Cell-based immunotherapy of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most aggressive and lethal primary glial brain tumor. It has an unfavorable prognosis and relatively ineffective treatment protocols, with the median survival of patients being ~15 months. Tumor resistance to treatment is associated with its cancer stem cells (CSCs). At present, there is no medication or technologies that have the ability to completely eradicate CSCs, and immunotherapy (IT) is only able to prolong the patient''s life. The present review aimed to investigate systemic solutions for issues associated with immunosuppression, such as ineffective IT and the creation of optimal conditions for CSCs to fulfill their lethal potential. The present review also investigated the main methods involved in local immunosuppression treatment, and highlighted the associated disadvantages. In addition, novel treatment options and targets for the elimination and regulation of CSCs with adaptive and active IT are discussed. Antagonists of TGF-β inhibitors, immune checkpoints and other targeted medication are also summarized. The role of normal hematopoietic stem cells (HSCs) in the mechanisms underlying systemic immune suppression development in cases of GBM is analyzed, and the potential reprogramming of HSCs during their interaction with cancer cells is discussed. Moreover, the present review emphasizes the importance of the aforementioned interactions in the development of immune tolerance and the inactivation of the immune system in neoplastic processes. The possibility of solving the problem of systemic immunosuppression during transplantation of donor HSCs is discussed.     

Dendritic cell immunotherapy for glioblastoma

Dendritic cell immunotherapy is emerging as a promising addition to the multimodal treatment of patients with glioblastoma multiform. Initial Phase I and II trials have demonstrated favorable outcomes with minimal toxicity. In this editorial, the current status and the future challenges of this therapy are discussed.     

免疫治疗联合放化疗治疗胶质母细胞瘤的研究进展

目前胶质母细胞瘤（glioblastoma,GBM）的标准治疗方法仍然是在最大安全程度的手术切除基础上辅以放化疗,但其5年生存率仍＜10%。免疫治疗如树突状细胞(dendritic cell,DC)疫苗、表皮生长因子受体突变体(EGFRvIII)疫苗、热休克蛋白（heat shock proteins,HSPs）疫苗等在临床试验中已经取得了巨大成就,临床III期试验也证明了免疫治疗与放化疗有协同作用。细胞毒性电离辐射是一种引起促炎信号级联免疫活化辅助细胞死亡的治疗方法,借此可以利用免疫治疗抗肿瘤。肿瘤免疫治疗的发展,使得免疫治疗可能成为继手术、放化疗后GBM治疗的另一个有效方法。寻找新的免疫治疗方法是未来GBM治疗的主要研究方向。本文就目前GBM的治疗策略及困境和放化疗联合免疫检查点抑制剂、DC疫苗以及EGFRvIII疫苗等免疫治疗研究进展作一综述。     

多发性骨髓瘤T细胞免疫治疗

多发性骨髓瘤(multiple myeloma,MM)是一种目前难以治愈的恶性浆细胞肿瘤,近年来蛋白酶体抑制剂和免疫调节剂的应用明显改善了治疗效果,但大部分MM患者最终难以逃脱复发的结局.越来越多的证据表明经体外活化或基因修饰的T细胞能在体内特异性的杀伤肿瘤细胞,有望治愈MM,尤其是近年来嵌合抗原受体修饰T细胞(chimeric antigen receptor-modified T cells,CAR-T)的发展,其能在体内以非MHC限制性方式特异杀伤表达特定抗原的肿瘤细胞,为MM的治疗提供了新的思路.本文将讨论T细胞免疫治疗的发展和技术改进,就目前较有前景的几种T细胞治疗方法做一综述.     

DC-CIK过继性细胞免疫治疗对卵巢癌患者的预后及免疫功能的影响

目的:探讨自体DC-CIK细胞疗法改善卵巢癌患者的预后及免疫功能的能力。方法:回顾性分析2014年1月至2015年12月在中国医科大学附属盛京医院均接受了规范的初始治疗、达到完全缓解（CR）的卵巢癌患者90例。分析比较其基本条件、免疫指标、无瘤生存时间及不良反应。结果:经DC-CIK生物免疫治疗的患者外周血CD3+、CD4+细胞百分率,CD3+、CD8+细胞百分率及CD4+/CD8+比值均上升,NK细胞即CD16+、CD56+细胞增多,同时随着疗程数的增多,升高的更加明显（P<0.05）。一线治疗后,单纯观察组平均无瘤生存时间为15.88±2.7个月明显少于DC-CIK生物免疫治疗组的22.5±4.13个月（P=0.039）。其中使用DC-CIK生物免疫治疗疗程≥5程的平均无瘤生存时间为27.25±4.53个月明显高于DC-CIK生物免疫治疗疗程≤4程的19.69±2.81个月,两者相比差异无明显统计学意义（P=0.412）。所有患者DC-CIK细胞治疗后血常规及肝肾功能检测与治疗前及单纯观察组患者相比无明显变化,也未见明显不良反应。结论:DC-CIK免疫治疗提高卵巢癌患者的免疫反应,改善卵巢癌患者预后,未见明显不良反应。     

