肿瘤全抗原致敏的恶性胸水中肿瘤浸润免疫细胞的体外抗癌作用

目的 观察恶性胸水中肿瘤浸润免疫细胞中浸润树突细胞(TIDC)和肿瘤浸润T淋巴细胞(TIL)经过IL-2活化负载CALU-6腺癌细胞肿瘤全抗原后的体外抗癌作用.方法 分离恶性胸水单个核细胞(PEMCs),采用两步贴壁法获得非贴壁细胞,TIDC及TIL是其主要功能细胞成分.SP法检测TIL亚群的数量及免疫功能,SP法S-100蛋白染色检测TIDC.IL-2活化肿瘤浸润免疫细胞并负载CALU-6肿瘤细胞全抗原,MTT法分别检测活化的免疫细胞对CALU-6和GLC-82腺癌细胞的体外杀伤作用.结果 IL-2活化培养7 d后TIDC和TIL数量明显增加.恶性胸水肿瘤浸润免疫细胞(主要包括TIL和TIDC)经过CALU-6腺癌细胞肿瘤全抗原负载后,相同浓度对于CALU-6腺癌细胞杀伤明显,而对于GLC-82腺癌细胞无明显杀伤作用,两者比较差异具有显著意义(P<0.01).结论 IL-2同时活化肿瘤微环境中TIL及TIDC,使其由一种无功能或功能低下状态恢复免疫监视功能,有效地负载肿瘤抗原,协同TIL等其他免疫细胞可以有效杀伤肿瘤细胞.活化恶性胸水免疫细胞自体回输治疗恶性胸水具有临床应用前景.     

胃癌细胞粒酶B表达与树突状细胞和活化T淋巴细胞浸润程度的关系及临床意义

粒酶B(granzyme B，GrB)是活化的细胞毒T淋巴细胞(CTL)和自然杀伤(NK)细胞发挥细胞毒作用的主要效应分子，也是其特异标志物，本研究采用免疫组化法，观察胃良／恶性上皮细胞是否表达GrB，同时检测组织中浸润S-100^ 树突状细胞(DC)和GrB^ 肿瘤浸润T淋巴细胞(TIL)，探讨GrB表达与胃癌生物学行为及局部免疫状态的相互关系。     

肿瘤浸润淋巴细胞在结直肠癌中的临床病理分析

目的:探讨结直肠癌中肿瘤浸润淋巴细胞(TIL)的临床病理和免疫组织化学特征, 及大量TIL与各病理因素的关系.方法:利用光镜和免疫组织化学法对370例结直肠癌中的肿瘤浸润淋巴细胞进行组织病理学观察, 并分析对比其与各临床病理因素的相关性.结果:81.9%的结直肠癌中可见淋巴细胞散在分布, 数量少, 18.1%却可见大量的淋巴细胞,并有一定的临床病理特征. 大量的TIL部分侵入肿瘤实质而部分位于肿瘤组织周围. 免疫组织化学染色显示, 肿瘤浸润淋巴细胞主要是由CD3+ T细胞、CD20+、CD79α+B细胞、浆细胞和CD56+NK细胞构成, 其中细胞毒性TIA-1均有较高的表达, 并伴有大量淋巴细胞浸润与肿瘤组织的分化程度、浸润深度及淋巴结转移有关(χ2 = 4.954, 11.240及12.768; P = 0.026,0.001及0.000).结论:结直肠癌组织中TIL的病理形态差异较大并与临床病理因素相关. 在结直肠癌中伴有大量肿瘤浸润淋巴细胞存在时有积极的意义.     

肿瘤浸润淋巴细胞治疗胆道癌研究进展

肿瘤浸润淋巴细胞(TIL)是存在于肿瘤实质和间质内的以T淋巴细胞为主的一类异质性淋巴细胞群体。将其从肿瘤组织消化和分离出来，经体外培养活化和扩增后，回输到患者体内可以起到杀伤肿瘤细胞的作用。TIL具有T淋巴细胞抗原受体的高度多样性、优异的浸润肿瘤部位能力以及低毒性等优势，被认为有希望用于治疗恶性实体肿瘤。目前，TIL疗法已在多种实体瘤中作为二线治疗进行临床试验并取得初步疗效。虽然目前尚无TIL应用于胆道癌单一病种的临床队列报道，近期的多癌种临床报道提供了少数接受TIL治疗的胆道癌患者的疗效信息，初步证实了安全性和有效性。但由于胆道癌一般被认为是大多数效应T淋巴细胞被隔离在肿瘤边缘的免疫排斥性肿瘤，TIL在胆道癌中的抗肿瘤作用仍难以预测。尝试与不同抗肿瘤方法的联合治疗以及开发新技术修饰细胞以增强TIL抗肿瘤能力是未来可能突破的发展方向。     

