胃癌的过继性免疫治疗研究进展

胃癌是目前世界上发病率及致死率较高的恶性肿瘤之一,在东亚地区尤其显著。针对胃癌的治疗手段仍是传统的手术联合化疗、放疗为主,尽管靶向药物治疗提供了新的选择,但其对晚期胃癌的疗效仍然有限。胃癌的免疫治疗作为独特的治疗手段,在近十多年发展较为活跃,特别是过继性免疫治疗手段不断有创新。过继性免疫治疗主要依赖回输具有抗肿瘤活性的细胞,目前回输的细胞由具有非特异性抗肿瘤作用向具有特异性抗肿瘤作用演变,特别是嵌合性抗原T细胞治疗的出现,为进展期胃癌患者提供了有一种潜在的选择。本文对胃癌过继性免疫治疗中采用的不同免疫活性细胞的作用机制、临床应用等进行总结,并针对其不足提出利用基因工程技术增强治疗靶向性、降低免疫逃逸的研究方向。     

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study

Purpose Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (γδ) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated γδ T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. Experimental design Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous γδ T cells every week or every 2 weeks, 6–12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. Results Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of γδ T cells in peripheral blood and also a high response to the antigen in vitro. Conclusions The results indicated that adoptive immunotherapy using in vitro-activated autologous γδ T cells was well tolerated and induced anti-tumor effects.     

Adoptive immunotherapy of breast cancer with lymph node cells primed by cryoablation of the primary tumor

Cryoablation of cancer leaves tumor-associated antigens intact in an inflammatory microenvironment that can stimulate a regional anti-tumor immune response. We examined whether cryoablated tumor draining lymph nodes (CTDLN) as adoptive immunotherapy may be an effective immunotherapeutic approach in the adjuvant treatment of breast cancer. BALB/c mice with MT-901 mammary adenocarcinoma tumors underwent cryoablation, resection or no treatment and tumor draining lymph nodes were harvested. Cryoablation resulted in only a mild increase in the absolute number of T-cells but a significant increase in the fraction of tumor-specific T-cells as evidenced on IFN-gamma release assay. FACS analysis demonstrated no significant relative shift in the proportion of CD4(+) or CD8(+) cells. The adoptive transfer of CTDLN resulted in a significant reduction of pulmonary metastases as compared to TDLN from either tumor-bearing mice or mice who underwent surgical excision. Cryoablation prior to surgical resection of breast cancer can be used as a method to generate effector T-cells for adjuvant adoptive cellular immunotherapy.     

采用不同方法制作胸腹水细胞块的效果比较及临床价值研究

目的:比较不同方法制作胸腹水细胞块的效果以优化胸腹水细胞块制作程序,并对其临床价值进行探讨。方法:以2014年3月至2015年3月我科收集的胸腹水标本120例为研究对象,将每样标本平均分成三组,使用三种不同方法(试管包埋法、直接离心法、细胞块试剂盒法)制作胸腹水细胞块。对不同方法制作细胞块的成功率、完整性及细胞切片恶性细胞的检出率进行考察与比较。结果:细胞块试剂盒法成功率最高,为96.67%,试管包埋法次之,成功率为92.50%,二者相比无显著差异(P0.05)。直接离心法制作成功率为80.83%,远远低于试管包埋法及细胞块试剂盒法,差异有统计学意义(P0.05)。细胞块试剂盒法制作的细胞块完整性最高,完整标本所占比例为96.67%,试管包埋法次之,完整率为94.17%,二者相比无显著差异(P0.05)。直接离心法制作完整率为68.33%,远远低于试管包埋法及细胞块试剂盒法,差异有统计学意义(P0.05)。三种方法恶性细胞检出率以细胞块试剂盒最高,与其他两种方法比较差异有统计学意义(P0.05)。结论:细胞块试剂盒法制作胸腹水细胞块具有最高的成功率及完整性,并且可以显著提高恶性细胞检出率,值得广泛使用。     

免疫细胞化学方法对胸腔积液中恶性肿瘤细胞的分类与诊断

目的应用免疫细胞化学对胸腔积液中的肺非小细胞癌分类与恶性间皮瘤的鉴别诊断。方法利用液基薄层细胞学自动涂片技术方法对筛查出的胸腔积液可疑瘤细胞及瘤细胞标本1158例进行细胞包埋连续切片,分别作肺非小细胞癌（NSCLC）肿瘤细胞标记物CK7、CK5＆6、TTF-1、E—Ca及恶性间皮瘤标记物MC（MesothelialCell,MC）、CR（Calfetinin,CR）、P53、Vimentin免疫细胞化学染色。结果1158例胸腔积液患者确诊为肺腺癌581例,鳞癌509例,腺鳞癌48例,恶性间皮瘤20例。TTF-1在腺癌中有明显高表达,阳性表达率为92．43％;CK58L6在鳞癌中有明显高表达,阳性表达率为97．45％;MC、CR在恶性间皮瘤中有明显高表达,阳性表达率为100．00％和95．00％。结论液基细胞学与免疫细胞化学技术相结合在胸腔积液鉴别诊断中有很重要的临床意义,CK7、CK58L6、TTF-1、E—ca联合应用可用于胸腔积液中NSCLC之间的分类与诊断,CK58L6、MC、CR、P53、Vimentin联合应用可用于胸腔积液中间皮瘤的定性诊断,值得在临床细胞病理学诊断中推广应用。     

