负载肝癌抗原树突细胞瘤苗对癌细胞免疫抑制作用

目的探讨人自体肝癌细胞裂解物致敏的树突细胞(DC)瘤苗对自体肝癌细胞免疫功能的影响。方法从肝癌患者外周血单个核细胞中诱导DC,用相关细胞因子及自体肝癌细胞刺激活化,制备DC瘤苗;应用流式细胞仪检测T淋巴细胞增殖情况,观察刺激术前及术后自体的T淋巴细胞以及产生的特异性的细胞毒性T淋巴细胞(CTL)对自身肿瘤细胞的抑制效果。结果肝癌DC瘤苗刺激术后自体T淋巴细胞增殖能力及抑制肿瘤细胞作用显著强于术前,T淋巴细胞增殖指数术后(1.86±0.15)较术前(1.18±0.13)显著增高、CD8+T细胞增殖指数术后(1.55±0.35)较术前(0.95±0.12)增加显著,术后与术前比较差异均有统计学意义(均P0.05)。肝癌DC瘤苗活化的术后自体CTL对自体肝癌细胞有较强杀伤作用,杀伤率由术前的(32.35±2.26)%增加到术后的(69.80±3.45)%。结论肝癌DC瘤苗活化的术后自体CTL对自体肝癌细胞杀伤作用比术前作用显著,DC瘤苗能有效地诱导抗肿瘤免疫反应,有望在肝癌术后的治疗中发挥作用。     

肝癌细胞裂解物致敏的树突状细胞瘤苗预防肝癌术后转移复发

目的探讨人自体肝癌细胞裂解物致敏的树突状细胞(D C)瘤苗预防肝癌术后转移复发的价值。方法从肝癌患者外周血单核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF) 和重组人白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏D C制备瘤苗。将3 0例肝癌术后患者随机分为D C瘤苗治疗组1 5例,对照组1 5例。对两组病例治疗前后免疫功能、临床疗效、肿瘤转移复发率及生存率进行观察比较。结果DC瘤苗治疗后外周血CD 3 、CD4 /CD 8 及自然杀伤细胞比率较治疗前明显升高(P<0.0 5),且明显高于对照组治疗后的相应水平(P<0.05)。DC瘤苗治疗后血清IL-1 0水平明显下降(P<0.0 5)。随访18个月,DC瘤苗治疗组的转移复发率为13.3 3%,明显低于对照组53.33%(P <0.05);而治疗组的生存率为93.33%,明显高于对照组60.00%(P<0.05)。结论肝癌术后行自体肝癌细胞裂解物致敏的DC瘤苗治疗,可改善患者的免疫功能,降低肿瘤转移复发率,提高患者术后生存率,为肝癌的免疫治疗开辟一条新途径。     

热休克肝癌细胞来源囊泡诱导细胞毒性T细胞的杀伤作用

目的研究热休克肝癌细胞来源囊泡(exosomes)刺激人树突状细胞(DC)能否诱导出肝癌细胞特异性细胞毒性T淋巴细胞(CTL)。方法 43℃热休克(水浴)1h培养肝癌细胞提取胞外囊泡,诱导DC刺激细胞毒性T淋巴细胞,MTT法测定CTL在体外对肝癌细胞的杀伤作用。结果热休克前后肝癌细胞胞外囊泡和肿瘤细胞裂解物刺激的DC均能促进对肝癌细胞的杀伤活性,热休克肝癌细胞胞外囊泡实验组较胞外囊泡和肝癌细胞冻融裂解物对照组有显著性差异(P0.05和P0.01)。结论热休克能够增强肝癌细胞胞外囊泡诱导的特异性抗肝癌细胞免疫反应。     

肝癌干细胞抗原致敏的DC-CTL对肝癌的抑制作用

目的研究肝癌干细胞抗原致敏的树突细胞(DC)-细胞毒性T淋巴细胞(CTL)对裸鼠肝癌的抑瘤作用。方法肝细胞性肝癌患者手术后取肝癌组织,根据肝癌干细胞表面标志CD133,用磁珠分选法初步获得肝癌干细胞,反复冻融提取法获得肝癌干细胞抗原;采集同一患者外周血,加入细胞因子体外诱导、扩增抗原致敏的DC和细胞因子诱导的杀伤(CIK),从CIK中分选出CD3~+CD8~+T细胞;将肝癌干细胞抗原致敏的DC与CD3~+CD8~+T细胞共培养后,获得抗原致敏的DC-CTL细胞;制备裸鼠肝癌模型,动物实验观察抗原致敏的DC-CTL对裸鼠肝癌的抑瘤作用。结果肝癌干细胞抗原致敏的DC-CTL细胞和肝癌细胞抗原致敏的DC-CTL细胞对裸鼠肝癌均有极显著的抑制作用,两组间比较差异有统计学意义(P<0.05),肝癌干细胞抗原致敏组的抑瘤率明显增高。结论肝癌干细胞抗原致敏的DC-CTL对肝癌具有显著抑制作用。     

