Induction of Specific Cytotoxic T Lymphocytes against Autologous Brain Tumor by Crossreactive Allo-tumor Cell Stimulation

Cytotoxic T lymphocytes (CTL) against autologous malignant brain tumor were generated in peripheral blood lymphoid cells (PBL) prepared from a patient with a malignant brain tumor by stimulation of the cultured PBL for 7 days with attenuated Crossreactive malignant melanoma (MM2) cells pretreated with mitomycin C. The Crossreactive MM2 cells were effective for antigen stimulation for CTL induction in place of autologous glioblastoma cells, which are difficult to expand in culture. The optimal ratio between nylon wool-passed T lymphocytes and nylon wool-adherent accessory cells to induce CTL in the patient's PBL was found to be 25 to 1. In vitro -activated CTLs induced by MM2 were cytotoxic not only to MM2, but also to the autologous tumor cells in an HLA class I-restricted manner, and their surface phenotype was found to be CD3⁺ and CD8⁺. CTL therapy using cross-reactive allogeneic tumor cells as the stimulator could be clinically valuable to treat malignant brain tumors.     

Direct Activation of Human CD8+ Cytotoxic T Lymphocytes by Interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8⁺ T cells present after 28 days in the induction culture. When purified CD8⁺ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8⁺ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

干扰肿瘤微环境对细胞毒性T淋巴细胞瘤体内归巢和杀伤活性的影响

目的 探讨应用低剂量环磷酰胺（cyclophosphamide, CYC）干扰荷瘤小鼠体内微环境对改善过继回输的细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）瘤体内归巢和杀伤活性的作用。方法 建立C57BL/6荷瘤小鼠模型并分为对照组、CYC注入（CYCI）组、0.9%NaCl溶液注入（NSI）+CTL组和CYCI+CTL组,每组40只,分别给CYCI组、CYCI+CTL组每只小鼠腹腔注入CYC （20mg/kg）,NSI+CTL组小鼠注入等量0.9%NaCl溶液。检测注射前后不同时间小鼠瘤体内调节性T细胞（Treg）、白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）水平变化。于注射后第4天回输CFSE-CTL,每只0.2 ml,含5×l0⁶个细胞。流式检测对比NSI+CTL组和CYCI+CTL组瘤体内CFSE-CTL比例。结果 NSI+CTL组小鼠Treg、IL-10、TGF-β水平随着瘤体增长而逐渐增高;CYCI+CTL组比NSI+CTL组回输的CFSE-CTL在肿瘤内归巢明显增加和持久（P<0.05）。各组肿瘤体积除对照组和CYCI组外,其余各组间差异均有统计学意义（P<0.05）。结论 提前应用低剂量CYC干预荷瘤小鼠体内微环境,可降低瘤体内Treg、IL-10和TGF-β水平,使过继回输的CTL在肿瘤中的归巢聚集明显增强,杀伤肿瘤效率提高。     

Identification of a Human T Cell Clone with the Cytotoxic T Lymphocyte and Natural Killer-like Cytotoxic Function against Autologous Mammary Carcinoma and K562 Line

Pleural exudative lymphocytes (PLED from a 60-year-old female patient showed high cytotoxicity against the autologous mammary tumor line, HMC-2, and NK-susceptible K562 cells, although PLEL demonstrated only weak cytotoxic potentials against several allogeneic tumor lines. We successfully obtained seven cytotoxic T cell clones from PLEL bulk populations, and assessed the possibility that these lymphocytes are simply natural killer (NK)-like cells or have the dual cytotoxic activity of cytotoxic T lymphocytes (CTL) and NK-like cells. These clones, designated as T_cHMC-2, showed strong cytotoxicity against both HMC-2 and K562 cells. In contrast, allogeneic human peripheral blood-derived NK cells could not kill HMC-2 targets. Furthermore, a blocking study of T_cHMC-2 cytotoxicity using monoclonal antibodies against CD3, CD8 and human MHC class I products showed that all of these antigen molecules were involved in the cytotoxicity of T_cHMC.2 clone against autologous HMC-2 cells, indicating MHC class I recognitive cytotoxicity. These data indicate that the T_cHMC-2 clone may have dual cytotoxicity with CTL- and NK-like activity against autologous HMC-2 mammary tumor and K562 cells, respectively.     

