无血清培养基与完全培养基对体外激活的A-NK细胞支持作用的比较

A-NK细胞由于其体外增殖和抗肿瘤能力强,对正常细胞无杀伤作用等优点,成为近年来肿瘤过继免疫疗法的研究热点.本研究多方面比较无血清培养基AIMV与完全培养基对体外A-NK细胞的支持作用,发现无血清培养基用于A-NK细胞的临床生物治疗,即不减少A-NK细胞的数量和活性,又能增加其临床应用的安全性.     

A-NK/IL-2抗肿瘤作用的新研究

目的研究A-NK/IL-2在完全培养基(CM)和无血清培养基(AIMV)中体外扩增及杀伤肿瘤细胞作用.同时探讨IL-12对于A-NK/IL-2治疗的辅助作用.方法用MIT法比较不同培养条件下的A-NK细胞体外增殖能力,并测定细胞体外杀伤肿瘤细胞的活性.通过做扫描和透射电镜,对A-NK细胞杀伤的肿瘤细胞进行形态学观察.结果不同培养条件下的A-NK细胞均可在短期内大量扩增(P＞0.05),且均有很强的杀伤肿瘤的活性(P＞0.05).被A-NK细胞杀伤的肿瘤细胞的死亡形式是溶解坏死(necrosis)和凋零坏死(apoptosis).结论 AIMV可替代CM用于A-NK细胞的培养.联合应用IL-12激活A-NK细胞可降低IL-2的使用剂量,从而避免IL-2带来的副作用.本研究为A-NK细胞的进一步研究和临床应用推广奠定了理论基础.     

无血清培养基与完全培养基对体外诱导免疫细胞支持作用的比较

目的：多方面比较无血清培养基AIMV与完全培养基对体外诱导免疫细胞支持作用。方法：分别用AIMV及完全培养基在IFN-γ、IL-2,及抗-CD3单抗存在的条件下培养人外周血单个核细胞,比较细胞的增殖能力、细胞表型、及细胞因子分泌能力,并比较不同培养基培养细胞回输体内后的抑制病毒效果。结果：与完全培养基培养细胞相比,无血清培养基AIMV培养细胞增殖能力与之相当;CD25的表达率增高,表达持续时间延长;细胞因子IFN-γ的分泌时间延长;回输体内后的抑制病毒作用更明显。结论：无血清培养基AIMV用于培养临床治疗用的免疫细胞,综合效果优于完全培养基。     

无血清培养基与完全培养基对体外诱导扩增CIK细胞及抗肿瘤活性的比较研究

目的:比较无血清培养基AIMV与完全培养基对体外诱导扩增CIK细胞的效果。方法:分别用AIMV及完全培养基加入4种细胞因子(IFNγ、IL2、IL1及OKT3)将脐带血单个核细胞(CBMNCs)诱导成CIK细胞,比较细胞的增殖能力、细胞表型、对肿瘤细胞的增殖抑制作用及其诱导肿瘤细胞凋亡等几个方面。结果:与完全培养基比较,无血清培养基AIMV培养的CIK细胞增殖高峰较晚(14～17d),但增殖倍数高(倍),在细胞表型、对三株肺癌细胞的生长抑制作用及不同效靶比诱导细胞凋亡方面两种培养基培养的CIK细胞差异无统计学意义,P>0.05。结论:无血清培养基AIMV可代替完全培养基。     

无血清培养基与完全培养基体外诱导扩增CIK细胞分泌细胞因子水平的比较

目的比较无血清培养基(AIMV)与完全培养基(CM)体外诱导扩增CIK细胞及CIK细胞分泌细胞因子水平。方法分别用AIMV及完全培养基加入4种细胞因子(IFN-γ、IL-2、IL-1及OKT3)将脐带血单个核细胞(CBMNCs)诱导成CIK细胞,比较细胞的增殖能力、细胞表型及分泌细胞因子水平。结果与完全培养基比较,无血清培养基培养的CIK细胞增殖高峰较晚(14~17天),但增殖倍数高,在细胞表型、分泌细胞因子水平方面两种培养基培养的CIK细胞均无明显的差别(P>0.05)。结论无血清培养基可代替完全培养基。     

