Adoptive cellular therapy with T cells specific for EBV-derived tumor antigens

Adoptive T-cell therapy is an attractive option for targeting tumors associated with Epstein-Barr virus. In immunogenic Type III latency tumors such as post transplant lymphoproliferative disease, EBV-specific CTL have been used successfully as prophylaxis and treatment. In Type II latency malignancies such as Hodgkin's disease and nasopharyngeal cancer, a more restricted array of EBV antigens are encoded and the clinical response rates after infusion of EBV-specific CTLs have been lower. Current strategies to increase response rates include targeting CTL to subdominant EBV antigens and genetically modifying CTL to increase their potency.     

Th9 Cells Represent a Unique Subset of CD4+ T Cells Endowed with the Ability to Eradicate Advanced Tumors

1. Download high-res image (200KB)
2. Download full-size image

     

Adoptive T cell therapy promotes the emergence of genomically altered tumor escape variants

Adoptive T cell therapy has been proven effective against melanoma in mice and humans. However, because most responses are incomplete or transient, cures remain rare. To maximize the efficacy of this therapy, it will be essential to gain a better understanding of the processes which result in tumor relapse. We studied these processes using B16ova murine melanoma and adoptive transfer of OT-I T cells. Transfer of T cells as a single therapy provided a significant survival benefit for mice with established subcutaneous tumors. However, tumors which initially regressed often recurred. By analyzing tumors which emerged in the presence of a potent OT-I response, we identified a novel tumor escape mechanism in which tumor cells evaded T cell pressure by undergoing major genomic changes involving loss of the gene encoding the target tumor antigen. Furthermore, we show that these in vivo processes can be recapitulated in vitro using T cell/tumor cell co-cultures. A single round of in vitro co-culture led to significant loss of the ova gene and a tumor cell population with rapidly induced and diverse karyotypic changes. Although these current studies focus on the model OVA antigen, the finding that T cells can directly promote genomic instability has important implications for the development of adoptive T cell therapies.     

肿瘤过继细胞治疗——老故事新演绎

肿瘤过继细胞治疗(adoptive cell therapy,ACT)较早被提出并一度盛行,但长期以来其疗效不佳,逐渐淡出人们视野。近年来,肿瘤ACT取得长足进步,利用T细胞过继免疫治疗黑素瘤获得令人鼓舞的成功;利用细胞因子诱导的杀伤细胞(cytokine induced killer cell,CIK)治疗癌症也在我国兴起,并取得可喜成绩。在肿瘤ACT领域,仍面临选择什么样的效应细胞、如何高效诱导筛选T细胞克隆、是否采用T细胞TCR转基因技术、如何通过武装化T细胞提高疗效、如何评判肿瘤ACT疗效等方面的问题。     

胃癌肿瘤微环境中Th17细胞的分布及临床意义

目的：探讨随着肿瘤的进展胃癌肿瘤微环境中Th17细胞的分布及与患者预后、生存之间的关系。方法采用流式细胞术检测60例胃癌患者Th17细胞在不同时期的胃癌肿瘤微环境中的阳性表达率及分布。通过免疫组织化学染色验证120例胃癌患者Th17细胞的表达情况,并分析Th17细胞的表达与患者预后生存之间的关系。结果60例新鲜胃癌组织标本中,Th17细胞在肿瘤组织中的阳性表达率（10.3%±7.2%）高于癌旁正常胃组织（4.6%±2.5%）,且组间差异具有统计学意义（P＜0.05）。而进展期胃癌组织中Th17细胞的阳性表达率（8.7%±6.8%）显著低于早期胃癌组织（12.4%±7.6%）,且组间差异具有统计学意义（P＜0.05）。120例胃癌根治术后的组织标本中,Th17细胞高表达组5年生存率为58.7%,低表达组为40.4%,二者的差异有统计学意义（χ2=4.634,P=0.031）。结论胃癌肿瘤微环境中Th17细胞的表达与疾病的进展及患者的预后密切相关,可以作为预测胃癌患者术后生存的重要免疫指标。     

调节性T细胞与恶性肿瘤的过继免疫治疗

过继免疫治疗利用自身免疫细胞进行体外扩增培养,然后回输到体内,它通过增强机体免疫力来对抗肿瘤,对肿瘤细胞具有靶向杀伤性作用,是最有可能彻底清除手术或放化疗后残存肿瘤细胞的治疗手段.但是,调节性T细胞(Regulatory T cells,Tregs)在多种恶性肿瘤患者外周血及微环境中普遍存在着比例上调的情况,这不仅会导致机体固有免疫的负性平衡,还会影响过继免疫治疗的疗效,使其不能发挥最大的抗肿瘤作用.通过对Tregs日渐深入的了解,人们已经研究出多种抑制及消除Tregs的治疗策略.     