前列腺素E2与腺苷协同抑制肿瘤浸润淋巴细胞活性的作用

肿瘤免疫过继治疗是近年来肿瘤免疫治疗的研究热点之一,主要是从肿瘤患者中分离免疫活性细胞,在体外进行扩增和功能鉴定,然后回输给患者.肿瘤浸润性淋巴细胞(TIL)是从肿瘤组织中分离出来的淋巴细胞,经离体培养,由IL-2诱导而成,具有特异性杀伤肿瘤的活性[1,2],其主要来源为手术切除所获得的实体肿瘤组织和浸润淋巴结等.目前是国际上研究和应用的主要免疫疗法.前列腺素E2(PGE2)在淋巴细胞发育与增殖等方面具有重要作用,腺苷则是公认的具有抑制细胞免疫功能的分子,两者都参与了机体的免疫调节,但对于TIL影响的报道甚少.我们通过PGE2与腺苷的协同作用,观察是否能够影响IFN-γ和TGF-β的分泌,抑制TIL的杀伤功能.     

恶性胸水中免疫学指标测定的临床意义

恶性胸水中免疫细胞所分泌的多种细胞因子共同组成了影响肿瘤免疫的微环境.分析细胞因子的变化,可能为恶性胸水的临床诊断及治疗提供线索.本综述主要探讨IL-2、IL-4、IL-6、IL-8、IL-10、T细胞亚群及癌胚抗原七种免疫学指标的临床意义.     

树突状细胞激活的肿瘤浸润性淋巴细胞抗胃癌活性的研究

目的　树突状细胞 (DC)是目前已知的功能最强的抗原提呈细胞 (APC) ,可以向包括肿瘤浸润性淋巴细胞 (TIL)在内的T淋巴细胞提呈抗原 ,并诱发细胞毒T淋巴细胞 (CTL)反应。该文探讨树突状细胞激活的肿瘤浸润性淋巴细胞体外对胃癌细胞 (SGC 790 1 )的杀伤活性。方法　从胃癌患者外周血获取DC ,应用粒 /巨噬细胞集落刺激因子 (GM CSF)、白介素 4(IL 4)和肿瘤抗原激活DC ,然后用DC激活TIL ,观察TIL在体外对自体胃癌细胞和人胃癌细胞株细胞的杀伤活性。结果　DC激活的TIL具有很高的对自体胃癌细胞杀伤活性 ,杀伤率为 (89.39± 3 .0 5) % ,明显高于未经DC激活的TIL、CD激活的T淋巴细胞和未经DC激活的T淋巴细胞对自体胃癌细胞的杀伤率 [杀伤率分别为 (54 .37±1 .50 ) % ,(53 .92± 1 .46) %和 (3 .55± 0 .2 5) % ]。而它们对SGC 790 1细胞的杀伤活性则相对较低。结论　胃癌患者外周血DC能诱导TIL产生高效而特异的抗胃癌免疫     

树突细胞负载骨髓瘤抗原介导的免疫反应

目的 研究负载肿瘤抗原的树突细胞能否诱导特异性细胞霉T淋巴细胞反应。方法 用多发性骨髓瘤患者骨髓CD34^ 细胞诱生树突细胞，并将骨髓瘤冻融物冲击致敏树突细胞。采用MTT法检测骨髓瘤抗原致敏及未致敏树突细胞诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞株)的杀伤率。结果 骨髓瘤冻融物致敏树突细胞诱导的自身T细胞对患者骨髓瘤细胞的杀伤远大于对K562细胞的杀伤。结论 患者骨髓瘤冻融物致敏的树突细胞能有效诱导自身T细胞发生特异性抗肿瘤免疫。     

参贞合剂对S180荷瘤小鼠T细胞免疫功能的影响

目的 研究参贞(人参、黄芪、女贞子)合剂对S180荷瘤小鼠T淋巴细胞免疫功能的调节作用.方法 小鼠胃饲参贞合剂,流式细胞术检测荷瘤小鼠T细胞亚群变化,MTT法检测荷瘤小鼠T淋巴细胞增殖能力,ELISA法检测IL-2诱生量.结果 胃饲参贞合剂使荷瘤小鼠的T淋巴细胞亚群、增殖能力和IL-2产生量均有明显提高(P＜0.05).结论 参贞合剂对荷瘤小鼠免疫功能有正向调节作用,将其研制成提高肿瘤患者免疫功能的药物具有良好的前景.     