A-NK细胞抗肝癌Walker256瘤株的实验研究

目的:观察A-NK细胞的体外生长与增殖,以及杀伤肿瘤细胞的能力,研究A-NK细胞局部注射的体内抗肿瘤作用。方法:用淋巴细胞分离液分离单个核细胞(PBMC),培养LAK细胞和A-NK细胞,分别将LAK细胞、A-NK细胞和Walker-256瘤株细胞接种于培养板中,培养24h后,用四甲基偶氮唑盐(MTT)方法测定吸光度,计算肿瘤杀伤率。复制鼠肝癌模型,分别将LAK、A-NK细胞经肝动脉注入鼠肝癌模型中,观察两组动物的生存时间。结果:A-NK细胞的增殖明显快于LAK细胞(P<0.05),A-NK细胞对肿瘤细胞的杀伤活性比LAK细胞强(P<0.01),A-NK细胞能显著延长肝癌动物模型的生存期,与LAK细胞比较,具有显著性差异(P<0.01)。结论:A-NK细胞具有增殖快,抗瘤活性强,在体内能抑制肿瘤生长,延长荷瘤动物的生存期。     

HSP70-TKD诱导的NK细胞杀伤肝癌细胞的实验研究

目的:探讨从人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中获得足够数量和高细胞毒活性的CD3-CD56 NK细胞的方法,并观察其对肝癌细胞HepG2杀伤效应.方法:以干细胞培养基(SCGM)为基础培养基,在不同浓度的抗CD3单抗、IL-2和PHA的培养条件下,从健康人PBMC中高效扩增获得CD3-CD56 NK细胞;以不同剂量的HSP70-TKD诱导NK细胞的活性,MTT法测定HSP70-TKD诱导的NK细胞对HepG2和榄香烯处理的HepGZ细胞的杀伤活性.结果:建立了从人PBMC体外扩增CD3-CD56 NK细胞体系:以SCGM为基础培养基,在600 U/mL IL-2、500 μg/L抗CD3单抗和50 mg/L PHA联合作用下,培养14 d细胞扩增了116倍,舍有(66.97±8.76)?3-CD56 NK细胞;HSP70-TKD诱导的NK细胞对HepG2杀伤活性低.与未经TKD诱导的NK细胞之间差异无统计学意义,P=0.298;而对税香烯处理过的HepG2杀伤活性高,诱导组与未诱导组之间差异有统计学意义,P=0.008;当TKD浓度为2.0 mg/L时,杀伤活性最高为(58.8±3.4)%.结论:以干细胞生长培养基为基础培养基,在抗CD3单抗和IL-2协同刺激下体外大量扩增NK细胞,HSP70-TKD诱导的NK细胞对细胞膜HSP70阳性的肿瘤细胞杀伤活性高,而对于细胞膜HSP70低表达的肿瘤细胞杀伤活性低.     

苯乙酸钠降低肿瘤细胞产生免疫抑制因子

本文报道用无毒性的活体荧光染料—钙黄绿素 AM标记的肿瘤细胞与人单核细胞共培养4小时,用Cyto Fluo 2300测定细胞粘附能力的荧光检测技术.结果表明IFN-γ和LPS激活的人单核细胞能迅速地与悬浮生长的人肿瘤细胞、B淋巴母细胞及淋巴细胞粘附.激活的人单核细胞表达更多的CD11a、CD11b、CD11c和CD18,对肿瘤细胞有更多的粘附性.抗体封闭及蛋白激酶抑制物处理细胞,粘附能力均受到明显的抑制.IFN-γ和LPS处理肿瘤细胞也能明显增加两种细胞间的粘附能力.上述结果提示人单核细胞粘附能力的增加与整合素及肿瘤细胞所表达的相关配体增加有关.     