High hydrostatic pressure induces immunogenic cell death in human tumor cells

Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high‐mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP‐treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA‐DR and the release of interleukin IL‐6, IL‐12p70 and TNF‐α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor‐specific T cells. DCs pulsed with HHP‐treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress‐mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase‐8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells.     

Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses

1. Download : Download high-res image (273KB)
2. Download : Download full-size image

     

宫颈癌HPV 分型与Th17和Treg细胞的相关性研究*

目的：检测感染人乳头状瘤病毒（human papilloma virus,HPV ）患者的外周血中CD4+IL 17+Th17、CD4+CD25+Foxp 3+Treg 细胞,分析Th17/Treg比值,比较血清中IL- 17、IL- 10、TGF-β 、IL- 23细胞因子的表达差异。方法：收集2014年9 月至2016年6 月119 例新疆医科大学第一附属医院HPV 感染未行治疗患者的资料,分为宫颈癌（cervical cancer ,CC）组46例、宫颈上皮内瘤变（cervicalintraepithelial neoplasia ,CIN）Ⅱ～Ⅲ组43例、慢性宫颈炎（chronic cervicitis ,CCS）组30例,根据检测HPV 分型将患者HPV 感染分为高危型80例与低危型39例,流式细胞仪检测Treg 及Th17细胞,ELISA 检测相关细胞因子。结果：高危型HPV 感染患者中CC组感染率为87％（40/ 46）,显著高于CIN Ⅱ～Ⅲ组77%（33/ 43）及CCS 组23%（7/ 30）。 CC组外周血中Th17、Treg 细胞及血清中相关细胞因子均明显高于其他两组,且随着宫颈病变加重,Th17/Treg比值升高更加显著。高危型HPV 感染患者外周血中Th17和Treg 细胞高于低危型HPV 感染患者（P < 0.05）,Th17/Treg比值在高危型HPV 感染患者中显著升高。高危型HPV 感染患者血清中IL- 17、IL- 10、TGF-β 、IL- 23细胞因子的表达较低危型HPV 感染患者均显著增高（P < 0.05）。 结论：Treg 、Th17细胞在高危型HPV 感染患者中存在增高现象,Th17/Treg比值的升高可能直接参与宫颈HPV 的感染及免疫逃逸过程,在宫颈癌的发生发展中起着重要作用。     

Anti-G-CSF treatment induces protective tumor immunity in mouse colon cancer by promoting NK cell,macrophage and T cell responses

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is highly expressed in human and mouse colorectal cancers (CRC). We previously reported that G-CSF stimulated human CRC cell growth and migration, therefore in this study we sought to examine the therapeutic potential of anti-G-CSF treatment for CRC. G-CSF is known to mobilize neutrophils, however its impact on other immune cells has not been well examined. Here, we investigated the effects of therapeutic anti-G-CSF treatment on CRC growth and anti-tumor immune responses. C57BL/6 mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS) to induce neoplasms were administered anti-G-CSF or isotype control antibodies three times a week for three weeks. Animals treated with anti-G-CSF antibodies had a marked decrease in neoplasm number and size compared to the isotype control group. Colon neutrophil and macrophage frequency were unchanged, but the number of macrophages producing IL-10 were decreased while IL-12 producing macrophages were increased. NK cells were substantially increased in colons of anti-G-CSF treated mice, along with IFNγ producing CD4⁺ and CD8⁺ T cells. These studies are the first to indicate a crucial role for G-CSF inhibition in promoting protective anti-tumor immunity, and suggest that anti-G-CSF treatment is a potential therapeutic approach for CRC.     

