The inability of adoptive immunotherapy to eradicate tumor cells in radiation-induced chimeric mice: the possible down-regulation of intratumor immune amplification

Tumors were eradicated following adoptive immunotherapy of (C57BL/6J X DBA/2J) F1 (B6D2F1) hybrid mice bearing the C57B1/6J (B6) sarcoma, MCA/76-9, by treatment with cyclophosphamide (CY) and adoptively transferred tumor-sensitized B6D2F1 T cells. Identical treatment of B6----B6D2F1 or DBA----B6D2F1 chimeric tumor-bearing mice resulted in temporary tumor regression only. Immunotherapy in both systems resulted in the appearance at the tumor site of large numbers of T lymphocytes and macrophages, which were shown to be derived from the adoptively transferred donor immune B6D2F1 cells or from the original reconstituting bone marrow respectively. The TAC and TAL isolated from either B6D2F1 or chimeric mice expressed potent toxicity in vitro towards tumor target cells. In addition, TAC from both systems inhibited tumor growth in a Winn assay in an immunologically specific manner. Suppressor cells detected in spleens 8 days after CY injection of normal B6, DBA, B6D2F1 hybrid and chimeric mice and also after adoptive immunotherapy of tumor-bearing B6, B6D2F1 and chimeric mice and were shown to persist in the spleens of chimeric mice for at least 31 days after adoptive immunotherapy. In contrast, spleen cells taken 31 days after therapy of B6D2F1 mice did not contain detectable suppressor cells and were able to induce tumor eradication when adoptively transferred to CY-treated tumor-bearing B6D2F1 mice. The possibility is discussed that two forms of suppressor mechanisms are induced after adoptive immunotherapy of chimeric mice and that they may down-regulate donor T cell responses at the tumor site.     

Treatment of an established graft-versus-host reaction in AKR mice by adoptive immunotherapy.

A model system in AKR mice for the induction and cure of a clinically evident graft-versus-host disease is reported. Graft-versus-host disease is inititated by i.p. injections of ecyclophosphamide (250 mg/kg body weitht ) into female AKR mice, on Day 0.This is followed by i.v. injections of 45 x 10-6 normal spleen cells (NSC) from male C57BL/6J mice. Median survival time for these mice is 33.4 plus or minus 4.5 days. Following the administration of C57BL/6J NSC, AKR mice were rescued from graft-versus-host disease by the following treatment protocol: (a) Day 6, 35 x 10-6 DBA/2 NSC given i.v.; (b) Day 10, cyclophosphamide i.p. (150 mg/kg body weight); (c) Days 11 and 26, 35 x 10-6 AKR NSC given i.v. These experiments demonstrate that graft-versus-host reaction can be elminiated by coupling a graft-versus-host reaction with a graft-versus-graft reaction and restoring the host by immunocompetent syngeneic cells.     

Th17细胞过继免疫治疗对荷弥漫大B细胞淋巴瘤小鼠肿瘤生长的影响

目的 探讨Th17细胞过继免疫治疗对荷弥漫大B细胞淋巴瘤(DLBCL)小鼠肿瘤生长的影响.方法 采用Mini MACS免疫磁珠分离纯化BALB/c小鼠脾来源的CD4+ CD62L+初始T细胞,采用"转化生长因子(TGF-β)、白细胞介素6(IL-6)、干扰素γ抗体(anti-IFN-γ)、白细胞介素4抗体(anti-IL-4)、白细胞介素23(IL-23)"因子组合体外诱导小鼠Th17细胞分化,采用ELISA法测定Th17细胞产生IL-17水平;采用DLBCL细胞株SUDHL-4接种SCID小鼠建立DLBCL荷瘤小鼠模型;选择0、8、18d(3组,5只小鼠/每组)对荷瘤小鼠进行Th17细胞过继免疫治疗,计算肿瘤体积,观察荷瘤小鼠生存期.结果 小鼠在接种SUDHL-4细胞8d左右均形成瘤结节,成瘤率为100％.与对照组小鼠相比,3个过继免疫治疗组小鼠肿瘤体积均明显缩小(P＜0.05),荷瘤小鼠生存期均显著延长(P＜0.05).结论 Th17细胞过继免疫治疗对DLBCL荷瘤小鼠具有抗肿瘤作用.     

GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice

To examine the behavior of tumor cells in tumors developing directly from mammary tissue in transgenic models, we have evaluated transgenic mice expressing green fluorescent protein (GFP). Using the mouse mammary virus promoter (MMTV) to directly drive expression of GFP, we find low levels of fluorescence in the mammary and salivary glands of transgenic animals. Using MMTV-Cre or WAP-Cre in combination with the Cre-activatable CAG-CAT-EGFP construct, we find stronger expression of GFP that is still tissue specific. These animals provide a range of expression of GFP that is suitable for analysis of transgenic mammary tumors and metastases in vivo at the single cell level of resolution.     

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells on tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented by in vivo prime and subsequent in vitro challenge with the streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy 1+, asialo GM1+, suggesting the activated NK cells (OK-NK cell). The culture supernatants of spleen cells with OK432 possessed the activity of IL-2 and IFN-gamma, and the IL-2 played a major role to induce the OK-NK cells via the production of IFN-gamma. In this study, we examined the effect of adoptive transfer of OK-NK cells on tumor-bearing mice. The mice were implanted SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously (s.c.) to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or i.t., adoptively. By the adoptive transfer of OK-NK cells, the 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was increased, significantly. The intratumoral remnants of 125I-labelled OK-NK cells were 61.27 and 8% after intratumoral transfer, respectively. By multiple transfer of OK-NK cells the anti-tumor effect was more augmented than that of a single transfer. Thus we recognized the anti-tumor effect of adoptive transfer of OK-NK cells on tumor-bearing mice, and suggested that OK-NK cells could be useful for the therapy of cancer patients.     

载脂蛋白C3 转基因小鼠脾脏免疫细胞表型及其活性分析

目的：分析载脂蛋白C3（Apo C3）转基因小鼠脾脏免疫细胞的表型及活性。方法：以12只ICR背景Apo C3转基因小鼠为研究模型,选取鼠龄/性别匹配的野生型小鼠作为对照,采集小鼠球后静脉丛血液,以酶反应显色比色法联合酶标仪测定血浆甘油三酯水平,选取血浆甘油三酯含量≥ 1 000 mg/dL （11.3 mmol/L）的小鼠用于后续实验。取小鼠脾脏制备成单细胞悬液,采用细胞膜表面分子、胞内分子及核内分子染色法,通过流式细胞术检测转基因小鼠和野生型小鼠脾脏免疫细胞频率和表型。再将转基因小鼠或野生型小鼠脾脏细胞与结肠癌细胞MC38分别以1：1、1：3、3：1效靶比共培养72 h后,通过流式细胞术检测CD8⁺ T细胞CD107a的表达。结果：Apo C3转基因小鼠血浆甘油三酯含量较野生型小鼠显著升高（P<0.01）,而体质量差异无统计学意义（P>0.05）。与野生型小鼠相比,Apo C3转基因小鼠脾脏CD3⁺CD8⁺、CD3⁺CD8⁺NKG2D⁺细胞频率明显增多,髓系抑制性Gr-1⁺CD11B⁺细胞频率增加,脾脏NK1.1⁺NK、NK1.1⁺NKG2D⁺细胞的频率明显减少（P均>0.05）;脾脏CD3⁺CD4⁺NKG2D⁺、CD3⁺CD4⁺IL-17⁺细胞频率以及调节性T细胞,NKT细胞,γδT细胞,B细胞和巨噬细胞频率均无明显变化（P均>0.05）。Apo C3转基因小鼠CD8⁺CD62L^-CD44⁺、CD8⁺CD44⁺KLGR1⁺频率升高,以效应性T细胞为主,且CD8⁺ T细胞分泌IFN-γ活性增强;转基因小鼠中CD8⁺ T淋巴细胞CD107a的表达增加差异均具有统计学意义（P均<0.05）。结论：Apo C3转基因小鼠呈现高水平血浆甘油三酯,其免疫细胞表型及活性具有显著变化,其中CD8⁺ T细胞频率及生物活性上调,髓系抑制性细胞频率升高,NK细胞频率降低;并且转基因小鼠中CD8⁺T淋巴细胞对靶细胞杀伤能力增强。     

肿瘤过继免疫治疗临床级抗原特异性T细胞的研究进展

抗原特异性T细胞是肿瘤过继免疫治疗的核心,靶向性强、杀伤活性高、毒性作用小、疗效卓越的临床级细胞的制备是提高治疗效果的关键。近年来,全封闭自动化T细胞分离系统提高了安全性,新型人工抗原提呈方法和γc家族细胞因子增加了T细胞扩增效率,低分化表型T细胞的选择提高了有效性,而T细胞受体和嵌合抗原受体基因修饰赋予T细胞抗原特异性杀伤性能,病毒基因转导和理化基因转染T细胞方法的不断改进为基因修饰重定向T细胞的抗原特异性提供了保障。越来越多的临床试验展示了令人鼓舞的治疗效果,为肿瘤过继免疫治疗注入了新的生命力。     