自体TIL联合IL-2治疗恶性胸水疗效观察

将恶性肿瘤并发胸腔积液（下称胸液）患者分两组治疗，治疗组用自体肿瘤浸润淋巴细胞（TIL）联合白介素-2（IL-2）疗法；对照组分别行胸穿抽液及胸腔闭式引流，并胸腔内注射顺铂。1个月后评价疗效和不良反应。结果治疗组总有效率85．7％，对照组75．0％，两组比较无显著统计学差异；但治疗组不良反应明显少于、轻于对照组（P〈0．05）。提示自体TIL联合IL-2治疗恶性胸液具有良好的临床应用价值。     

Morphological observation of tumor infiltrating immunocytes in human rectal cancer

AIM: To investigate the morphological characterization of tumor infiltrating dendritic cells (TIDCs) and tumor infiltrating lymphocytes (TILs) in human rectal cancer. METHODS: Light and electron microscopy as well as im-munohistochemistry were used to observe the distributive and morphological changes of TIDCs and TILs. RESULTS: TIDCs were mainly located in tumor-surrounding tissue. The number of TIDCs in the earlier stage was higher than that in the later stage (P<0.01). TILs were mainly seen in adjacent tissue of cancers and tumor-surrounding tissue. There were more TILs in the earlier stage than that in the later stage (P<0.01). Under electron microscope, TIDCs were irregular in shape and exhibited many dendritic protrusions. It isn't obvious that cancer cells perforated the basement membrane and TILs were arranged along the basement membrane in the earlier stage. In the later stage, it is explicit that cancer cells perforated the basement membrane and surrounded by TILs. There were contacts among TIDCs, TILs and tumor cell. One TIDCs contacted one or several TILs which clustered around TIDCs. Glycogen granules were seen between TIDCs and TILs. CONCLUSION: The number of TIDCs and TILs is related with tumor progression There exist close relationships among TIDCs, TILs and tumor cell.     

Heparanase expression, degradation of basement membrane and low degree of infiltration by immunocytes correlate with invasion and progression of human gastric cancer

AIM： To disclose the mechanisms that accelerate or limit tumor invasion and metastasis in gastric cancer patients. METHODS： The heparanase expression, continuity of basement, degree of infiltration by dendritic cells and lymphocytes in gastric cancer tissues from 33 the early and late stage patients were examined by immunohistochemistry, in situ hybridization and transmission electron microscopy. RESULTS： Heparanase mRNA expression in the late stage patients with gastric cancer was stronger than that in the early stage gastric cancer patients. In the early stage gastric cancer tissues, basement membrane （BM） appeared intact, whereas in the late stage, discontinuous BM was often present. The density of Sl00 protein positive tumor infiltrating dendritic cells （TIDC） in the early stage gastric cancer tissues was higher than that in the late stage. The infiltrating degree of tumor infiltrating lymphocytes （TIL） in the early stage patients whose tumor tissues contained a high density of TIDC was significantly higher than that in the late stage gastric cancer tissues patients with a low density of TIDC. There were few cancer cells penetrated through the continuous BM of cancer nests in the early stage gastric cancers, but many cancer cells were found outside of the defective BM of cancer nests in the late stage. CONCLUSION： Our results suggest that strongheparanase expression is related with the degradation of BM which allows or accelerates tumor invasion and metastasis. However, high density of TIDC and degree of infiltration by TIL are associated with tumor progression in human gastric cancers.     

树突状细胞与肺癌的研究进展

树突状细胞是体内最为强大的专职抗原递呈细胞,可激活机体初始免疫应答。已有的研究发现,侵入到肺癌组织的突状细胞存在成熟障碍,未成熟树的突状细胞不能有效地向肿瘤特异性T细胞递呈肿瘤抗原,同时通过诱导调节性T细胞的增殖参与肺癌的免疫逃逸。肺癌组织中的树突状细胞与患者临床预后密切相关,基于树突状细胞的肿瘤疫苗已作为肺癌生物治疗的手段之一得以深入研究并取得了令人满意的前期效果。     

Dendritic cell-based tumor vaccine for cervical cancer I: in vitro stimulation with recombinant protein-pulsed dendritic cells induces specific T cells to HPV16 E7 or HPV18 E7