负载肝癌抗原树突细胞瘤苗抑制胶原合成免疫作用

目的探讨人自体肝癌细胞裂解物致敏的树突细胞(DC)瘤苗对自体免疫功能的影响。方法从肝癌患者外周血单个核细胞中诱导DC,用相关细胞因子及自体肝癌细胞刺激活化,制备DC瘤苗;观察刺激术前及术后CTL对自身胶原合成的抑制效果。结果肝癌DC瘤苗活化的术后自体CTL对自体血清中胶原蛋白有较强抑制作用,抑制率由术前的(835±116)mg/ml减少到术后的(412±218)mg/ml。结论肝癌DC瘤苗活化的术后自体CTL对自体胶原蛋白抑制作用比术前作用显著。     

体外负载冻融抗原的脐血树突状细胞诱导的抗肝癌效应

目的评价体外应用肝癌细胞冻融抗原负载的脐血树突状细胞（DC）所诱导的抗肝癌效应。方法采集健康足月剖宫产孕妇胎盘端脐血,分离脐血单个核细胞（CBMNC）及T淋巴细胞,用GM-CSF、IL-4及TNF-α联合诱导CBMNC分化为DC,观察形态学变化并以流式细胞术鉴定,选培养的第3天以肝癌细胞冻融抗原负载的CBMNC-DC,以负载抗原的DC刺激自体淋巴细胞活化为自体细胞毒性T淋巴细胞（CTL）,并用CTL对肝癌细胞进行杀伤,MTT法测定活化的自体淋巴细胞的相对数量和CTL对肝癌细胞的杀伤率。结果体外负载肿瘤冻融抗原的脐血DC可诱导显著的自体效应淋巴细胞增殖及抗肝癌效应。结论体外负载抗原的脐血DC可诱导显著的抗肝癌效应,是具有临床应用前景的肝癌疫苗。     

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     

树突状细胞致敏的肿瘤疫苗对胰腺癌细胞的杀伤效应

目的:研究胰腺癌细胞冻融物致敏树突状细胞(DC)诱导的细胞毒性T细胞(CTL)对原代培养的自体胰腺癌细胞的杀伤作用．方法:从6例手术切除的胰腺癌组织中分离胰腺癌细胞,反复冻融获得肿瘤抗原;以该肿瘤抗原致敏外周血DC,诱导T细胞转变为CTL;采用Cr51释放法观察CTL对原代培养的自身胰腺癌细胞的杀伤活性,分别以来源于胰腺癌细胞株Pancl的肿瘤抗原致敏DC和未致敏DC刺激的CTL作为抗原对照和阴性对照．结果:实验组CTL对自身细胞的杀伤活性为69．05%±15．79%→88．05%±15．34％,抗原对照组CTL的杀伤活性为43．08％±6．92％→67．30％±8．91％,两组CTL杀伤率均显著高于阴性对照组(P<0．01);而实验组与抗原对照组相比,前者的杀伤活性显著高于后者者(P<0．05)．结论:胰腺癌细胞冻融物致敏的DC疫苗可以诱导T细胞产生高效的针对自体癌细胞的细胞毒效应;新鲜肿瘤组织来源的胰腺癌细胞比传代的Pancl细胞具有更好的抗原性．     

原发性肝癌肿瘤浸润性淋巴细胞的细胞表型及杀伤活性

本文用间接免疫荧光染色流式细胞计数分析技术和LDH释放法检测了11例肝癌患者肿瘤浸润性淋巴细胞（TIL）的细胞表型及杀伤活性。结果培养初期TIL细胞特征是以CD3为主，CD4／CD8为0．83，NK细胞23．3±3．6％。经重组白细胞介素2（rIL—2）激活20天后，CD3减少，CD4／CD8为1.85，NK细胞数量增至39．2±9．8％，P＜0．01。激活后的TIL对自体肝癌细胞杀伤率比培养初期高3．74倍，亦比对同时培养的BEL—7204肝癌细胞杀伤率高1．75倍。对自体和异体肝癌细胞杀伤作用均显著高于LAK细胞，且具有靶细胞特异性。结果表明肝癌TIL对肝癌细胞杀伤作用，可能与NK细胞数量增多，导致肝癌细胞凋亡有关。     