In vivo and in vitro effect of adoptive immunotherapy of experimental murine brain tumors using lymphokine-activated killer cells

Adoptive immunotherapy for the experimental murine brain tumor was investigated by using lymphokine-activated killer (LAK) cells both in vitro and in vivo. Supernatants of 48-h culture medium of spleen cells from Wistar rats in the presence of concanavalin A were used as interleukin 2 (IL-2). LAK cells were generated by cocultivation of spleen cells from Fischer rats with IL-2 with the peak reactivity on Day 2 or 3 of culture. Lytic activity was observed against not only syngenic tumor cells but also allogenic and xenogenic tumor cells, while no lytic activity was observed against normal brain cells. The cell depletion test, dye exclusion test, and immunofluorescence method using monoclonal antibodies revealed that LAK cells partially belonged to the population of the activated T-cell group, but the precursor cells did not react with any monoclonal antibodies used. On the basis of these results in vivo study was performed. LAK cells and immune spleen cells were adoptively transferred to the rats i.v. or intratumorally (i.t.) on the seventh day after the inoculation of T9, a gliosarcoma induced by methylcholanthrene from Fischer rats, into the right basal ganglia. Then the survival rate and necrotic foci were compared between the groups treated with those cells and the control. The survival rate of the groups treated with LAK cells was significantly higher than that of the control (administered i.v.; P less than 0.01, administered i.t.; P less than 0.05). But the treatment with immune spleen cells was not effective. The incidence and area of necrotic foci in the tumors treated with LAK cells were greater than those of the others. Microautoradiography was also performed using [3H]thymidine-labeled LAK cells, which were administered i.v. to the models on the 14th day after the inoculation of T9. It was revealed that LAK cells accumulated in the lung shortly after the administration and then in the liver and spleen, especially in the white pulp. IL-2 inhibitor activity of the sera from the tumor-bearing rats was greater than that of normal rats (P less than 0.001), but it was depressed markedly by cyclophosphamide (P less than 0.01). The adoptive transfer of LAK cells may be one of the effective treatments of malignant brain tumor. The nature of IL-2 inhibitors is necessary to be clarified for more effective immunotherapy.     

A new approach to generating antitumor effectors for adoptive immunotherapy using human adherent lymphokine-activated killer cells

Lymphocytes from human peripheral blood incubated with interleukin-2 (IL2) develop lymphokine-activated killer (LAK) activity with the ability to kill a wide variety of tumor cells in a non-major histocompatibility complex-restricted manner. Adoptive immunotherapy with LAK cells and IL2 has been reported to lead to a regression of solid tumors in some patients with advanced malignancies. Aiming to improve the effectiveness of clinical adoptive immunotherapy, we developed a procedure for selective enrichment from human blood mononuclear cells (MNC) of IL2-activated antitumor effector cells. These cells, termed adherent LAK (A-LAK) cells because of their characteristic property of adherence to plastic, demonstrated both higher proliferative potential and greater antitumor cytotoxicity than unseparated MNC. Human A-LAK cells represented only 1 to 4% of IL2-activated MNC at 24 h but expanded from 130- to 1100-fold in 20 days. They comprised a population highly enriched in CD3-Leu19+ effector cells with antitumor activity against fresh human solid tumor cells and established cell lines. A-LAK cells retained antitumor activity for up to 14 days when cultured in the presence of IL2. They also mediated antibody-dependent cytotoxicity. Large-scale generation of A-LAK cells from the blood of patients with cancer proved feasible and should yield populations that are effective in vivo at lower doses than those required with unseparated LAK cells. This offers the potential for improving the antitumor effects, reducing the toxicity, and facilitating the administration of adoptive immunotherapy in humans. A Phase I/II clinical trial utilizing A-LAK cells and IL2 in patients with melanoma and renal cell carcinoma is now in progress.     

