The Induction of Cytotoxic T Lymphocytes against HLA-A Locus-matched Lung Adenocarcinoma in Patients with Non-small Cell Lung Cancer

To induce cytotoxic T lymphocytes (CTL) against non-small cell lung cancer (NSCLC) efficiently, the induction of CTL was attempted using HLA-A locus-shared allogeneic NSCLC cells. T cells derived from either tumor tissue specimens or the regional lymph nodes of patients with NSCLC were stimulated twice or three times with an HLA-A2/A24-positive NSCLC cell line (PC-9), and thereafter the cytotoxic activity was examined by ⁵¹Cr-release assay. In patients with HLA-A24/ adenocarcinoma, anti-PC-9 cytotoxicity was induced in all 6 patients tested. Anti-PC-9 cytotoxicity was induced in 2 out of 5 patients with HLA-A2 (A24⁻)/adenocarcinoma, in 2 out of 4 patients with HLA-A24/squamous cell carcinoma, and 1 of 2 patients with HLA-A2/squamous cell carcinoma. The cytotoxic activity was observed to kill PC-9 selectively, not other NSCLC lines, and the activity was substantially blocked by anti-MHC class I antibody, but not by anti-MHC class II antibody. The PC-9-specific CTL produced γ-interferon in response to autologous tumor cells. These results indicated that the anti-PC-9 cytotoxicity was mediated by cytotoxic T lymphocytes that may recognize the T cell epitope(s) shared and presented by HLA-A2 and/or HLA-A24-positive NSCLC.     

肺癌患者红细胞与淋巴细胞的协同抗肿瘤免疫作用

将肺癌患者的红细胞和淋巴细胞按一定步骤与经血清致敏的癌细胞作用,观察肺癌患者红细胞与淋巴细胞围攻癌细胞情况.结果显示:肺癌患者红细胞与淋巴细胞不仅可各自单独围攻癌细胞,且具有协同抗肿瘤免疫作用,肺癌患者红细胞对淋巴细胞免疫粘附癌细胞的促进作用较正常人降低.本文对这些现象进行了初步的分析.     

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.     

Autologous Tumor-specific Cytotoxic T Lymphocytes in a Patient with Lung Adenocarcinoma: Implications of the Shared Antigens Expressed in HLA-A24 Lung Cancer Cells

Human lung adenocarcinoma-specific cytotoxic T lymphocytes (CTL) were generated by multiple stimulations with autologous tumor cells (named A110L) from regional lymph node lymphocytes and tumor-infiltrating lymphocytes expanded by solid-phase anti-CD3 monoclonal antibody (mAb) and recombinant interleukin-2. The CTL lysed A110L but failed to kill either autologous B lymphocytes immortalized by the Epstein-Barr virus or K562. The killing activity of the CTL against autologous A110L was inhibited by anti-MHC class I mAb (W6/32), but not by anti-MHC class II mAb. The CTL produced interferon-γ and GM-CSF in response to A110L and the production was completely blocked by the addition of anti-MHC class I mAb. The HLA type of the CTL was HLA-A2/A24, B52/B54, Cw1/-. Allele-specific deletion of HLA-A2 molecules was observed in A110L by staining with anti-HLA-A2 mAb. A partial blocking effect on the cytokine production from the CTL was also obtained with anti-CD8, and anti-HLA-A24 mAbs, but not with anti-MHC class II, anti-CD4 and anti-HLA-A2 mAbs. To analyze further the mechanism of antigen recognition by the CTL, the cross reactivity of the CTL against several HLA-A locus-matched (HLA-A24+) and mismatched allogeneic tumor cells (HLA-A24-) was investigated. The A110L-specific CTL showed a weak but significant cytotoxicity against some HLA-A24 positive lung cancer cell lines, such as Sq-1 (HLA-A11/A24, squamous cell carcinoma) and PC-9 (HLA-A2/A24, adenocarcinoma), but failed to kill HLA-A locus-mismatched allogeneic tumors. This cross reactivity of the CTL against Sq-1 and PC-9 was blocked by anti-MHC class I mAb. These results thus demonstrate that shared common tumor antigens might exist among lung cancer cells in the context of HLA-A24.     