Gene-modified dendritic cells for immunotherapy against cancer

Dendritic cells (DCs) are described as professional antigen-presenting cells because of their superior T-cell stimulatory capacity. For this reason, attention is being focused on using DCs for clinical applications to treat cancer patients. Although preclinical studies are promising, the majority of clinical studies with DCs have not fulfilled the expectations, yet. The field of DC biology has progressed rapidly over the past years, leading to several options for the improvement of vaccination. Among the different parameters to investigate, this review focuses on the efficiency and biological and functional consequences of different gene transfer methods into different subsets of human DCs. Another important consideration for DC-based vaccination is the elucidation of the role of maturation and apoptosis during DC differentiation.     

NK细胞在肿瘤过继性免疫治疗中的临床应用与研究进展

目的 自然杀伤(natural killer,NK)细胞是固有免疫细胞,是机体抗肿瘤的第一道防线.NK细胞无需预先致敏即可杀伤肿瘤细胞,具有良好的安全性和抗肿瘤作用.NK细胞作为抗肿瘤过继免疫细胞治疗制剂在世界范围内广泛应用.本文总结NK细胞在肿瘤过继性免疫治疗中的临床应用与研究进展.方法 应用PubMed、Elsevier、Springer、Wiley Online Library和CNKI文献检索系统,以“NK细胞,NK细胞与肿瘤生物治疗,NK细胞与肿瘤过继性免疫治疗”为关键词,检索1964-06-2015-12的有关文献.纳入标准:1)与NK细胞生物学相关的文献;2)与NK细胞体外扩增相关的文献;3)与NK细胞过继治疗血液性肿瘤及实体瘤的Ⅰ期及Ⅱ期临床研究相关的文献;4)与未来NK细胞临床应用研究方向相关的文献.根据纳入标准分析文献81篇.结果 NK细胞对肿瘤的杀伤活性主要取决于细胞表面活化性受体和抑制性受体间的动态平衡.目前,广泛认为基于NK细胞的过继性免疫治疗是具有良好潜力的肿瘤治疗方案.能否在GMP水平体外激活、扩增获得大量的NK细胞对肿瘤的过继治疗至关重要.用于体外扩增的NK细胞的来源包括外周血、脐带血以及干细胞.用于刺激NK细胞扩增的方法也不尽相同,包括可溶性细胞因子、抗体以及其他分子等.目前,自体或同种异体NK细过继疗法在血液或实体肿瘤治疗中广泛应用,但国外更倾向于应用同种异体NK细胞过继治疗.结论 NK细胞在肿瘤的过继性免疫治疗中具有重要临床意义和应用潜力,同时也面临诸多技术难题有待突破.     

Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.     

癌相关CD8+记忆T细胞在过继免疫治疗中的应用

针对癌症患者的过继免疫治疗是临床肿瘤治疗研究中的热点话题。癌症患者体内天然存在着能够对恶性肿瘤细胞进行识别、攻击和杀伤的癌相关CD8+记忆T细胞,它们具有对少量肿瘤细胞产生快速免疫应答和持续长期发挥抗癌作用的优点。癌相关CD8+记忆T细胞主要存在于骨髓中,如将骨髓中的这部分T细胞分离出来,并以特定手段激活并回输体内,将可发挥强大的免疫治疗作用。为了帮助临床癌症治疗研究者们更加清晰地认识癌相关CD8+记忆T细胞的生物学特性、肿瘤杀伤能力和临床治疗效果,并妥善处理实际应用过程中遇到的各种问题,本文对近年有关癌相关CD8+记忆T细胞在肿瘤免疫治疗中应用的研究进展加以介绍。     

Isolation and expansion of allogeneic myeloma-specific interferon-gamma producing T cells for adoptive immunotherapy

Adoptive immunotherapy is a promising approach in the treatment of multiple myeloma. We have tested the identification, separation, and expansion of allogeneic myeloma-specific T cells in vitro. Irradiated myeloma cell line ARH 77 has been used to stimulate allogeneic CD4⁺ and CD8⁺ T lymphocytes. Activated myeloma-specific T cells that produced interferon-gamma were isolated using immunomagnetic beads and further expanded in vitro to numbers of up to 400×106 T cells. Specificity of the T lymphocytes was tested using a 5-(6-)carboxyfluoresceine diacetate succinimidyl ester (CFSE)—based cytotoxicity test. This study demonstrates the feasibility of identification and isolation of tumor-specific T cells from allogeneic donors that can be expanded in vitro to numbers useful for clinical applications.     