Tumor-specific granulocyte/macrophage colony-stimulating factor and interferon γ secretion is associated with in vivo therapeutic efficacy of activated tumor-draining lymph node cells

In this study, cytokine release by tumor-draining lymph node cells sensitized in vitro (IVS-TDLN) was examined and correlated with therapeutic efficacy in adoptive immunotherapy. Mice bearing immunologically distinct MCA 207 and MCA 205 sarcoma tumors were utilized in criss-cross experiments. IVS-TDLN obtained from mice bearing 10-day subcutaneous (s. c.) tumors mediated immunologically specific regression of established 3-day pulmonary metastases, but demonstrated non-specific cytolytic reactivity against both tumors in a 4-h⁵¹Cr-release assay. By contrast, these IVS-TDLN cells were found specifically to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon (IFN) when restimulated in vitro with irradiated tumor cells. To determine the predictive value of tumor-specific cytokine release with in vivo therapeutic efficacy, a kinetic analysis of antitumor activities of TDLN obtained from animals bearing MCA 207 tumors for increasing lengths of time was performed. IVS-TDLN cells from mice bearing day-7, -10 and-14 s. c. tumors manifested tumor-specific release of GM-CSF and IFN, and mediated significant antitumor reactivity in vivo. In contrast IVS-LN cells from day-0 and day-21 tumor-bearing animals did not release significant amounts of GM-CSF and IFN, and were not therapeutically efficacious in vivo. Day-4 IVS-TDLN released high levels of GM-CSF and IFN non-specifically, and were not therapeutic in adoptive immunotherapy at doses effective for day-7 and day-14 IVS-TDLN cells. In other experiments, IVS cells generated from different lymph node groups in animals bearing 10-day established s. c. tumors were examined and found to have unique profiles of cytokine release. In these studies, the ability of IVS cells to release specifically both cytokines as opposed to one was associated with greater therapeutic efficacy on a per cell basis. Our findings suggest that the tumor-specific releases of GM-CSF and IFN are useful parameters to assess the in vivo therapeutic efficacy of immune lymphocytes.     

683 例胸腔积液病因分析

目的：目前医学证实有很多疾病的分布与民族及种族有关,而维吾尔族是新疆特有的少数民族,本文探讨了2010~2012 年入住我院的诊断明确的所有683 例胸腔积液（PE）患者与其中276 例维吾尔族PE 患者的病因分布的差异,从而指导临床工作。方法：回顾性分析2010~2012 年入住我院的683 例PE 患者及其中276 例维吾尔族PE 患者的临床资料,分析维吾尔族PE 患者与入我院总的PE 患者二者之间病因分别的差异。结果：入我院总的PE 患者与其中维吾尔族PE 患者最常见的病因相同,均为结核性PE、恶性PE、心功能不全性PE、肺炎旁PE,但二者之间这四种病因的分布有差异,前者病因分布顺序为恶性PE、结核性PE、心功能不全性PE、肺炎旁PE,而后者病因的分布顺序为结核性PE、心功能不全性PE、恶性PE、肺炎旁PE;二者在不同年龄段的病因分布也具有差异,后者在≤40 岁组与41~59 岁组比较、≤40 岁组与≥60 岁组比较有统计学差异,但41~59 岁组与≥ 60 岁组比较无统计学差异,但前者在上述三个年龄段比较均有统计学差异。结论：入我院总的PE 患者与维吾尔族PE患者的最主要的病因相同,但二者这四个病因分布的顺序不同,且二者在不同年龄段的病因分布也存在差异,前者在三个年龄段的病因分布均存在差异,而后者仅在二个年龄段的病因分布存在差异。     