Cytotoxicity Tests against Cultured Human Lung Cancer Cells With Autologous Lymphocytes Activated in vitro by Mitomycin C-Treated Tumor Monolayers in the Presence of T-Cell Growth Factor

Human peripheral blood or lymph node lymphocytes, obtained frompatients with a variety of lung cancer, were incubated in vitrowith mitomycin C-treated tumor monolayers in the presence ofT-cell growth factor. The cytotoxicity of these lymphocytesfor autologous tumor cells (autologous killer activity) wasassessed by a 4-hr ⁵¹Cr-release assay. Cytotoxic activity wasobserved in 14 out of a total of 20 cases. Lymphocytes frompatients with squamous cell carcinoma, large cell carcinomaand carcinoid exhibited positive activity levels of 11.1 ±1.8, 16.3 and 23.9% respectively. Nine out of 13 patients withadenocarcinoma exhibited positive activity with a mean valueof 8.8 ± 6.8%. No lymphocyte activity against small cellcarcinoma was observed. Natural killer (NK) activity did not always correlate with autologouskiller (AK) activity. Treatment of lymphocytes with monoclonalanti-human lymphocyte antibody revealed differences in effectorcell populations concerning these two activities; AK activitywas abrogated only by treatment with anti-human Lyt 3 antibodyand complement, whereas NK activity was abrogated by anti-humanLyt 1, 2 and 3 and partially by anti-human la antibody. Theseresults indicate that AK activity is mediated exclusively byT cells, but that NK activity is mediated by several subpopulationsof lymphocytes such as T cells, null cells and others.     

Inhibition of Cytotoxicity to Autologous Tumor Cells by the Regional Lymph Node Cells of Patients with Primary Lung Cancer

The regional lymph node (RL) cells of patients with primarylung cancer exhibited no cytotoxicity to autologous tumor cellsin a 4-hr ⁵¹Cr-release assay, but when cultured in the presenceof Interleukin 2 (IL2), the RL cells did become cytotoxic tothose target cells. When RL cells were included in a cytotoxictest of IL2-activated RL cells (autologous killer T cells; AK.Tcells) and autologous target cells, the cytotoxicity of theAKT cells was significantly inhibited in 27 out of a total of42 cases, but this suppression was observed against neitherallogeneic effector cells (seven out of nine cases) nor naturalkiller cells (all seven cases tested). The cytotoxicity of AK.Tcells to allogeneic target cells was inhibited by RL cells inthree out of six cases. Nylon-wool column separation indicatedthat the cell population adhering to the nylon wool mediatedthe sup-pressive effect of the RL cells. These data suggestedthe presence of nylon-wool-adherent suppressor cells in theregional lymph nodes of patients with primary lung cancer whichsuppress the cytotoxicity of autologous killer lymphocytes toautologous tumor cells.     

Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer - Reduction after Surgical Resection

Background: The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. Materials andMethods: We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim-3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controlsby flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patientcohort. Results: It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 andp=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevatedTim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages(III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression wassignificantly reduced on after surgical resection of the primary tumor (p<0.01). Conclusions: Tim-3 expressionin natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lungcancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.     

The Proangiogenic Phenotype of Natural Killer Cells in Patients with Non-Small Cell Lung Cancer

The tumor microenvironment can polarize innate immune cells to a proangiogenic phenotype. Decidual natural killer (dNK) cells show an angiogenic phenotype, yet the role for NK innate lymphoid cells in tumor angiogenesis remains to be defined. We investigated NK cells from patients with surgically resected non-small cell lung cancer (NSCLC) and controls using flow cytometric and functional analyses. The CD56⁺CD16^- NK subset in NSCLC patients, which represents the predominant NK subset in tumors and a minor subset in adjacent lung and peripheral blood, was associated with vascular endothelial growth factor (VEGF), placental growth factor (PIGF), and interleukin-8 (IL-8)/CXCL8 production. Peripheral blood CD56⁺CD16^- NK cells from patients with the squamous cell carcinoma (SCC) subtype showed higher VEGF and PlGF production compared to those from patients with adenocarcinoma (AdC) and controls. Higher IL-8 production was found for both SCC and AdC compared to controls. Supernatants derived from NSCLC CD56⁺CD16^- NK cells induced endothelial cell chemotaxis and formation of capillary-like structures in vitro, particularly evident in SCC patients and absent from controls. Finally, exposure to transforming growth factor-β₁ (TGFβ₁), a cytokine associated with dNK polarization, upregulated VEGF and PlGF in peripheral blood CD56⁺CD16^- NK cells from healthy subjects. Our data suggest that NK cells in NSCLC act as proangiogenic cells, particularly evident for SCC and in part mediated by TGFβ₁.     

Histocompatibility Leukocyte Antigen-A2402-restricted Cytotoxic T Lymphocytes Recognizing Adenocarcinoma in Tumor-infiltrating Lymphocytes of Patients with Colon Cancer

To cast light on T cell-mediated specific immunity at the tumor site of colon cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) from colon cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted cytotoxicity against adenocarcinoma. IL 2-activated TIL from all four HLA-A24 patients examined lysed HLA-A2402+ adenocarcinomas, but not HLA-A2402 tumors. Those of two of the four cases also lysed HLA-A2402⁺ squamous cell carcinomas. CD8⁺ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2402⁺ adenocarcinomas were established from one CTL line. This CTL line produced IFN-γ upon recognition of an HLA-A2402^- adenocarcinoma transfected with HLA-A2402 cDNA. These results suggest the presence of HLA-A2402-restricted CTL recognizing adenocarcinoma at the tumor site of colon cancer. Furthermore, HLA-A31-restricted CTL activity was found in IL-2-activated TIL from one of two HLA-A31⁺ patients, suggesting the existence of HLA-class I-restricted CTL involving an allele other than A24     