DC/CIK维持治疗晚期非小细胞肺癌的临床观察

目的 评价树突状细胞联合自体细胞因子诱导杀伤细胞（DC/CIK）维持治疗晚期非小细胞肺癌（NSCLC）的疗效和不良反应。方法 根据治疗情况将2010年1月至2014年1月120例含铂方案化疗4～6个周期后疾病稳定的晚期NSCLC患者分为对照组（n=65）和研究组（n=55）,仅研究组化疗后接受DC/CIK维持治疗。对两组进行生存随访并比较中位无进展生存期（PFS）和总生存期（OS）,同时记录不良反应情况。结果研究组的中位PFS为5.0个月,高于对照组的3.5个月,差异有统计学意义（P＜0.05）;研究组的中位OS为8.5个月,与对照组（8.0个月）的差异无统计学意义（P＞0.05）;两组化疗后获不同疗效亚组中位PFS和OS的差异均无统计学意义（P＞0.05）。常见不良反应为骨髓抑制、发热、乏力和关节酸痛,两组不良反应发生率的差异无统计学意义（P＞0.05）。结论DC/CIK维持治疗可延长化疗后疾病稳定晚期NSCLC患者的进展时间,且不良反应轻,患者可耐受。     

miR-145调控肝癌腹水模型Th9细胞升高的机制研究

目的:探讨miR-145调控肝癌腹水模型Th9细胞升高的作用机制。方法:构建小鼠H22肝癌腹水模型。造模两周后处死小鼠并分离出脾脏组织,流式细胞术分析肝癌腹水组（MA组）与正常对照组（Control 组）小鼠脾脏Th9细胞表达水平。ELISA法检测脾脏IL-9表达水平。RT-PCR法检测脾脏miR-145表达水平。Western Blot法检测脾脏中PI3K/Akt/mTOR/P70S6K/HIF-1α相关蛋白表达水平。分选小鼠脾脏CD4+T细胞,随机分为miR-145 mimics组和NC组,分别应用miR-145-5P mimics、阴性对照寡核苷酸（negative control,NC）进行转染,RT-PCR检测各组miR-145、HIF-1α mRNA及IL-9 mRNA表达水平。结果:与Control 组相比,MA组Th9细胞及其细胞因子IL-9表达均升高（P<0.05）,miR-145表达降低（P<0.05）,p-PI3K、p-Akt、mTOR、p-mTOR、p-P70S6K、HIF-1α蛋白均升高（P<0.05）。与NC组相比,miR-145 mimics组miR-145显著升高,HIF-1α mRNA及IL-9 mRNA表达下降。结论:肝癌腹水中Th9细胞升高可能与miR-145下降导致的PI3K/Akt/mTOR/P70S6K/HIF-1α通路激活有关。     

Primary systemic therapy does not eradicate disseminated tumor cells in breast cancer patients

Introduction The presence of disseminated tumor cells in the bone marrow of breast cancer patients has proven to be an independent prognostic factor. The aim of this study was to investigate the status of tumor cell dissemination after primary systemic therapy in relation to therapy response. Methods Bone marrow aspirates were obtained from 120 patients after completion of primary systemic therapy. Disseminated tumor cells were detected by immunocytochemistry using the APAAP method. Bone marrow status was correlated with clinicopathological factors as well as tumor response to primary systemic therapy. Results Sixty out of 120 patients had disseminated tumor cells in their bone marrow aspirates (50%). Response rates were 18% for pathologic complete remission, 52% for partial remission, 28% for no change and 3% for progression. Despite complete remission, 36% of these patients were bone marrow positive. In the partial remission group, the positivity rate was 48%. About 61% of patients with stable disease had disseminated tumor cells in their bone marrow. A trend to higher positivity rates was observed in the poor responder group compared to responders (61% vs. 38%, P = 0.1). Conclusion Primary systemic therapy does not completely eradicate disseminated tumor cells in the bone marrow of breast cancer patients. The biological role of persistent disseminated tumor cells needs to be further investigated to optimize current and future therapeutic strategies.     