Augmentation of Human Cytotoxic T Lymphocytes against Autologous Tumor by a Factor Released from Human Monocytic Leukemia Cell Line

A human acute monocytic leukemia cell line, THP-1, releases a factor which activates human cytotoxic (killer) T lymphocytes (CTL) against autologous tumor in vitro . The factor, named cytotoxic (killer) T cell activating factor (KAF), is an acidic protein of 70,000 to 100,000 dalton molecular size. Peripheral blood leukocytes from two patients, bearing epithelioid sarcoma or malignant schwannoma, were cultured for 7 days with individual autologous tumor to induce CTL directed to the corresponding tumor. Monocyte-depleted peripheral leukocytes generated lesser CTL activity than the monocyte-containing leukocyte population. However, the KAF was able to replace the monocyte function. The KAF acted at the CTL generation phase as well as the effector phase. The KAF-activated killer cells possessed CD4^-8⁺ surface phenotype. The CTL killed autologous tumor or other unrelated tumor cell lines only when they shared some of the HLA class I antigens. It was also demonstrated that the KAF does not activate killer cells without proper antigenic stimuli, because the KAF-augmented CTL possess specificity against autologous tumor or other HLA-A or -B matched tumor cell lines. The therapeutic applicability of human KAF for anti-tumor CTL therapy against autologous tumor is discussed.     

Pilot Study of Local Autologous Tumor Infiltrating Lymphocytes for the Treatment of Recurrent Malignant Gliomas

A prospective pilot study was performed in order to assess the safety of treating recurrent malignant gliomas (MGs) with locally infused autologous tumor infiltrating lymphocytes (TILs) and recombinant interleukin-2 (rIL-2). Six patients were entered between June 27, 1994 and June 2, 1995 and followed until July 1, 1998. At surgery an Ommaya reservoir was placed for later infusion of TILs and rIL-2. Following surgery, autologous TILs were expanded in vitro in the presence of rIL-2 and infused on treatment days 1 and 14, with concurrent rIL-2 infusions performed three times each week for one month. Following completion of immunotherapy all patients were offered chemotherapy. Phenotypic analysis demonstrated TILs to be T-lymphocytes (87–99% CD3+). Of these, 4 of 6 cases (67%) phenotyped as cytotoxic/suppressor T-lymphocytes (CD8+) and 2 of 6 cases (33%) phenotyped as helper/inducer T-lymphocytes (CD4+). TILs demonstrated limited selective cytotoxicity, with dose dependent cytotoxicity against autologous tumor, allogenic tumor and long term MG cell lines.There were no significant (Grade 3 or 4) complications. One patient developed transient low grade fevers, and 2 developed asymptomatic hydrocephalus. All patients developed transient and asymptomatic cerebral swelling, noted on the immediate post-treatment imaging studies.At three and six month follow-up, 3 patients responded with partial response, 2 demonstrated stable disease and 1 patient progressed. At long term follow-up, 1 patient had a complete response (45 month follow-up), 2 had a partial response (48 and 47 month follow-up) and 3 patients expired as a result of progressive disease (at 12, 12 and 18 months following immunotherapy). A relationship between subsequent chemotherapy or extent of resection to outcome was not appparent but could not be excluded.This pilot study demonstrated that locally infused autologous TILs and rIL-2 could be delivered without serious toxicity. Further studies are indicated to determine the safety and long term efficacy of TIL immunotherapy.     

大肠癌CA-Hb3抗原体外可诱导特异性CTL反应

在5例大肠癌患者的瘤细胞诱导自身外周血淋巴细胞的CTL反应中,有2例诱导成功,其肿瘤细胞CA-Hb_3抗原的表达呈强阳性;另3例无诱导作用,其肿瘤细胞CA-Hb_3的表达呈弱阳性.借助脂质体将CA-Hb_3导入3例弱阳性的肿瘤细胞内,免疫组化证实有2例瘤细胞的CA-Hb_3的表达转呈强阳性,该瘤细胞能诱导出CTL反应.阻断细胞内HLA-Ⅰ类分子与CA-Hb_3结合及在细胞膜上的表达皆能抑制CTL的杀伤反应.结果表明用大肠癌CA-Hb-3抗原可以增强肿瘤细胞的免疫原性,所诱导的CTL具有抗原特异性及MHC-Ⅰ类分子限制.     