Serial assessment of circulating T lymphocyte phenotype and receptor repertoire during treatment of non-muscle invasive bladder cancer with adoptive T cell immunotherapy

Recurrence and progression of non-muscle-invasive bladder cancer (NMIBC), frequent despite the availability of multiple treatment modalities, may be partly explained by the presence of immunosuppressive cell populations. We hypothesized that progression of disease could be prevented by the administration of an activated T cell immunotherapy (ACT) at time points when immunosuppressive populations increased in peripheral blood. In an N-of-1 study, a patient with multiple primary bladder high grade urothelial carcinomas, previously treated with standard local resection and chemotherapy but with evidence of progression, received ACT consisting of dendritic cells mixed with cytokine induced killer cells (DC/CIK), intravenously 18 times over a 6 year period at indicated time of observed increases in peripheral blood immunosuppressive CD8⁺/CD28^- cells. Peripheral blood was analyzed for T cell phenotype by flow cytometry, T cell receptor (TCR) repertoire, and circulating tumor DNA (ctDNA) by next generation sequencing (NGS) at the time of each infusion. Cystoscopy and pelvic CT scans were performed at routine intervals to assess clinical status of disease. There has been no recurrence or metastasis of urothelial carcinoma. Peripheral blood cytotoxic T cells and unique TCR clones increased and suppressive T cell populations decreased after DC/CIK infusions evidenced by the two more proof-of concept cases. ctDNA analysis detected mutations in six genes (ARID1B, MYCN, CDH23, SETD2, NOTCH4 and FAT1) which appeared at different times, but all of them disappeared after the DC-CIK infusions. These data suggest that DC/CIK infusions may be associated with beneficial changes in T cell phenotype, TCR repertoire, decreases in circulating tumor DNA and sustained recurrence-free survival.     

以PD-1/PD-L1为靶点的肺癌免疫治疗临床研究进展

免疫检查点的研究在近几年实现突破性进展,PD-1/PD-L1信号通路与免疫逃逸机制密切相关,针对阻断PD-1/PD-L1 通路免疫检查点的治疗在肺癌中取得明显效果。从Checkmate-017、Checkmate-057研究到KEYNOTE-010研究和OAK研究,逐 步奠定了PD1/PD-L1抑制剂作为化疗失败晚期NSCLC的标准治疗的地位;PD-1/PD-L1抑制剂可联合其他治疗方式,包括放疗、 化疗、靶向治疗以及其他免疫治疗等方式,在肺癌综合治疗中起到协同作用,从而提高了疗效。免疫检查点抑制剂带来了肺癌治 疗模式的改变,也对肿瘤疗效评价模式、治疗相关不良反应的处理带来挑战。另外,免疫检查点抑制剂的研发也有力地推动了精 准医疗的进展。     

巨噬细胞过继免疫治疗的实验研究

目的 研究脂质体瘤苗激活小鼠巨噬细胞的过继免疫治疗作用.方法 制备脂质体H22肝癌瘤苗,脂质体S180瘤苗,Freund's完全佐剂H22肝癌瘤苗以及对照脂质体瘤苗并免疫小鼠.用Hanks液清洗免疫鼠的腹腔收集糖原诱导的腹腔巨噬细胞.实验鼠腹腔注射1×105个H22肝癌细胞24小时后再腹腔给予各供体巨噬细胞5×106个/只.结果 脂质体H22瘤苗和Freund's佐剂H22瘤苗与对照组相比较,其免疫鼠提供的巨噬细胞具有较强的过继免疫治疗作用.结论 脂质体瘤苗激活的小鼠巨噬细胞具有特异性的可过继转移的抗瘤作用.     