急性髓性白血病细胞体外培养及其生物学特性的研究

目的:研究急性白血病细胞体外培养的方法及其在体外培养过程中生物学特性的变化.方法:分别收集10例急性髓性白血病细胞,用含细胞因子无血清液体培养基进行培养2～4周,并对培养前后细胞进行细胞计数,流式细胞仪检测CD33、CD13、CD34及CD14表面抗原鉴定细胞分化,半固体培养法对培养前后的白血病细胞进行集落形成能力检测.结果: 含细胞因子无血清液体培养基能有效支持AML细胞进行体外短期培养及增殖, 14 d时细胞得到明显增殖,达(25 1±11 7)倍,优于培养前,P<0 05;培养28 d时细胞数较前明显减少,P<0 05.集落形成单位培养14 d后与培养前比较差异无统计学意义,P>0 05;但培养28 d后显著低于培养前,P<0 05.在体外悬浮培养14 d时,CD33、CD13、CD34及CD14的表达率与培养前差异无统计学意义(P>0 05),但至28 d时,CD33、CD13及CD14的表达率高于培养前,而CD34表达率比培养前降低,P<0 05.结论: 含细胞因子无血清液体培养基能有效支持急性髓性白血病细胞短期体外培养并维持其生物学特性.     

HLA-G反义基因增强免疫效应细胞杀伤活性的研究

目的:研究HLA-G反义寡核苷酸逆转肿瘤细胞免疫逃逸,提高免疫效应细胞识别杀伤活性的作用和机制.方法:采用反义核酸技术合成HLA-G反义寡核苷酸(ASODN),硫代化修饰与脂质体形成复合物,转导入高表达HLA-G的绒毛膜癌细胞系JEG-3.采用RT-PCR方法检测HLA-G mRNA表达水平的变化;流式细胞术检测细胞表面HLA-G蛋白表达水平的变化;MTT法检测:NK杀伤活性、CD3AK杀伤、增殖活性的变化;ELISA方法探讨ASODN调节CD3AK细胞因子IFN-γ产生的变化.结果:HLA-G ASODN可显著抑制HLA-G mRNA和蛋白水平的表达,逆转可溶型HLA-G分子对NK细胞杀伤活性的抑制作用;可部分逆转HLA-G分子对CD3AK细胞产生IFN-γ的抑制作用,并增强CD3AK的增殖活性,增强JEG-3细胞对CD3AK的杀伤敏感性.结论:HLA-G ASODN通过抑制肿瘤细胞HLA-G mRNA和蛋白水平的表达,增强NK,CD3AK的杀伤作用和机制,发挥逆转肿瘤细胞免疫逃逸作用.     

ANTITUMOR AQCTIVITY OF REGIONAL ADMINISTRATION OF A-NK CELLS IN VIVO

Objective： To observe the proliferation of A-NK in vitro and antitumor activity in vivo. Methods： The growth curve of A-NK and NA-NK cells was drawn in vitro. In the rat model, we compared the regional administration of A-NK-/IL-2 with the systemic administration. Results： The expansion of A-NK cells reached to climax on day 10 in the culture, increased 16.08 folds compared with the only 3.36 folds for NA-NK cells. In the rat model, we found that the regional administration of A-NK/IL-2 was better than systemic administration or administration of NA-NK/IL-2 not only in tumor infiltration and antitumor response, but also in the survival rate of rats （P〈0.05）. Conclusion： A-NK cells is a new immune effecter cells with high expansibility and high antitumor activity in vivo and in vitro.     

Targeting of products of genes to tumor sites using adoptively transferred A-NK and T-LAK cells

Despite successes in animals, cytokine gene expression selectively in human tumors is difficult to achieve owing to lack of efficient delivery methods. Since interleukin (IL)-2-activated natural killer (A-NK) and phytohemagglutinin and IL-2 activated killer T (T-LAK) cells, as previously demonstrated, localize and accumulate in murine lung tumor metastases following adoptive transfer, we transduced them to test their ability to deliver products of genes selectively to tumors. Assessments of transduction efficiency in vitro demonstrated that adenoviral transduction consistently resulted in high (>60%) transduction rates and substantial expression of transgenes such as GFP, Red2, luciferase, beta-galactosidase and mIL-12 for at least 4 days. In vivo experiments illustrated that Ad-GFP transduced A-NK and Ad-Red2 (RFP) transduced T-LAK or mIL-12 transduced A-NK cells localized 10-50-fold more or survived significantly better than mock transduced cells, respectively, within lung metastases than in the surrounding normal lung tissue. Most importantly, mIL-12 transduced A-NK cells provided a significantly greater antitumor response than non-transduced A-NK cells. Thus, adoptive transfer of A-NK and T-LAK cells represents an efficient method for targeting products of genes to tumor sites.     

Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases

The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 microg kg(-1) MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 microg kg(-1) free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection.     

Infiltration of interleukin-2-inducible killer cells in ascitic fluid and pleural effusions of advanced cancer patients

Using ascitic fluid or pleural effusion obtained from 13 ovarian or metastatic breast cancer patients, we separated tumor cells from effusion-associated lymphocytes (EAL) with Percoll density centrifugation. Lymphocytes were incubated with recombinant interleukin 2 (IL-2) for 3-4 days and then assessed for tumoricidal activity in a 51chromium-release assay. The IL-2-activated EAL were found to lyse autologous fresh tumor cells, as well as allogeneic fresh tumor cells and FMEX tumor cells, a melanoma cell line which is resistant to natural killer cell activity but is sensitive to lysis by lymphokine-activated killer cells. There was little or no tumoricidal activity seen in freshly isolated EAL or in EAL which were cultured in medium without IL-2. Phenotypically, the IL-2-activated EAL were largely CD3-, although some cytolytic activity was found in CD3+ populations. Also, most activity was found in cells positive for CD2 (OKT11) and CD16 (Leu 11b), and negative for the monocyte marker Leu M3. These results indicate that the activated cell types found in EAL were predominantly natural killer/lymphokine-activated killer-like with a small contribution from T-cells. Finally, EAL were readily activated by IL-2 in medium containing autologous effusion fluid, indicating that in situ activation of tumoricidal activity by IL-2 can occur in the face of potentially inhibitory substances or cells that may exist in the effusions. Direct introduction of IL-2 may therefore be a potential therapeutic modality of effusion-forming cancers.     

Antitumor effect of large doses IL-2-activated HLA haploidentical peripheral blood stem cells on refractory metastatic solid tumor treatment

OBJECTIVE: The traditional immunotherapy for patients with refractory metastatic solid tumors is limited because tumors induce immunosuppression. New treatment is, therefore, needed. The aim of this study was to evaluate the clinical efficacy of infusion of high-dose interleukin (IL)-2-activated allogeneic haploidentical peripheral blood stem cells (haplo-PBSCTs) on patients with an advanced stage of refractory solid tumors. METHODS: This study involved 11 patients with refractory metastatic tumors and haploidentical relatives as donors for haplo-PBSCs. The therapeutic outcome of the IL-2-activated haplo-PBSC infusion and patients' cytokine levels were evaluated. The cytotoxicity of IL-2-activated haplo-PBSCs for tumor cells was determined using in vitro cytotoxicity assays. RESULTS: A range from 2.5 to 5.6 x 10(10) of activated haplo-PBSCs were harvested after exposure to rhIL-2, along with a significant increase in the proportion of natural killer (NK) cells and activated lymphocytes (CD69+ and CD25+), and enhanced cytotoxicity of haplo-PBSCs for several tumor cell lines. Following treatment, 1 (1/11) patient achieved a partial response (PR), 1 (1/11) achieved a mild response (MR), 6 (6/11) achieved stable disease (SD), and 3 (3/11) achieved progressive disease (PD). For all of the 11 patients, the median progression-free survival (PFS) was 5 months (3-14 months). We also observed the phenomenon of Th2 shifted to Th1, which played a crucial role in cancer immunotherapy. CONCLUSIONS: The adoptive transfusion of IL-2-activated haplo-PBSCs has potent antitumor effects both in vitro and in vivo. This finding suggests that IL-2-activated haplo-PBSCs may serve as an alternative therapy for advanced-stage solid tumors, especially for those patients who are refractory or ineligible for chemo- or radiotherapy.     

Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells

We investigated the in vitro effects of combining interleukin-18 (IL-18) and IL-2 on human lymphocytes. The combined use of these two cytokines synergistically enhanced the proliferation, cytolytic activity, and interferon-gamma production of peripheral blood mononuclear cells. Phenotypic analysis revealed a preferential expansion of CD56+CD3- cells and an up-regulation of IL-2 receptor-alpha expression on natural killer cells. Isolated natural killer cells showed a substantial increase in proliferation and cytotoxicity compared with CD4+ and CD8+ T cells. The combined use of IL-18 and IL-2 should be considered a viable strategy to induce an antitumor response in vivo.     

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium （supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor） maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum- containing medium-cultured U251 cells （24%-35% vs. 8%-11%, P 〈 0.001）. Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of BT-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium （P 〈 0.01）. Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express BT-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.     

Increased cytotoxicity against B-chronic lymphocytic leukemia by cellular manipulations: potentials for therapeutic use

B-cell chronic lymphocytic leukemia (CLL) is characterized by profound immune dysfunction and a marked resistance to apoptosis. Understanding the cellular biology of immune effector cells from CLL patients as well as leukemic target cells is essential to developing immune mediated therapeutic strategies for CLL. In this study, an immortal CLL cell line called WSU-CLL has been used to study the characteristics of B-cell CLL as a tumor target for natural killer (NK), activated natural killer, and lymphokine activated killer (LAK) cells. The WSU-CLL cells were significantly less (p<0.001) susceptible to NK cell mediated cytotoxicity compared to K562, a standard tumor target cell line. In vitro activation of effector cells with either short term, low dose IL-2 or long term, high dose IL-2 significantly increased the susceptibility of CLL cells for cell mediated killing. The addition of CD1a+/CD3-/CD4+/CD80+/CD83+ dendritic cells derived from human umbilical cord blood increased the cytotoxicity of LAK cells against WSU-CLL. There is an increased expression of Bcl-2 and decreased expression of Fas on WSU-CLL cells as determined by RT-PCR techniques indicating possible roles for these genes in exerting resistance to immune cell mediated lysis. When Bcl-2 expression was downregulated in WSU-CLL cells using gene specific antisense oligonucleotides, the susceptibility of WSU-CLL cells to the cytotoxicity of chemotherapeutic agent Fludarabine was increased. Thus, our results suggest that in vitro activation with cytokines, addition of accessory cell populations such as dendritic cells and/or manipulation of key gene expression i.e. down regulation of Bcl-2 might be potential strategies to increase the antitumor cytotoxicity against CLL cells.     

Immunotherapy with effector cells and IL-2 of lymph node metastases of human squamous-cell carcinoma of the head and neck established in nude mice.

We have previously reported that immune anti-tumor effector cells, both cytotoxic T lymphocytes (CTLs) and IL-2-activated natural killer (A-NK) cells, are effective at eliminating human head-and-neck cancer (HNC) targets in vitro and in vivo in xenograft models. In this study, these 2 types of human effector cell were compared for the ability to prevent the development of lymph node metastases in a metastasis model of human squamous-cell carcinoma of the head and neck (SCCHN) established in nude mice. A tumor cell line, OSC-19, was injected into the floor of the mouth in nude mice, and the tumor grew progressively and metastasized to cervical lymph nodes by day 21. As effector cells, a human HLA-A2-restricted CTL line recognizing a shared antigen on OSC-19 and human non-MHC-restricted A-NK cells were used. Both types of effector cell mediated high levels of lysis against OSC-19 targets in 4-hr (51)Cr-release assays. Administration of human CTLs or A-NK cells and IL-2 to the site of tumor growth in mice with 7-day OSC-19 tumors resulted in significant reduction of the number of lymph node metastases relative to untreated or sham-operated controls or to mice treated with IL-2 without the effector cells. Our results suggest that in a xenograft model of human SCCHN implanted in the oral cavity of nude mice, the development of lymph node metastases can be successfully controlled by adoptive transfer of human SCCHN-specific CTLs or SCCHN-reactive A-NK cells plus IL-2.