Locoregional adoptive immunotherapy using LAK cells and IL-2 against liver metastases from digestive tract cancer

We investigated the clinical efficacy of locoregional and systemic adoptive immunotherapy (AIT) with or without interleukin-2 (IL-2) against solid metastatic lesions from digestive tract cancer. Eighteen patients, who were treated with more than 10(10) lymphokine-activated killer (LAK) cells, were enrolled in this study. Seven of the 18 patients received hepatic arterial infusion (HAI) of LAK cells with or without IL-2 against metastatic liver tumors (locoregional therapy group). The remaining 11 patients received systemic transfer of LAK cells with IL-2 against metastatic lesions located in organs other than the liver (systemic therapy group). Three of 7 locoregional therapy group patients showed clinically significant tumor regressions that were evaluated as being equivalent to partial response (PR). Two of the 11 systemic therapy group patients showed significant tumor regressions, but this response rate was much lower than that of the locoregional therapy group. The 2 effective cases in the systemic therapy group were esophageal cancer patients. Locoregional AIT with or without IL-2 against liver metastases from digestive tract cancer could be an effective therapeutic modality in some patients who are refractory to conventional therapies (e.g., chemotherapy and radiotherapy). It is necessary to find a new way to augment the anti-tumor effect of this therapy in combination with prior or concomitant chemotherapy.     

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.     

Potentiation of tumor eradication by adoptive immunotherapy with T-cell receptor gene-transduced T-helper type 1 cells

Adoptive immunotherapy using antigen-specific T-helper type 1 (Th1) cells has been considered as a potential strategy for tumor immunotherapy. However, its application to tumor immunotherapy has been hampered by difficulties in expanding tumor-specific Th1 cells from tumor-bearing hosts. Here, we have developed an efficient protocol for preparing mouse antigen-specific Th1 cells from nonspecifically activated Th cells after retroviral transfer of T-cell receptor (TCR)-alpha and TCR-beta genes. We demonstrate that Th1 cells transduced with the TCR-alpha and -beta genes from the I-A(d)-restricted ovalbumin (OVA)(323-339)-specific T-cell clone DO11.10 produce IFN-gamma but not interleukin-4 in response to stimulation with OVA(323-339) peptides or A20 B lymphoma (A20-OVA) cells expressing OVA as a model tumor antigen. TCR-transduced Th1 cells also exhibited cytotoxicity against tumor cells in an antigen-specific manner. Moreover, adoptive transfer of TCR-transduced Th1 cells, but not mock-transduced Th1 cells, exhibited potent antitumor activity in vivo and, when combined with cyclophosphamide treatment, completely eradicated established tumor masses. Thus, TCR-transduced Th1 cells are a promising alternative for the development of effective adoptive immunotherapies.     

Local adoptive immunotherapy of human head and neck cancer xenografts in nude mice with lymphokine-activated killer cells and interleukin 2

The efficacy of local adoptive immunotherapy with human lymphokine-activated killer cells and recombinant interleukin 2 (rIL-2) in growth inhibition of established squamous cell carcinoma of the head and neck (SCCHN) was evaluated in a nude mouse model. The model of xenografted SCCHN was established by s.c. injections of in vitro maintained tumor cells (2-10 x 10(6) cells/mouse) into the flank of splenectomized animals pretreated with cyclophosphamide (200 mg/kg). The SCCHN line used was tumorigenic in 95% of the appropriately conditioned nude mice. Inhibition of tumor growth by locally administered effector cells was the end point of the study, since the tumors did not metastasize within 6 weeks of tumor challenge. Either i.p. or local administration of rIL-2 alone (1000 units/day) to the tumor site daily for 2 weeks resulted in a significant inhibition of tumor growth. In the absence of detectable natural killer activity in these mice, a modest dose of rIL-2 had a direct antitumor effect on SCCHN cells in vivo. In addition, complete inhibition of tumor growth was achieved with 3 times weekly injections of 5-10 x 10(6) lymphokine-activated killer cells delivered to the tumor site and 1000 units of rIL-2 administered locally every day for 2 weeks. Our data indicate that local or systemic immunotherapy with rIL-2 alone or local adoptive immunotherapy with an adequate dose of lymphokine-activated killer cells plus rIL-2 may be effective in preventing the growth of established SCCHN tumors in vivo.     