以肿瘤干细胞为靶的免疫治疗

肿瘤干细胞(cancer stem cell,CSC)是肿瘤发生和转移的种子细胞,CSC具有自我更新、增殖和不完全分化的能力。CSC对化疗/放疗的抵抗以及免疫逃逸成为肿瘤复发的根源,要提高肿瘤治愈率必须彻底清除CSC。认识CSC的标记特征和"干性"调节关键分子才能有效和特异地攻击CSC。免疫治疗具有抗原识别的靶向性和时空效应性,是以CSC为靶的治疗的基础;以单克隆抗体和致敏的免疫细胞为主要治疗技术的免疫治疗清除CSC具有可实践性和挑战性。     

Regulatory T cells are not a strong predictor of survival for patients with glioblastoma

Background

Regulatory T cells (Tregs) are potentially prognostic indicators in patients with glioblastoma. If differences in frequency of Tregs in tumor or blood account for substantial variation in patient survival, then reliably measuring Tregs may enhance treatment selection and improve outcomes.

Methods

We measured Tregs and CD3+ T cells in tumors and blood from 25 patients with newly diagnosed glioblastoma. Tumor-infiltrating Tregs and CD3+ T cells, measured by quantitative DNA demethylation analysis (epigenetic qPCR) and by immunohistochemistry, and peripheral blood Treg proportions measured by flow cytometry were correlated with patient survival. Additionally, we analyzed data from The Cancer Genome Atlas (TCGA) to correlate the expression of Treg markers with patient survival and glioblastoma subtypes.

Results

Tregs, as measured in tumor tissue and peripheral blood, did not correlate with patient survival. Although there was a correlation between tumor-infiltrating Tregs expression by epigenetic qPCR and immunohistochemistry, epigenetic qPCR was more sensitive and specific. Using data from TCGA, mRNA expression of Forkhead box protein 3 (FoxP3) and Helios and FoxP3 methylation level did not predict survival. While the classical glioblastoma subtype corresponded to lower expression of Treg markers, these markers did not predict survival in any of the glioblastoma subtypes.

Conclusions

Although immunosuppression is a hallmark of glioblastoma, Tregs as measured in tissue by gene expression, immunohistochemistry, or demethylation and Tregs in peripheral blood measured by flow cytometry do not predict survival of patients. Quantitative DNA demethylation analysis provides an objective, sensitive, and specific way of identifying Tregs and CD3+ T cells in glioblastoma.     

Rapid enrichment of human papillomavirus (HPV)-specific polyclonal T cell populations for adoptive immunotherapy of cervical cancer

The majority of cervical cancers are caused by human papillomavirus type 16 (HPV16). Cervical cancer is associated with an ineffective host immune response against the HPV16 oncoproteins, characterized by the lack of the strong E6-specific T-helper type 1 (Th1) immunity that is generally present in healthy individuals, the presence of improperly polarized HPV16E6- and E7-specific CD4(+) T cells and increased numbers of regulatory T cells. Therefore, immunotherapeutic intervention is likely to require a modality that deletes the regulatory T cell component and enhances the HPV16-specific Type 1 T cell response. HLA-matched allogeneic stem cell transplantation may offer such a modality, because it involves the eradication of host immune cells and enables the transfer of donor derived tumor-specific T cells to the patient. As a first step in the development of such a treatment, we evaluated the success rate of a protocol for enrichment of HPV16E6-specific CD4(+) T cells from healthy donor PBMC on the basis of their IFNgamma secretion. After a short in vitro stimulation with overlapping 30 amino acid long HPV16E6 peptides, we enriched the IFNgamma secreting cells by magnetic cell sorting. The obtained polyclonal CD4(+) T cell populations recognized distinct epitopes within HPV16E6, as well as E6 protein, processed and presented by autologous professional antigen presenting cells. The described protocol proved successful in PBMC from more than half of the healthy adult blood donors. These HPV16E6-specific CD4(+) T cells may turn out to be an essential component of future adoptive T cell therapy for advanced cervical cancer, by orchestrating CTL dependent and independent tumoricidal mechanisms.     

Vascular heterogeneity and targeting: the role of YKL-40 in glioblastoma vascularization

Malignant glioblastomas (GBM) are highly malignant brain tumors that have extensive and aberrant tumor vasculature, including multiple types of vessels. This review focuses on recent discoveries that the angiogenic factor YKL-40 (CHI3L1) acts on glioblastoma-stem like cells (GSCs) to drive the formation of two major forms of tumor vascularization: angiogenesis and vasculogenic mimicry (VM). GSCs possess multipotent cells able to transdifferentiate into vascular pericytes or smooth muscle cells (PC/SMCs) that either coordinate with endothelial cells (ECs) to facilitate angiogenesis or assemble in the absence of ECs to form blood-perfused channels via VM. GBMs express high levels of YKL-40 that drives the divergent signaling cascades to mediate the formation of these distinct microvascular circulations. Although a variety of anti-tumor agents that target angiogenesis have demonstrated transient benefits for patients, they often fail to restrict tumor growth, which underscores the need for additional therapeutic tools. We propose that targeting YKL-40 may compliment conventional anti-angiogenic therapies to provide a substantial clinical benefit to patients with GBM and several other types of solid tumors.     