Purpose Human papillomavirus (HPV) type 16 and 18 are the most prevalent genotypes in cervical cancers. The viral oncoproteins E6 and E7 are considered to be tumor-specific targets for immunotherapy. HPV E7 antigen-loaded dendritic cells (DC) were evaluated as cellular tumor vaccine.Methods Autologous monocyte-derived DCs loaded with recombinant HPV16 or HPV18 E7 oncoprotein were used to induce in vitro a specific T cell response. Specificities of activated T cells were determined.Results E7-specific T cells could be identified in 18/20 T cell lines from healthy blood donors. CD4⁺ T cell responses (13/16) were found by proliferation assay. CD8⁺ CTLs (12/18) were detectable by interferon-gamma (IFN-) ELISpot analysis. Seven donors reacted in both assays and only 2/20 T cell lines did not react in any assay. Thus, specific T cells could be activated in >80% of healthy individuals. T cell lines from suitable donors were specific for HLA-A*0201-restricted epitopes. Furthermore, HPV E7 antigen-loaded DC stimulated specific responses in freshly isolated tumor infiltrating lymphocyte (TIL) populations of cervical cancer patients.Conclusion Autologous dendritic cells loaded with HPV E7 protein can induce T cell responses in healthy individuals by in vitro stimulation and evoke responses in TIL from cervical cancer biopsies. Since there are no limitations with respect to specific HLA-haplotypes, these findings may be a basis for the development of a therapeutic protein-based DC tumor vaccine against cervical cancer for HPV16- and HPV18-positive patients.Abbreviations B-LCL B lymphoblastoid cell line - CTL Cytotoxic T lymphocyte - DC Dendritic cell - IFN- Interferon-gamma - IL Interleukin - GM-CSF Granulocyte/macrophage-colony stimulating factor - HPV Human Papillomavirus - HSA human serum albumin - PBL Peripheral blood lymphocytes - PBMC Peripheral blood mononuclear cells - SD standart deviation - TIL Tumor infiltrating lymphocytes - TNF- Tumor necrosis factor-alpha     

Differentiation of antitumor-specific cytotoxic T lymphocytes from autologous tumor infiltrating lymphocytes in non-Hodgkin's lymphomas.

The present study describes a new culture protocol allowing the activation and proliferation of autologous tumor infiltrating T lymphocytes (TIL), and the generation of antitumor specific CTL in non-Hodgkin's lymphoma (NHL). Cells from eight patients with indolent NHL were used. We performed 3-week co-cultures of TIL with irradiated autologous malignant B cells in the presence of low doses of IL-1beta, IL-2 and IL-12. The proliferation, phenotype and cytotoxicity, and antitumor specificity of T cells recovered were studied. T-cell clonality was analyzed using TCRgamma gene rearrangement amplification by a multiplex PCR. Under these culture conditions, TIL proliferated, and the CD8+ T lymphocytes that were in a minority at the beginning of the culture increased dramatically in 6 out of 8 cases. In two cases, CD4+ T lymphocytes expanded. We showed that an oligoclonal selection of reactive T cells occurred in culture. Specific cytotoxicity developed against autologous malignant B cells in the 6 cases where there was an expansion of CD8+ T lymphocytes. Inhibition experiments performed with mAb directed against HLA class I and II molecules, CD4, CD8 and TCRgammadelta showed that the cytotoxic effector cells were CD8+ T lymphocytes probably expressing TCRalphabeta+. Cytokine secretion was analyzed in culture medium, and we detected significant levels of IFN-gamma, TNF-alpha, and IL-10 and no IL-4 (except in one case). Our results demonstrate that memory T cells from lymphoma patients can be amplified and differentiated into antitumor cytotoxic cells using a combination of the cytokines IL-1beta, IL-2, and IL-12 in association with non modified tumor cells.     

Elevated soluble interleukin-2 receptor and interleukin-2 receptor positive cells in carcinomatous pleural effusions

Soluble interleukin-2 receptor (IL-2R) level and IL-2R positive (IL-2R+) cells were studied in twenty patients with carcinomatous pleural effusions. The mean value of soluble IL-2R level in carcinomatous pleural effusions was 2930 +/- 1722 U/ml and that in sera was 965 +/- 610 U/ml. Soluble IL-2R level in carcinomatous pleural effusions was found to be significantly higher (p less than 0.001) than that in sera of patients with carcinomatous pleural effusions and that in transudates. Serum soluble IL-2R level in patients with carcinomatous pleural effusions was found, to be significantly higher (p less than 0.001) than that in normal controls (264 +/- 70 U/ml). We also studied IL-2R+ cells in pleural fluids and peripheral blood of patients with carcinomatous pleural effusions. The mean percentage of IL-2R+ cells in carcinomatous pleural fluid lymphocytes was 22.8 +/- 17.8%, as compared with 3.0 +/- 2.2% in peripheral blood lymphocytes of normal controls (p less than 0.001). No significant differences were observed among the cell types of lung cancer examined (adenocarcinoma, squamous, small cell and large cell carcinoma) and no correlation among levels of soluble IL-2R and IL-2R+ cell in either pleural fluid or blood. Our results suggest that in patients with carcinomatous pleural effusions, T cell-mediated active immune mechanisms (IL-2/IL-2R system) against cancer cells are more active in pleural fluid than in peripheral blood.     