CIK细胞用于中晚期非小细胞肺癌一线维持治疗的临床观察

目的 探讨自体细胞因子诱导的杀伤（CIK）细胞免疫治疗在中晚期非小细胞肺癌（NSCLC）一线维持治疗中的疗效及安全性。方法 选取本院2009年6月至2011年3月接受一线治疗且疗效评价稳定（SD）或以上的NSCLC患者112例,随机分为治疗组（n=56）和对照组（n=56）。在接受一线化疗方案原药或减药的维持化疗基础上,治疗组加用CIK细胞免疫治疗,检测CIK细胞治疗前后外周血T细胞亚群的变化;根据药物毒副反应判定标准NCI-CTC 4.0,观察两组患者的毒副反应情况并随访远期生存。结果 CIK治疗后较治疗前T细胞亚群状态改善,未见3～4级不良反应。治疗组的中位无进展生存期（PFS）为8.6个月,高于对照组的7.5个月（P＜0.05）,中位生存时间（OS）分别为19.2个月和18.6个月,差异无统计学意义（P＞0.05）;治疗组中一线疗效≥50％PR-CR者（靶病灶最大径之和缩小50％～100％）的中位PFS为11.5个月,高于疗效＜50％PR-SD者（靶病灶最大径之和缩小0～50％）的79个月（P＜005）,但中位OS的差异无统计学意义（P＞0.05）;对照组中一线疗效≥50％PR-CR者和疗效＜50％PR-SD者中位PFS及OS的差异均无统计学意义（P＞0.05）;一线疗效≥50％PR-CR者中,治疗组的中位PFS为11.5个月,亦高于对照组的88个月（P＜0.05）,但中位OS的差异无统计学意义（P＞0.05）。结论 CIK细胞免疫治疗在中晚期NSCLC维持治疗中,可以改善患者免疫功能,提高自身抗肿瘤能力,延长PFS,而且具有良好的安全性,可提高NSCLC维持治疗的疗效。     

Photodynamic therapy of melanoma with new,structurally similar,NIR-absorbing ruthenium (II) complexes promotes tumor growth control via distinct hallmarks of immunogenic cell death

Cancer therapies that generate T cell-based anti-cancer immune responses are critical for clinical success and are favored over traditional therapies. One way to elicit T cell immune responses and generate long-lasting anti-cancer immunity is through induction of immunogenic cell death (ICD), a form of regulated cell death that promotes antigenicity and adjuvanticity within dying cells. Therefore, research in the last decade has focused on developing cancer therapies which stimulate ICD. Herein, we report novel photodynamic therapy (PDT) compounds with immunomodulatory and ICD inducing properties. PDT is a clinically approved, minimally invasive anti-cancer treatment option and has been extensively investigated for its tumor-destroying properties, lower side effects, and immune activation capabilities. In this study, we explore two structurally related ruthenium compounds, ML19B01 and ML19B02, that can be activated with near infrared light to elicit superior cytotoxic properties. In addition to its direct cell killing abilities, we investigated the effect of our PSs on immunological pathways upon activation. PDT treatment with ML19B01 and ML19B02 induced differential expression of reactive oxygen species, proinflammatory response-mediating genes, and heat shock proteins. Dying melanoma cells induced by ML19B01-PDT and ML19B02-PDT contained ICD hallmarks such as calreticulin, ATP, and HMGB1, initiated activation of antigen presenting cells, and were efficiently phagocytosed by bone marrow-derived dendritic cells. Most importantly, despite the distinct profiles of ICD hallmark inducing capacities, vaccination with both PDT-induced dying cancer cells established anti-tumor immunity that protected mice against subsequent challenge with melanoma cells.     

Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment

Many types of tumors are organized in a hierarchy of heterogeneous cell populations. The cancer stem-like cells (CSCs) hypothesis suggests that tumor development and metastasis are driven by a minority population of cells, which are responsible for tumor initiation, growth and recurrences. The inability to efficiently eliminate CSCs during chemotherapy, together with CSCs being highly tumorigenic and invasive, may result in treatment failure due to cancer relapse and metastases. CSCs are emerging as a promising target for the development of translational cancer therapies. Ideal panacea for cancer would kill all malignant cells, including CSCs and bulk tumor cells. Since both chemotherapy and CSCs-specific therapy are insufficient to cure cancer, we propose combination therapy with CSCs-targeted agents and chemotherapeutics for improved breast cancer treatment. We generated in vitro mammosphere of 2 breast cancer cell lines, and demonstrated ability of mammospheres to grow and enrich cancer cells with stem-like properties, including self-renewal, multilineage differentiation and enrichment of cells expressing breast cancer stem-like cell biomarkers CD44⁺/CD24^?/low. The formation of mammospheres was significantly inhibited by salinomycin, validating its pharmacological role against the cancer stem-like cells. In contrast, paclitaxel showed a minimal effect on the proliferation and growth of breast cancer stem-like cells. While combination therapies of salinomycin with conventional chemotherapy (paclitaxel or lipodox) showed a potential to improve tumor cell killing, different subtypes of breast cancer cells showed different patterns in response to the combination therapies. While optimization of combination therapy is warranted, the design of combination therapy should consider phenotypic attributes of breast cancer types.     

Response of cultured 9L cells to spirohydantoin mustard and X-rays

Rat brain tumor cells (9L) were treated in vitro for 1 hr with 2 μg/ml (～30% survival) of spirohydantoin mustard (SHM) 4 and 24 hr before, during, and 4 and 24 hr after graded doses of radiation (0–20 Gy). The resulting survival curves generated from colony formation data and corrected for drug kill indicate that for all schedules SHM enhanced the radiation response, particularly for surviving fractions greater than 0.5. Analysis of the combination data using isobolograms indicates the effects of the separate agents were additive in producing either 1, 2, or 3 log cell kills. Therefore, the enhancement of the radiation response by SHM can be explained solely on the basis of the shapes of the single agent survival curves.     

食管鳞癌循环肿瘤细胞在新辅助放化疗中动态变化及预后意义

目的 循环肿瘤细胞(circulating tumor cells,CTCs)被认为是肿瘤复发转移的重要因素之一,食管鳞癌患者放疗后CTCs阳性率较治疗前下降并和预后相关.本研究探讨进展期食管鳞癌新辅助化疗中CTCs的状态,以及CTCs与食管癌临床特征及预后的相关性.方法 回顾性分析十堰市太和医院(湖北医药学院附属医院)2013-01-10-2014-03-27收治的96例食管鳞癌患者,采用免疫磁珠富集联合免疫荧光法检测患者外周血中CTCs,对比分析患者CTCs状态与临床特征及2年生存率的关系.同时分析患者新辅助放化疗前后CTCs状态的变化,以期明确新辅助治疗的意义及适应人群.计数资料采用χ2检验或Fisher精确概率法,生存分析采用Kaplan-Meier法和Cox模型法.结果 96例患者随访至2016-03-27,随访率100%,中位随访时间25个月.患者CTCs状态与肿瘤的病变长度、T分期、N分期及临床分期相关(P<0.05),与患者的年龄、性别及病变部位无关,P>0.05.首诊CTCs阳性患者的中位生存时间约19.2个月;而CTCs阴性患者的中位生存时间约26.8个月,两者差异有统计学意义,P=0.038.Cox模型结果显示,Ⅲ~ⅣA期是影响CTCs不同状态患者的独立危险因素.首诊CTCs阳性的患者Ⅱ期中位生存时间为28.2个月,与CTCs阴性患者的30.2个月比较,差异无统计学意义,P=0.189;但Ⅲ期的中位生存时间分别为19.9和24.6个月,差异有统计学意义,P=0.044;ⅣA期的中位生存时间分别为11.2和21.2个月,差异有统计学意义,P=0.045.新辅助治疗后,有21例(34.42%)患者由CTCs阳性转为阴性,转阴率差异有统计学意义,P=0.002.新辅助治疗后,CTCs仍为阳性的患者中位生存时间约16.5个月,与CTCs转阴的患者(23.4个月)和首诊CTCs阴性的患者(26.8个月)比较,差异有统计学意义,P<0.05;但后两者差异无统计学意义,P>0.05.结论 CTCs可反映食管癌患者的疾病进展程度,并可作为判断预后的指标.此外,新辅助放化疗可改善首诊CTCs阳性患者的中位生存时间.