683例胸腔积液病因分析

目的：目前医学证实有很多疾病的分布与民族及种族有关,而维吾尔族是新疆特有的少数民族,本文探讨了2010～2012年入住我院的诊断明确的所有683例胸腔积液（PZ）患者与其中276例维吾尔族PE患者的病因分布的差异,从而指导临床工作。方法：回顾性分析2010-2012年入住我院的683例PE患者及其中276例维吾尔族PE患者的临床资料,分析维吾尔族PE患者与入我院总的PE患者二者之间病因分别的差异。结果：入我院总的PE患者与其中维吾尔族PE患者最常见的病因相同,均为结核性PE、恶性PE、心功能不全性PE、肺炎旁PE,但二者之间这四种病因的分布有差异,前者病因分布顺序为恶性PE、结核性PE、心功能不全性PE、肺炎旁PE,而后者病因的分布顺序为结核性PE、心功能不全性PE、恶性PE、肺炎旁PE;二者在不同年龄段的病因分布也具有差异,后者在≤40岁组与41-59岁组比较、≤40岁组与≥60岁组比较有统计学差异,但41～59岁组与≥60岁组比较无统计学差异,但前者在上述三个年龄段比较均有统计学差异。结论：入我院总的PE患者与维吾尔族PE患者的最主要的病因相同,但二者这四个病因分布的顺序不同,且二者在不同年龄段的病因分布也存在差异,前者在三个年龄段的病因分布均存在差异,而后者仅在二个年龄段的病因分布存在差异。     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10¹¹ of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.Abbreviations PWM pokeweed mitogen - LAK lymphokine-activated killer - IL-2 interleukin 2 - PEL peripheral blood lymphocytes - TIL tumor-infiltrating lymphocytes - GVHD graft-versus-host disease - HLA human leukocyte antigen     

嵌合抗原受体T细胞免疫疗法的研究进展

嵌合抗原受体T(CAR-T)细胞是经基因工程修饰后表达靶向肿瘤细胞CAR分子的T细胞。CAR-T细胞治疗在血液肿瘤治疗中展现了显著的疗效而备受关注,是一种新的肿瘤免疫疗法。目前,CAR-T细胞治疗在实体瘤治疗中还面临着很大的挑战。而且,在临床应用的过程中仍会出现一些不良反应,如细胞因子释放综合征、神经毒性、归巢障碍、溶瘤综合征及脱靶效应等。本文就CAR-T疗法的基本原理及临床应用与不良反应展开做一综述。     

肝癌免疫治疗的研究进展

肝细胞肝癌是全球发病率和死亡率最高的恶性肿瘤之一,发病率和死亡率呈逐年上升趋势。我国是肝癌大国,每年肝癌的死亡病例数位居全球第一。免疫治疗是继手术、化疗和放疗之后新兴的癌症治疗手段,其通过解除肿瘤微环境对免疫细胞的抑制作用并激活机体免疫功能,实现控制和杀伤肿瘤细胞。常用的免疫治疗的方法有免疫检查点治疗、过继免疫治疗和肿瘤疫苗治疗等。与传统治疗手段相比,免疫治疗因具有增强机体免疫功能、延缓肿瘤进展、延长患者生存时间等优点,逐渐成为基础和临床研究的热点。文中就免疫治疗在肝癌领域的研究进展作一综述。     

肺癌性胸腔积液中生存素基因检测的诊断意义探讨

目的：生存素基因（survivin）是一种新近发现的抗凋亡基因,在肿瘤组织中呈现表达。本文旨在探讨和比较肺癌性胸腔积液和结核性胸腔积液中生存素基因的表达情况,以及其联合细胞学检查对判断肺癌性胸腔积液的敏感度。方法：应用逆转录酶-聚合酶链反应法（RT-PCR）检测2007年06月～2008年03月42例肺癌患者癌性胸腔积液标本,及同时期28例结核性胸腔积液标本的生存素mRNA表达情况,并联合细胞学检查结果进行对比分析。结果：42例肺癌患者胸腔积液标本中生存素mRNA的阳性率为52138％（22／42）;癌细胞的检出率为30．95％（13／42）;生存素mRNA检测联合细胞学检查诊断肺癌的敏感性为61．90％（26／42）,显著高于单独胸腔积液细胞学检测的敏感性（P〈0．001）。28例结核性胸腔积液标本的生存素mRNA阳性率为7．14％（2／28）,显著低于肺癌患者胸腔积液标本生存素mRNA的阳性率（P〈0．001）。结论：运用RT—PCR方法检测胸腔积液中生存素mRNA的表达在判断肺癌性胸腔积液中具有一定的敏感性和特异性,可能作为肺癌辅助诊断的一个新检测指标。     

阿霉素处理的肿瘤抗原致敏树突状细胞的抗肿瘤免疫研究

研究表明化疗药物作用于肿瘤细胞后可有效激发免疫应答,这与肿瘤细胞的性质和化疗药物有关。该研究主要探讨阿霉素（adriamycin,ADM）处理小鼠宫颈癌u14细胞获得的肿瘤抗原致敏树突状细胞（dendriticceils,DCs）的免疫应答及对肿瘤的杀伤效应。分别应用ADM和反复冻融法处理小鼠宫颈癌U14细胞,取其离心上清液,致敏小鼠骨髓来源的DC,观察DC诱导的淋巴细胞增殖反应和细胞毒性T淋巴细胞（cytotoxicTlymphocyte,CTL）对宫颈癌细胞的细胞毒效应。结果显示：ADM处理的U14细胞抗原致敏后的DC组所激发和扩增的T细胞数及对宫颈癌细胞的杀伤效果显著高于对照组（P〈0．05）。因而提示ADM处理的肿瘤抗原能有效地致敏DC并产生抗肿瘤免疫效应。     