脑胶质瘤患者自体免疫治疗前后T细胞亚群的变化

 目的 探讨脑胶质瘤患者自体免疫治疗的临床应用，观察该治疗对T细胞亚群水平的影响。方法 试验组21例脑胶质瘤患者接受自体树突状细胞免疫治疗，对照组19例接受常规治疗，进行临床随访并检测两组T细胞亚群水平的变化。结果 试验组中位生存时间（29个月）远大于对照组（10个月），两组生存曲线分布差异有显著性意义（P〈0．05）。脑胶质瘤患者外周血中CD^3＋含量、CD^4＋含量以及CD^4＋／CD^8＋比值明显低于正常对照组（P〈0．01），免疫治疗后患者外周血CD^3＋、CD^4＋、CD^8＋含量以及CD^4＋／CD^8＋比值与对照组比较均增加（P〈0．05），但CD^4＋／CD^8＋比值仍低于健康成人组（P〈0．05）。结论 脑胶质瘤存在免疫功能抑制，自体树突状细胞肿瘤疫苗治疗可一定程度重建、增强机体肿瘤免疫应答，抑制脑胶质瘤的复发和进展，从而有效地延长脑胶质瘤患者的生存时间。     

Adoptive Immunotherapy for Small Cell Lung Cancer by Expanded Activated Autologous Lymphocytes: a Retrospective Clinical Analysis

Background: To investigate the clinical efficacy of expanded activated autologous lymphocytes (EAAL) inpatients with small cell lung cancer (SCLC). Materials and Methods: A total of 32 SCLC patients were selectedand randomly divided into EAAL treatment and control groups, 16 cases in each. EAAL were obtained byproliferation of peripheral blood mononuclear cells (PBMCs) of patients followed by phenotype determination.Clinical data of all patients were recorded. Patients of both groups were followed up and the overall survival(OS) were compared retrospectively. Results: After culture and proliferation in vitro, the percentages of CD3+,CD3+CD8+, CD45RO+, CD28+, CD29+, CD8+CD28+ and CD3+CD16+/CD56+ cells increased markedly (p<0.05).The OS of the EAAL treatment group was longer than that of control group, but the difference was not statisticallysignificant (p=0.060, HR=0.487, 95%CI 0.228~1.037). 1- to 3-year survival rates in EAAL treatment group werelonger than those in control group, but there was still no significant difference (p>0.05). COX multivariateregression analysis showed that the number of chemotherapy cycles and the application of EAAL immunotherapywere independent prognostic factors for SCLC patients. The OS in females and chemotherapy≤6 cycles wereobviously prolonged after EAAL immunotherapy. Conclusions: In vitro induction and proliferation of EAALis easy and biologically safe. Generally, EAAL adoptive immunotherapy can evidently prolong the OS of SCLCpatients.     

恶性实体瘤患者自体CIK细胞的体外扩增及初步临床应用

目的观察恶性实体瘤患者外周血来源的细胞因子诱导杀伤细胞(CIK)在自体血浆中的增殖特性,回输治疗的安全性及近期疗效。方法用淋巴细胞分离液分离获取40例恶性实体瘤患者外周血单个核细胞,在含10%自体血浆的RPMI-1640培养液中添加rhIFN-γ、Anti-CD3mAb和rhIL-2联合诱导单个核细胞为CIK细胞。在培养第13天进行细胞计数,通过流式细胞术检测细胞免疫表型,并将细胞进行自体回输。结果在培养第13天,CIK细胞得到大量扩增,细胞数增加30.0±5.2倍。CD3 和CD3 CD16 CD56 细胞在培养第13 d分别为(93.0±6.1)%和(25.8±12.2)%。40例恶性实体瘤患者回输自体CIK细胞后,6例部分缓解;13例好转;9例病情稳定;12例进展。治疗总有效率为15%。治疗过程中主要出现畏寒、发热,未观察到其它明显的毒副作用。结论恶性实体瘤患者自体血浆可以高效扩增CIK细胞,回输治疗安全有效,且无明显的毒副作用。     

Autologous tumor cell vaccination combined with adoptive cellular immunotherapy in patients with Grade III/IV astrocytoma