肺癌患者Th1/Th2细胞的变化

[目的]观察肺癌患者血浆及外周血单个核细胞(PBMC)诱生IL 2、IL 4水平 ,进而反映患者Th1和Th2细胞功能。[方法]运用细胞因子诱生及ELISA技术 ,测定48例肺癌术前患者和24例术后患者血清及外周血单个核细胞诱生的IL 2、IL 4水平。以IL 2水平反映Th1细胞功能 ,以IL 4水平代表Th2细胞功能。[结果]肺癌患者血清及PBMC诱生产生IL 2水平减低 ,而IL 4水平升高。PBMC诱生及血清IL 2、IL 4水平在不同分期、不同病理类型肺癌病人间比较无差别 ,行肺癌根治术后的患者血清及PBMC诱生IL 2水平高于未手术肺癌患者水平。[结论]肺癌患者体内Th1/Th2细胞亚群功能失调 ,这可能是肺癌组织在患者体内进行性生长及免疫逃避的机制之一 ;肺癌患者Th1/Th2亚群失调在肺癌不同分期、不同病理类型患者间无差别 ,而手术可改善肺癌病人Th1/Th2亚群功能失调状态。     

肺癌患者肺泡巨噬细胞细胞因子及其细胞杀伤活性的研究

目的：了解肺癌患者肺泡巨噬细胞（alveolar macrophage,AM)产生细胞因子的情况及其细胞因子的细胞杀伤活性,同时观察γ干扰素（γ interferon,γ－INF）和脂多糖（lipoplysac charide,LPS)对AM活性的影响。方法：经肺泡灌洗获AM,分离,γ－INF和／或LPS刺激培养,分别检测AM产生NO、IL－1和TNF－α的量以及其细胞杀伤活性。结果：肺癌患者的部分AM,即使未经刺激,亦分泌细胞因子并具有细胞杀伤活性;经γ－INF和LPS刺激后,两者均使AM分泌细胞因子增多和增强杀伤活性,且联合刺激较单独刺激对AM的作用影响大。结论：肺癌患者临近肿瘤组织的部分AM活性增强;γ－INF和LPS能刺激AM产生细胞因子并提高其细胞杀伤活性,且两者具有协同作用。     

肿瘤干细胞标志物与非小细胞肺癌患者生存的相关性分析

摘 要：［目的］ 探讨肿瘤干细胞（cancer stem cells,CSCs）标志物与非小细胞肺癌（non-small cell lung cancer,NSCLC）患者预后的关系。［方法］ 入组53例2017—2018年在上海市胸科医院进行手术并病理确诊的ⅢA期NSCLC患者,通过单因素和多因素回归方法分析临床因素与NSCLC患者总生存期（overall survival,OS）的相关性。通过免疫组化检测CSCs标志物（OCT4、SOX2、CD44、ALDH1A1）在NSCLC中的表达,并进行免疫组织化学评分。将CSCs标志物高H评分、年龄和微乳头成分6项指标进行累加评分,定义为肿瘤干性指数。利用Kaplan-Meier法分析肿瘤干性评分与NSCLC患者OS的关系。［结果］ 多因素回归分析结果显示年龄(HR=1.948,95%CI：1.092～2.474,P=0.031）、微乳头成分（HR=2.720,95%CI：1.267～5.842,P=0.011）和CEA水平（HR=1.008,95%CI：1.000~1.015,P=0.040）与OS 显著性相关。免疫组化结果显示,OCT4在肺腺癌和肺鳞癌中均有较高的表达。在肺腺癌中,OCT4高表达组预后较差（P=0.004）。SOX2、CD44、ALDH1A1对肺癌预后的影响不明显。肿瘤高干性评分与OS显著性相关(HR=2.212,95%CI：1.245~3.676,P=0.024)。［结论］在肺腺癌患者中,OCT4高表达和肿瘤高干性指数可能与预后差相关。     

肺癌患者外周血循环肿瘤细胞检测的研究进展

肺癌是我国发病率和死亡率均较高的恶性肿瘤,预后较差.患者5年生存率还有待进一步提高,患者致死的主要原因是肺癌的复发和转移.血行播散是肺癌转移的重要途径,肿瘤细胞可以通过血液循环发生远处转移.肿瘤细胞进入血液循环是肿瘤发生远处转移的关键步骤之一,因此对肺癌患者循环肿瘤细胞检测的研究已受到越来越多的重视.在此,我们就相关研究进展加以概述.     