Lymphokine activated killer cells

Summary Various subpopulations of human leukocytes may be induced by lymphokines to exert cytotoxic activity. In man major histocompatibility complex non-restricted tumor cell lysis by interleukin-2 (IL-2) induced peripheral blood lymphocytes is attributed mainly to natural killer cells. These T cell receptor negative large granular lymphocytes are called lymphokine activated killer (LAK) cells. In order to explore the potential of LAK cells in tumor therapy, several clinical studies have been conducted, using IL-2 alone or in combination with ex vivo IL-2-activated peripheral blood lymphocytes. Objective responses have reproducibly been achieved only in renal cell carcinoma and malignant melanoma and were associated with considerable toxicity. In view of restricted efficacy and increasing doubts as to whether LAK cells indeed account for the in vivo observed responses, more recent strategies focus on tumor antigen specific cytotoxic T cells or tumor infiltrating lymphocytes. Successful translation of this approach into clinical practice, however, may be dependend on some basic problems of tumor immunology to be solved which were thought to be by-passed by the LAK cell approach.     

Clinical significance of programmed death 1/programmed death ligand 1 pathway in gastric neuroendocrine carcinomas

BACKGROUND Recently, more and more studies have demonstrated the pivotal role of programmed death 1/programmed death ligand 1(PD-1/PD-L1) pathway in the immune evasion of tumors from the host immune system. However, the role of PD-1/PD-L1 pathway in gastric neuroendocrine carcinomas(G-NECs) remains unknown.AIM To investigate the expression of PD-1/PD-L1 and role of PD-1/PD-L1 pathway in G-NECs, which occur rarely but are highly malignant and clinically defiant.METHODS We investigated the expression of PD-L1 on tumor cells and PD-1^+, CD8^+, and FOXP3^+ T cell infiltration by immunohistochemistry in 43 resected G-NEC tissue specimens. The copy number alterations of PD-L1 were assessed by qRT-PCR.RESULTS Most of the G-NECs tumor cells exhibited a near-uniform expression pattern of PD-L1, while some showed a tumor-stromal interface enhanced pattern. Of the 43G-NECs, 21(48.8%) were classified as a high PD-L1 expression group, and the high expression of PD-L1 was associated with poor overall survival(OS). The high expression of PD-L1 was correlated with abundant PD-1^+ tumor infiltrating lymphocytes(TILs) instead of CD8^+ TILs and FOXP3^+ regulatory T cells(Tregs).Our analysis also suggested that the infiltration of CD8^+ TILs tended to be a favorable factor for OS, although the difference did not reach the statistical significance(P = 0.065). Meanwhile, PD-L1 was significantly overexpressed in cases with copy number gain as compared with those without.CONCLUSION Our data demonstrated for the first time that high expression of PD-L1 in GNECs is associated with a poor prognosis, while the high expression may be due to the copy number variation of PD-L1 gene or stimulation of TILs. These results provide a basis for the immunotherapy targeting PD-1/PD-L1 pathway in GNECs.     

Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo

This article describes a DNA-based vaccination strategy evaluated ex vivo with human cells. The vaccine was prepared by transferring tumor-derived genomic DNA to PCI-13 cells, a highly immunogenic tumor cell line ("recipient cell"), which had been genetically modified to secrete IL-2 (PCI-13/IL-2). PCI-13 cells expressed class I MHC determinants (HLA-A2) shared with the tumor from which the DNA was obtained as well as allogeneic determinants. DNA from a gp100(+) melanoma cell line was transduced into gp100(-) PCI-13/IL-2 cells (PCI-13/IL-2/DNA). A T cell line specific for the gp100 epitope responded to PCI-13/IL-2/DNA cells by IFN-gamma-secretion measured in enzyme-linked immunospot assays. The T cell line also recognized the gp100 epitope presented by dendritic cells that ingested PCI-13/IL-2/DNA cells, which had been induced by UVB irradiation to undergo apoptosis. After up-take and processing of apoptotic PCI-13/IL-2/DNA cells, the dendritic cells primed normal peripheral blood lymphocytes to generate effector T cells specific for the tumor donating the DNA. The results indicate that tumor epitopes encoded in such DNA are expressed in recipient cells and can induce tumor-specific T cells. The findings support translation of this vaccination strategy to a phase I trial in patients with cancer.