Summary Brain tumors are highly resistant to treatment. Their diffuse infiltrative nature and the relative inaccessibility of the brain to blood and lymph are barriers to surgical and cytotoxic treatments alike. Preclinical animal studies demonstrated that intravenously administered tumor antigen-specific T lymphocytes will reject tumors growing in the brain. Specifically activated effector T lymphocytes may be generated by in vivo immunization followed by restimulation of antigen-primed T cells with autologous tumor cells in vitro. In order to apply these findings to humans, feasibility studies of combined active immunization and specific adoptive cellular immunotherapy were performed on fifteen patients with recurrent astrocytoma. The objective was to determine whether; 1) T cells could be grown from peripheral blood of patients immunized with autologous tumor cells, and 2) whether stimulated cells could be safely readministered to patients. Patients were immunized with a combination of their own irradiated tumor cells and Bacillus of Calmette and Guerin. Two weeks later, a mononuclear cell-rich fraction of blood was obtained by leukapheresis. Mononuclear cells were cultured with irradiated autologous tumor cells and interleukin-2. Selective expansion of CD4⁺ and CD8⁺ T lymphocytes occurred. Intravenous transfer of stimulated cells to the fifteen patients on twenty-four separate occasions with or without systemic administration of interleukin-2 was tolerated with limited toxicity. The studies established the feasibility of conducting controlled studies of the anti-tumor effects of tumor antigen-specific cellular immunotherapy.     

Interleukin-12 Augments the Generation of Autologous Tumor-reactive CD8+ Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes

Human tumor-infiltrating lymphocytes (TIL) were obtained from breast cancer, renal cancer or neuroblastoma to investigate the generation of autologous tumor-reactive CD8⁺ cytotoxic T lymphocytes (CTL). When TIL were cultured with interleukin (IL)-2 (100 U/ml), the growth of TIL peaked around 8–10 days after the initiation of culture. In contrast, the proliferation of TIL cultured with IL-2 plus IL-12 peaked around 4–5 days after culture and tumor cells rapidly disappeared from the culture. To determine the generation of autologous tumor-reactive CD8⁺ CTL, TIL-derived CD8⁺ T cells were separated by FACStar. Both IL-2-activated and IL-2 plus IL-12-activated TIL-CD8⁺ T cells showed the same level of lymphokine-activated killer activity against a variety of tumor cells. However, TIL-CD8⁺ T cells activated with IL-2 plus IL-12 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. The autologous tumor cell-killing activity of TIL-CD8⁺ CTL was significantly inhibited by the addition of F(ab)₂ anti-CD3 monoclonal antibody, indicating that these CTL recognize autologous tumor antigen through T cell receptor. These results imply that IL-12 is a novel cytokine which facilitates the generation of autologous tumor-reactive CD8⁺ CTL from TIL.     

IL-2基因修饰的细胞毒 T淋巴细胞抗肿瘤效应的研究

目的：了解IL－2基因修饰的细胞毒T淋巴细胞（cytotoxic T lymphocytes,CTL)的增殖和杀伤活性,探索细胞因子基因疗法及被动免疫治疗脑胶质瘤的新途径。方法：用昆明种小鼠的脾细胞体外诱导CTL,用腺病毒载体转染IL－2基因,观察其体外增殖活性和样伤活性,再用G422小鼠胶质母细胞瘤细胞建立肺转移瘤模型,36h后经过继回输,2周后计数肺的肿瘤结节,观察IL－2基因转染的CTL对实验性肺转移瘤的治疗作用。结果：重组腺病毒载体在MOI（multipliciy of infection)为100时,转染率达96．8％,IL－2基因修饰CTL增殖活性、IL－2的分泌（248u)和体外杀伤活性（36．4％）明显增强,对实验性肺转移瘤的治疗作用都显著增强,肺转移结节数（28）显著减少（P＜0．01）。结论：IL－2基因转染的CTL过继回输,可直接杀伤和诱导激活机体抗肿瘤免疫反应,使体内抗肿瘤效果显著增强,有效抑制实验性肺转移瘤的生长,为胶质瘤的过继免疫治疗提供了新的思路和实验依据。     

影响人脑恶性胶质瘤预后因素的研究

背景与目的:影响人脑恶性胶质瘤预后的因素众多,本文通过总结我们的临床病例,分析影响人脑恶性胶质瘤预后的因素。方法:收集自2003年10月至2004年4月在南方医科大学附属南方医院神经外科手术治疗的67例恶性胶质瘤患者的资料。生存分析单因素使用Kaplan-Meier法计算生存率并采用对数秩(log-rank)检验;多因素分析使用Cox比例风险模型,采用逐步回归分析。结果:单因素分析结果显示术前癫痫、肿瘤分化程度、肿瘤范围、术中病理标示肿瘤向周边侵袭性生长程度、手术切除程度及术后个体化综合治疗等因素与患者预后有关(P<0.05);多因素分析示手术切除范围、术后个体化综合治疗、肿瘤侵袭性生长程度、肿瘤的分化程度则是影响无瘤生存的独立因素。年龄、性别、KPS评分、肿瘤体积等因素两种分析均未发现与预后有关。结论:手术切除范围、术后个体化综合治疗、肿瘤侵袭性生长程度、肿瘤的分化程度对预后影响较大;而术前癫痫、肿瘤范围对判断预后价值有限;患者的年龄、性别、KPS评分、肿瘤体积与预后无关。     