Selection of Radioresistant Cells by Vitamin A Deficiency in a Small Cell Lung Cancer Cell Line

Radiation sensitivity of a human small cell lung cancer cell line, Lu-134-B cells, cultured in serum-supplemented medium and of cells transferred to and cultured in delipidized serum-supplemented (vitamin A-deficient) medium was studied. The cells cultured in serum-supplemented medium showed the phenotype of classic small cell lung cancer sensitive to radiation, while cells transferred to delipidized serum-supplemented medium showed partial squamous cell differentiation and became resistant to radiation. These results suggest that some small cell lung cancer cells in vitro change their morphology and radiosensitivity depending on the culture conditions. The change in radiosensitivity was reproducible, and was not reversible by culture of the radioresistant cells in delipidized serum-supplemented medium with addition of retinoic acid (vitamin A-sufficient medium) for two months, although squamous cells disappeared. Acquisition of radioresistancy was considered to occur as the result of clonal selective growth in delipidized medium of a minor cell population in the original cell culture, based on a study of chromosome number. It was also found that there was no association of myc -family oncogenes with the changes of radiosensitivity in this cell line.     

Ⅲ～Ⅳ期肺癌患者外周血CIK细胞水平及临床意义研究

目的 :探讨Ⅲ～Ⅳ期肺癌患者外周血CIK细胞水平变化及临床意义。 方法 :流式细胞术检测57例Ⅲ～Ⅳ期肺癌患者及27例健康体检者外周血CIK细胞水平,接受化疗的35例患者治疗后2周再次行外周血CIK细胞水平测定。 结果 :肺癌组外周血CIK细胞水平明显高于正常对照组外周血CIK细胞水平;肺癌组外周血CIK细胞水平与病理类型无关,随临床分期进展而升高;35例接受化疗患者化疗后外周血CIK细胞水平明显低于化疗前。 结论 :Ⅲ～Ⅳ期肺癌患者外周血CIK细胞水平显著升高,有较强抗肿瘤作用;与病理类型无关,受临床分期及治疗干预影响,可作为病情监测的一项指标。     

肺癌患者染色体稳定性及诱变剂敏感性的研究

目的 探讨细胞染色体不稳定性与肺癌的关系。方法 采用微量全血培养法对２４例经病理证实但未治疗的原发性肺癌患者、２０例非癌性患者和２０名健康志愿者外周血淋巴细胞（ＰＢＬ）进行自发和诱发的染色体畸变和微核检测。结果人肺癌患者ＰＢＬ自发及诱发染色体畸变率和微核率明显高于非癌性肺病患者和健康者;肺癌患者具有较高的染色体畸变率和微核率。肺癌患者染色体稳定性较差。结论 细胞染色体稳定性评价对肺癌的早期诊断、鉴     

K-ras 抗原致敏的DC诱导CTL 对胰腺癌体内杀伤活性的研究*

目的:研究K-ras多肽的致敏树突状细胞（DC）活化的特异性细胞毒性T 淋巴细胞（CTL）对胰腺癌的体内外杀伤作用。方法:联合应用粒细胞- 巨噬细胞集落刺激因子和白细胞介素-4 诱导培养外周血DC。表达K-ras突变体的胰腺癌细胞株全瘤、单纯K-ras突变体多肽和K-ras突变体表位肽阳离子纳米颗粒分别致敏DC。致敏DC刺激T 淋巴细胞得到肿瘤抗原特异的细胞毒性T 淋巴细胞（CTL）。 Patu 8988、SW1990细胞系制备荷瘤裸鼠模型评价CTL 体内抗肿瘤活性。结果:负载全瘤抗原的DC其诱导产生的CTL 对胰腺癌有较好的抑制,负载单纯K-ras（12-Val ）突变体多肽、K-ras（12-Val ）突变体表位肽阳离子纳米颗粒的DC其诱导产生的CTL 对表达K-ras（12-Val ）突变体阳性（Patu 8988）的胰腺癌有较特异的抑制作用,而对K-ras（12-Val ）突变体阴性（SW1990）的胰腺癌的抑制作用与对照组比较无显著性差异。结论:负载肿瘤抗原的DC诱导的CTL 可显著提高对荷瘤裸鼠的生存时间,抑制肿瘤的生长速度,并显示其可增加抗肿瘤特异性。      

The CTC-Chip: An Exciting New Tool to Detect Circulating Tumor Cells in Lung Cancer Patients

Circulating tumor cells (CTCs) are rare cells that originate from a malignancy and circulate freely in the peripheral blood. The ability to capture and study CTCs is an emerging field with implications for early detection, diagnosis, determining prognosis and monitoring of cancer, as well as for understanding the fundamental biology of the process of metastasis. Here, we review the development and initial clinical studies with a novel microfluidic platform for isolating these cells, the CTC-chip, and discuss its potential uses in the study of lung cancer.