Gene gun application in the generation of effector T cells for adoptive immunotherapy

We utilized the gene gun to transfect subcutaneous D5 melanoma and MT-901 mammary carcinoma tumors in situ with a granulocyte/macrophage-colony-stimulating factor (GM-CSF) plasmid complexed to gold particles. There was diminished tumor growth following bombardment with GM-CSF plasmid, which was apparent only during the period of administration. Transgenic GM-CSF was produced by the skin overlying the tumors and not by the tumors themselves. GM-CSF plasmid bombardment resulted in increased cell yields within tumor-draining lymph nodes (TDLN) with at least a 12-fold increase in the percentage of dendritic cells (8.9%) compared to controls (0.7%). Secondarily activated TDLN cells from animals transfected with GM-CSF demonstrated enhanced cytokine release (interferon γ, GM-CSF and interleukin-10) in response to tumor stimulator cells compared to controls, and had an increased capacity to mediate tumor regression in adoptive immunotherapy. There was a small, but detectable, non-specific immune adjuvant effect observed with gold particle bombardment alone, which was less than with GM-CSF plasmid. The adjuvant effect of GM-CSF plasmid required peri-tumoral transgene expression since gene bombardment away from the tumor was ineffective. Received: 27 April 1999 / Accepted: 27 August 1999     

Immunotherapeutic approaches in Hepatocellular carcinoma: Building blocks of hope in near future

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic cancer and is among the major causes of mortality due to cancer. Due to the lack of efficient conventional therapeutic options for this cancer, particularly in advanced cases, novel treatments including immunotherapy have been considered. However, despite the encouraging clinical outcomes after implementing these innovative approaches, such as oncolytic viruses (OVs), adoptive cell therapies (ACT), immune checkpoint blockades (ICBs), and cancer vaccines, several factors have restricted their therapeutic effect. The main concern is the existence of an immunosuppressive tumor microenvironment (TME). Combination of different ICBs or ICBs plus tyrosine kinase inhibitors have shown promising results in overcoming these limiting factors to some extent. Combination of programmed cell death ligand-1 (PD-L1) antibody Atezolizumab and vascular endothelial growth factor (VEGF) antibody Bevacizumab has become the standard of care in the first-line therapy for untestable HCC, approved by regulatory agencies. This paper highlighted a wide overview of the direct and indirect immunotherapeutic strategies proposed for the treatment of HCC patients and the common challenges that have hindered their further clinical applications.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture     

Therapeutic use of a long-term cytotoxic T cell line recognizing a common tumour-associated antigen: The pattern of in vitro reactivity predicts the in vivo effect on different tumours

Summary A long-term-cultured cytotoxic T lymphocyte (CTL) line (E/88) was obtained from splenic lymphocytes of BALB/c (H-2 ^d) mice bearing the weakly immunogenic colonic carcinoma C26. This line was shown to be /TCR⁺V6⁺CD3⁺CD8⁺CD4^– and to recognize a common tumour-associated antigen on syngeneic carcinomas and sarcomas in a major-histocompatibility—complex-restricted and T-cell-receptor(TCR)-mediated fashion. The assessment of cytotoxic activity on a panel of 30 normal and neoplastic target cells of differing etiology and histotype showed that E/88 CTL lysed syngeneic colon carcinomas and some fibrosarcomas but not leukemias, lymphomas or mammary carcinomas. Clones derived from the E/88 line exhibited the same lytic pattern. Moreover, anti-T3, anti-Lyt2.2, anti-/TCR and anti-V6 mAbs as well as anti-H-2^d antisera abolished cytotoxicity when used in blocking experiments. The therapeutic activity of E/88 CTL upon in vivo transfer was assessed in mice bearing either experimental or spontaneous metastases of C26. In both models therapy with E/88 lymphocytes in combination or not with interleukin-2 was highly effective. Adoptive immunotherapy carried out with two clones obtained from line E/88 showed comparable therapeutic effects. In addition, treatment of syngeneic mice bearing experimental metastases of in vitro E/88-lysable or E/88-resistant tumours, showed that E/88 CTL can eradicate metastases of the former but not of the latter neoplasms. These data indicate that long-term CTL lines recognizing common tumour-associated antigens can be derived from tumourbearing animals and used in adoptive immunotherapy of tumours previously shown to be lysed in vitro by these effectors.