Successful Induction of Tumor-specific Cytotoxic T Lymphocytes from Patients with Non-small Cell Lung Cancer Using CD80-transfected Autologous Tumor Cells

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80-transfected tumor cells as stimulators of the in vitro induction of autologous tumor-specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non-small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80-transfected tumor cells (CD80-AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non-transfected tumor cells (AT). AT-stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80-AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT-stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)-class I-restricted cytokine production in response to AT, while the MHC-class I-restricted responses were found in CD80-AT-stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.     

BCR-ABL Fusion Peptides and Cytotoxic T Cells in Chronic Myeloid Leukaemia

The BCR-ABL gene that arises in chronic myeloid leukaemia (CML) is a neoantigen. Peptides derived from the BCR-ABL fusion junction may therefore be immunogenic, if appropriately presented to the immune system. This article reviews data demonstrating that certain junctional peptides will bind to HLA molecules, and that these peptides will elicit specific T-lymphocyte responses in vitro, in both normal subjects and in CML patients. The clinical relevance of these observations is discussed.     

A Pilot Study of Autologous Cancer Cell Vaccination and Cellular Immunotherapy Using Anti-CD3 Stimulated Lymphocytes in Patients with Recurrent Grade III/IV Astrocytoma

The study objectives were to determine; (1) whether activated T cells could be generated from peripheral blood of patients immunized with their own cancer cells, (2) whether adoptive transfer of the activated T cells to patients had toxic effects and (3) whether the infused cells produced clinical responses. Study patients had recurrent, surgically accessible grade III/IV astrocytomas. The patients were tapered off steroids after total surgical resection and immunized with autologous cancer cells plus Bacillus, Calmette and Guerin (BCG). Peripheral blood mononuclear cells were activated with anti-CD3, expanded with interleukin-2 (IL-2) and reinfused to patients. The number of activated T cells that was given back to patients varied between 10¹⁰ and 10¹¹. Side effects that were observed following immunization and adoptive cell transfer included mainly transient flu-like symptoms. One patient's tumor partially regressed, but there was no effect on survival. Two other patients' tumors regressed, and the patients are apparently disease-free more than 5 and 4 years later. The other six patients' tumors were apparently unaffected by the treatment. Patient age, tumor grade and CD4/CD8 composition of infused cells were positively correlated with clinical responses. Cellular immunotherapy is feasible and is associated with minimal toxicity. Additional appropriately controlled studies will be required to determine whether cellular immunotherapy could be used as a treatment for central nervous system malignancy. Additional studies also will be required to determine the underlying immunological mechanisms.     

Histocompatibility Leukocyte Antigen-A2-restricted and Tumor-specific Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes of a Patient with Testicular Embryonal Cancer

T lymphocytes play an important role in tumor rejection. To understand T cell-mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxicity. We established a CD3⁺CD4^-CD8⁺ cytotoxic T lymphocyte (CTL) line from the IL-2-activated TIL of a 37-year-old patient with testicular embryonal cancer. A 6 h ⁵¹Cr-release assay was performed to measure the cytotoxicity of the CTL. The CD3⁺CD4^-CD8⁺ CTL line showed cytotoxicity against HLA-A2⁺ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA-A2" tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA-A2-restricted and tumor-specific CTL at the tumor site of testicular embryonal cancer.     

MicroRNAs在胶质瘤治疗中的作用及研究进展

越来越多证据表明microRNAs在神经肿瘤的发生发展中扮演着重要角色。随着组织和血浆中提取和测定microRNAs技术的改进．某些特定的胶质母细胞瘤或其他恶性肿瘤的microRNAs作为临床生物标志物已为时不远,但应用microRNAs治疗恶性胶质瘤仍有较大困难。本文通过microRNAs在恶性胶质瘤的表达、分子靶向治疗及microRNAs的给药途径三个方面对microRNAs治疗恶性胶质瘤的研究进展做一综述。