前列腺癌树突细胞瘤苗的制备及其应用

目的 构建前列腺癌树突细胞（DC）瘤苗,并探讨其体内外抗前列腺癌的作用。方法 分离C57BL／6小鼠骨髓前体细胞制备DC,光镜下观察DC的形态学特征,混合淋巴细胞试验、辣根过氧化物酶（HRP）吞噬试验观察DC的生物学特性。经RM-1前列腺癌细胞裂解产物致敏构建DC瘤苗,将DC瘤苗皮下注射于18只前列腺癌模型小鼠。结果成熟DC突起多而长,胞内囊泡少,刺激T细胞增殖能力强;DC瘤苗分泌IL-12能力增强;小鼠应用DC瘤苗后,其脾脏T细胞对RM-1细胞具有特异性杀伤作用;荷瘤小鼠肿瘤生长缓慢,坏死明显,瘤体内及肿瘤周围有大量炎细胞浸润。结论小鼠骨髓单核细胞在粒细胞／巨细胞集落刺激因子（GM-CSF）和IL-4诱导下可转化为DC。RM-1前列腺癌细胞裂解物致敏构建的DC瘤苗能诱导T细胞对RM-1细胞产生特异性杀伤作用,且能分泌更多的IL-12,可用于前列腺癌的免疫治疗。     

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。     

Induction of specific cytolytic T lymphocytes using fusions of hepatocellular carcinoma (HCC) patient-derived dendritic cells and allogeneic HCC cell line

BACKGROUND/AIMS: To assess the ability of hepatocellular carcinoma (HCC) patient-derived dendritic cells (DCs) fused with allogeneic HCC cell line to activate autologous lymphocytes to generate specific cytotoxic T lymphocytes (CTL) in vitro. METHODOLOGY: DCs were obtained by culturing adherent peripheral blood mononuclear cells (PBMC) from HCC patients in the presence of 100 microg/L recombinant human granulocyte/ macrophage- colony stimulating factor (rhGM-CSF) and 20 microg/L interleukin-4 (rhIL-4) for 1 week in vitro. DCs were fused with allogeneic HCC cell line HepG2 cells using polythyleneglycol (PEG), and the fusion cells were designated as DCs/HepG2. By labeling DCs and HepG2 with green and red fluoresceins, respectively, the cellular fusion was examined under fluorescence microscope. The ability of DCs/HepG2 to stimulate proliferation and differentiation of autologous lymphocytes was assessed by MTT method, and the specific killing efficacy of DCs/HepG2-induced CTL against HepG2 was evaluated. RESULTS: HCC patient-derived DCs expressed a certain level of CD1a, HLA-DR, CD54, CD80 and CD86. Fluorescence microscopic examination demonstrated that co-incubation of DCs and HepG2 in the presence of PEG lead to generation of DCs/HepG2. In the mixed lymphocyte reaction assay, DCs/HepG2 had a significantly greater ability to activate proliferation of autologous lymphocytes, as compared with DCs alone, DCs plus HepG2, HepG2 alone and medium control (P<0.05). The DCs/HepG2-activated CTL showed a potent specific killing efficacy against HepG2 cells. CONCLUSIONS: Fusions of HCC patient-derived DCs and allogeneic HCC cell line could efficiently stimulate autologous lymphocytes to generate tumor-specific CTL in vitro. It might represent a promising approach of immunotherapy for HCC.     

A study of the proteins responsible for stimulating B-CLL-specific T-cell responses by autologous dendritic cells pulsed with tumour cell lysate

We have previously shown that autologous dendritic cells (DCs) pulsed with tumour cell lysate and cultured with autologous T cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) stimulate significant increases in proliferation, IFN-gamma secretion and specific HLA class II-restricted cytotoxicity to B-CLL targets. In this study, normal and tumour cell lysates were analysed by reducing SDS-PAGE, and protein bands of interest eluted from the polyacrylamide gel by electroelution. The eluted protein fractions and whole-cell lysate were then pulsed onto autologous DCs and cocultured with autologous T cells. Finally, the stimulatory fractions were sequenced. B-CLL cell lysates revealed protein bands at 65, 42, 35 and 25 kDa, while normal B-cell lysates only showed a single protein band at 65 kDa. Both the 65 kDa band and 42 kDa bands were capable of stimulating comparable amounts of IFN-gamma secretion and specific cytotoxicity as whole lysate, while the other protein bands were not. Sequencing of the 65 kDa fraction showed a dominant peptide that matched human serum albumin, while sequencing the 42 kDa fraction showed three dominant peptides which matched human actin and another unidentified protein. The significance of these findings remains unclear.     