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。     

DCIK细胞治疗急性髓细胞性白血病的疗效观察

摘 要　目的: 评价DCIK细胞过继免疫治疗急性髓细胞性白血病（acute myeloid leukemia，AML）的临床疗效。方法：选取苏州大学附属第一医院2006年1月至2007年12月所收治的10例确诊AML患者，经常规化疗后处于完全缓解中，经院伦理委员会批准和患者知情同意，用血细胞分析仪分离外周血中单个核细胞(peripheral blood mononuclear cells，PBMC)，在体外诱导培养成DCIK细胞；以流式细胞术检测DCIK细胞的表型，以MTT法检测DCIK细胞对人红白血病细胞K562的杀伤活性，确认质量合格后回输给患者进行抗肿瘤免疫治疗；治疗后评价其近期临床疗效、免疫学活性及不良反应等。结果：体外成功诱导培养DCIK细胞，其对白血病细胞株K562的杀伤率为(58.3±3.3)%；DCIK细胞群中，CD3+CD56+ 占(38.4±9.42)%。10例患者经治疗后，7例持续完全缓解（continuous complete remission，CCR），占70%；患者回输DCIK后外周血中的CD4+ 、CD8+ 、CD56+ 的比例均有明显提高(P<0.05或P<0.01)；无一患者出现严重不良反应。结论：DCIK能诱导机体产生特异性的免疫反应，对急性髓细胞性白血病的治疗有较好的临床疗效。     

CIK细胞联合白介素-2对鼻咽癌病人免疫功能及EB病毒指标的影响

目的：细胞因子诱导杀伤细胞（cytokine-induced killer cells，CIK细胞）是目前抗肿瘤过继细胞免疫治疗最为有效的方案。本研究通过治疗30例鼻咽癌病人，探讨CIK细胞联合IL-2对鼻咽癌病人免疫学功能及EB病毒指标的影响。方法：将符合条件的30例病人配对分为两组。治疗组病人接受CIK细胞过继细胞免疫治疗，每周一次，连续4周与对照组比较。免疫功能及EB病毒学指标分别通过测定外周血T淋巴细胞亚群，EB病毒VCA—IgA、EA—IgA、EDAb、DNA免疫荧光定量进行评估。结果：随访8—18个月，中位随访时间12个月。CIK治疗1个月后患者CD4^＋CD25^＋％明显下降，EB病毒VCA—IgA、EA—IgA、DNA免疫荧光定量下降显著。结论：CIK细胞可改善鼻咽癌病人的免疫功能，加速EB病毒抗体及DNA拷贝数下降。提示CIK治疗可消灭肿瘤微小残留灶及转移灶，可作为一种鼻咽癌的辅助治疗。     

CIK细胞联合白介素-2对鼻咽癌病人免疫功能及EB病毒指标的影响

目的:细胞因子诱导杀伤细胞(cytokine-inducedkillercells,CIK细胞)是目前抗肿瘤过继细胞免疫治疗最为有效的方案。本研究通过治疗30例鼻咽癌病人,探讨CIK细胞联合IL-2对鼻咽癌病人免疫学功能及EB病毒指标的影响。方法:将符合条件的30例病人配对分为两组。治疗组病人接受CIK细胞过继细胞免疫治疗,每周一次,连续4周与对照组比较。免疫功能及EB病毒学指标分别通过测定外周血T淋巴细胞亚群,EB病毒VCA-IgA、EA-IgA、EDAb、DNA免疫荧光定量进行评估。结果:随访8～18个月,中位随访时间12个月。CIK治疗1个月后患者CD4 CD25 %明显下降,EB病毒VCA-IgA、EA-IgA、DNA免疫荧光定量下降显著。结论:CIK细胞可改善鼻咽癌病人的免疫功能,加速EB病毒抗体及DNA拷贝数下降。提示CIK治疗可消灭肿瘤微小残留灶及转移灶,可作为一种鼻咽癌的辅助治疗。     

基因修饰T细胞肿瘤过继免疫治疗安全性的研究进展

基因修饰T细胞(CAR-T,TCR-T)赋予其肿瘤靶向性、更强的杀伤活性和持久的生命力,在恶性血液肿瘤和实体瘤的临床试验中取得了可喜的效果.然而,不断积累的的临床资料也发现了安全性担忧:炎症细胞因子级联反应引发的肿瘤靶向毒性;抗原识别相关的脱靶效应、非肿瘤靶向效应、移植物抗宿主病等,不仅难以预见而且复杂难治,促使研究人员以毒性发生机制为切入点,尝试应用自杀基因开关、细胞内凋亡酶调控、分离式CAR受体、抑制性CAR受体等调控策略,实现对基因修饰T细胞时间和空间的精准控制,预防毒性反应的发生.本文综述基因修饰T细胞的安全问题及应对策略.