DC-CIK过继性免疫疗法联合化疗治疗转移性结直肠癌患者的疗效

目的： 研究DC-CIK（dendritic cell-cytokine induced killer cell）过继性免疫治疗联合化疗对转移性结直肠癌（metastatic colorectal cancer,mCRC）的疗效及安全性。 方法： 选取2010年11月至2011年11月在大连市中心医院治疗的80例mCRC患者,40例行DC-CIK治疗联合化疗（联合组）,40例行单纯化疗（化疗组）,评价两组患者治疗后免疫功能、疗效、毒副反应和生活质量（quality of life,QOL)。 结果： 共完成了160周期DC-CIK治疗,联合组治疗前后外周血T细胞亚群无显著变化（P>0.05）,化疗组治疗后外周血中CD3+、CD3+CD4+、CD3+CD8+、CD3-CD56+细胞比例较治疗前显著下降,且明显低于联合组（P<0.05）;联合组3周期治疗后CD4+ T细胞中IFN-γ水平较治疗前显著升高（P<0.05）,化疗组治疗后IFN-γ、IL-2、TNF-α水平著下降,且明显低于联合组（P<0.05）。联合组和化疗组总有效率（response rate, RR）未见明显差异（37.5% vs 22.5%,P>0.05）;联合组疾病控制率（disease control rate,DCR）明显高于化疗组（77.5% vs 50.0%,P<0.05）。联合组Ⅲ～Ⅳ度白细胞减少及Ⅲ～Ⅳ度迟发性腹泻的发生率明显低于化疗组（17.5% vs 42.5%,5.0% vs 25.0;均P<0.05）,其他相关不良反应无显著性差异,而且对症治疗后均可缓解。联合组患者的中位无进展生存（progression-free survival,PFS）较化疗组患者长（6.5个月 vs 4.5个月,P<0.05）,联合组和化疗组患者的总生存（overall survival,OS）比较差异无统计学意义（P>0.05）。联合组在躯体功能、情绪方面较治疗前明显改善,而且明显好于化疗组（P<0.05）。 结论： DC-CIK过继性免疫治疗联合化疗可以明显改善mCRC患者的免疫功能,提高总体疗效,减轻化疗不良反应,延长无进展生存,改善mCRC患者生活质量。     

Curcumin analog GO-Y030 boosts the efficacy of anti-PD-1 cancer immunotherapy

Regulatory T cells (Tregs) in the tumor microenvironment regulate tumor immunity. Programmed cell death protein 1 (PD-1) is known to be expressed on Tregs and plays crucial roles in suppressing tumor immunity. However, the immune checkpoint inhibitor, anti-PD-1 antibody, is known to promote the proliferation of the Treg population in tumor-infiltrating lymphocytes, thereby restricting the efficacy of cancer immunotherapy. In this study, we focused on the curcumin analog GO-Y030, an antitumor chemical. GO-Y030 inhibited the immune-suppressive ability of Tregs via metabolic changes in vitro, even in the presence of immune checkpoint inhibitors. Mechanistically, GO-Y030 inhibited the mTOR-S6 axis in Tregs, which plays a pivotal role in their immune-suppressive ability. GO-Y030 also controlled the metabolism in cultured CD4⁺ T cells in the presence of TGF-β + IL-6; however, it did not prevent Th17 differentiation. Notably, GO-Y030 significantly inhibited IL-10 production from Th17 cells. In the tumor microenvironment, L-lactate produced by tumors is known to support the suppressive ability of Tregs, and GO-Y030 treatment inhibited L-lactate production via metabolic changes. In addition, experiments in the B16-F10 melanoma mouse model revealed that GO-Y030 helped inhibit the anti-PD-1 immune checkpoint and reduce the Treg population in tumor-infiltrating lymphocytes. Thus, GO-Y030 controls the metabolism of both Tregs and tumors and could serve as a booster for anti-immune checkpoint inhibitors.