｔｉｃｄｅｎｄｒｉｔｉｃｃｅｌ,ＨＤＣ）前体的方法。采用经门静脉插管胶原酶二步循环灌流法及Ｐｅｒｃｏｌ不连续密度梯度离心法分离非肝细胞,得到含ＨＤＣ前体组分,将其分三组培养：第一组加入重组小鼠粒细胞／巨噬细胞集落刺激因子（ＭｒＧＭＣＳＦ）,待培养９天后,将细胞转移至鼠尾胶原上继续培养。第二组加入ＭｒＧＭＣＳＦ培养,但细胞不转移。第三组为对照组,仅加完全培养液。结果显示：第一、二组细胞于培养４天可见有许多细胞集落形成,第一组细胞转移至鼠尾胶原上培养１天后,细胞集落减少,而培养液中ＤＣ密度明显增加,光镜和扫描、透射电镜观察证明培养ＨＤＣ纯度达９５％,第二组则无上述细胞释放现象。实验结果提示,ＨＤＣ前体在体外受细胞因子刺激能大量扩增,但必须在胶原存在下才能分化成熟     

An immunotherapy approach with dendritic cells genetically modified to express the tumor-associated antigen,HER2

Dendritic cells (DC), genetically modified to express ovalbumin by the retroviral vector GCDNsap, can elicit stronger anti-tumor immunity than those loaded with the peptides. To assess the clinical feasibility of the strategy, such DC were prepared by differentiation of hematopoietic progenitor cells transduced with the human epidermal growth factor receptor 2 (HER2). When inoculated in mice, the DC primed both HER2-specific cytotoxic T lymphocytes and type 1 T helper lymphocytes, resulting in production of HER2-specific antibody. Of importance is that the antibody mediated antibody-dependent cellular cytotoxicity and opsonization. The potent anti-tumor effects were also confirmed by results of experiments using HER2-transgenic mice. Inoculation of HER2-transduced DC resulted in longer disease-free survival of treated mice that showed significant reduction of primary and metastatic tumors. Interestingly, footpad inoculation resulted in stronger anti-tumor effects compared to subcutaneous administration and induced higher levels of the HER2-specific antibody, suggesting that an important role of humoral immunity in anti-tumor effects for malignancies with membrane-type tumor-associated antigens (TAA). Taken together, vaccination of the TAA-transduced DC may represent a promising form of therapy for breast cancers expressing HER2.     

MYCN as a target for cancer immunotherapy

MYCN is a potential target for cancer immunotherapy by virtue of its overexpression in numerous human malignancies and its functional role in tumour progression. Here we show limited expression of MYCN in normal human tissues indicating that anti-MYCN immune responses are unlikely to cross react with self tissues. An HLA-A2 restricted ten amino acid peptide epitope from MYCN, VILKKATEYV, was used to stimulate cytotoxic T cell lines from the peripheral blood of normal blood donors, and from a patient with MYCN amplified neuroblastoma. Strong and specific activity was seen against each MYCN overexpressing cell line and against autologous tumour cells. We generated two CTL clones capable of killing cells pulsed with as low as 0.5 nM of VIL peptide. Therefore strong and specific immune responses against MYCN expressing tumours are possible in patients with the most common HLA class 1 type in the Caucasian population.     

Successful induction of clinically competent dendritic cells from granulocyte colony-stimulating factor-mobilized monocytes for cancer vaccine therapy

Recent studies have suggested that dendritic cell (DC)-based immunotherapy is one promising approach for the treatment of cancer. We previously studied the clinical toxicity, feasibility, and efficacy of cancer vaccine therapy with peptide-pulsed DCs. In that study, we used granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood monocytes as a cell source of DCs. However, previous investigations have suggested that G-CSF-mobilized peripheral blood monocytes produce reduced levels of proinflammatory cytokines such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α. These T helper (Th)-1-type cytokines are thought to promote antitumor immune response. In this study, we assessed the functional abilities of DCs generated from G-CSF-mobilized monocytes obtained from 13 patients with CEA-positive advanced solid cancers. Peripheral blood mononuclear cells were obtained from leukapheresis products collected before and after systemic administration of G-CSF (subcutaneous administration of high-dose [5–10 μg/kg] human recombinant G-CSF for five consecutive days). In vitro cytokine production profiles after stimulation with lipopolysaccharide (LPS) were compared between monocytes with and without G-CSF mobilization. DCs generated from monocytes were also examined with respect to cytokine production and the capacity to induce peptide-specific T cell responses. Administration of G-CSF was found to efficiently mobilize peripheral blood monocytes. Although G-CSF-mobilized monocytes (G/Mo) less effectively produced Th-1-type cytokines than control monocytes (C/Mo), DCs generated from G/Mo restored the same level of IL-12 production as that seen in DCs generated from C/Mo. T cell induction assay using recall antigen peptide and phenotypic analyses also demonstrated that DCs generated from G/Mo retained characteristics identical to those generated from C/Mo. Our results suggest that G-CSF mobilization can be used to collect monocytes as a cell source for the generation of DCs for cancer immunotherapy. DCs generated in this fashion were pulsed with HLA-A24-restricted CEA epitope peptide and administered to patients safely; immunological responses were induced in some patients.