慢性乙型肝炎患者外周血树突状细胞诱导特异性T淋巴细胞应答

目的 探讨慢性乙型肝炎患者外周血树突状细胞(Dc)是否诱导特异性T细胞应答。方法(1)将研究对象分为慢性乙型肝炎患者组、急性乙型肝炎痊愈组、健康志愿者组,分离各组研究对象的外周血单个核细胞(PBMC),细胞内细胞因子染色方法检测其对细胞毒性T淋巴细胞(CTL)特异表位多肽乙型肝炎病毒核心抗原(HBcAg)18-27的记忆性免疫应答;(2)培养慢性乙型肝炎患者DC,将负载有乙型肝炎抗原表位多肽的DC诱导特异的T细胞应答。采用细胞内细胞因子染色方法检测诱导的T细胞分泌的细胞因子,乳酸脱氢酶释放法测定诱导的T细胞杀伤活性。结果(1)急性乙型肝炎患者PBMC对HBcAg 18-27 CTL特异表位多肽存在记忆的免疫应答,其分泌干扰素-γ的CD8+T细胞占CD8+T细胞总数的(4.3±2.5)％,分泌白细胞介素-2的占总细胞数的(4.8±2.2)％,分泌肿瘤坏死因子-α占总细胞数的(4.6±2.3)％。而慢性乙型肝炎患者和健康志愿者对其记忆应答很低,与急性乙型肝炎患者比较差异有显著性,t值为2.508-3.305,P<0.05。(2)用多肽共孵育过的慢性乙型肝炎患者DC多次诱导的T细胞慢性乙型肝炎患者组,加肽孵育的靶细胞比例为30:1、10:1、3:1时,其杀伤率分别为(57.0±20.3)％、(49.5±20.2)％、(21.8±12.9)％,均高于对照组,表明慢性乙型肝炎患者DC可以诱导特异的T     

Down-regulation of β-centractin might be involved in dendritic cells dysfunction and subsequent hepatocellular carcinoma immune escape: a proteomic study

Aims Proteomic study was used to clarify the mechanism of hepatocellular carcinoma (HCC) immune escape concerning Dendritic cells (DCs’) dysfunction and their association with HCC invasion. Methods Human peripheral blood mononuclear cells (PBMCs) derived DCs from healthy donors were pulsed with soluble cell lysates prepared from different metastatic potential human HCC cell lines. The total protein of these DCs was analyzed by two-dimensional electrophoresis and Electro-Spray Mass Spectrometry. The allostimulatoy capacity and phenotype of these DCs were also evaluated. The clinical significance of β-centractin, one of the largest quantitative changed spot, down-regulation in DCs was further evaluated in autologous PBMCs derived DCs pulsed with auto-tumor lysates in 26 HCC patients. Results The expression of β-centractin was found to be considerably lower either in DCs pulsed with HCCLM6 (high metastatic potential HCC cell line) lysates, accompanied by down-regulation of CD86 molecule and impaired allostimulatory capacity, than those of DCs pulsed with lysates from HCC cell lines with low or without metastatic potential or in DCs pulsed with lysates from HCC with invasiveness than those without invasiveness. Conclusions The down-regulation of β-centractin in DCs pulsed with high metastatic potential HCC lysates might associate with DCs dysfunction and HCC invasiveness. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Yong-Qiang Weng and Shuang-Jian Qiu contributed equally to the work.     

Dendritic cells fused with allogeneic hepatocellular carcinoma cell line compared with fused autologous tumor cells as hepatocellular carcinoma vaccines

Purpose: To investigate the specific antitumor responses against autologous hepatocellular carcinoma (HCC) cells of dendritic cells (DCs) fused with allogeneic HCC cell line, and evaluated the feasibility of BEL7402 as an alternative strategy to deliver shared HCC antigens to DCs. Methods: Previous studies demonstrated fusions of patient‐derived DCs and autologous tumor cells could induce T‐cell responses against autologous tumors. These fusion cells require patient‐derived tumor cells, which are not, however, always available. Here, we report the fusing of autologous DCs with allogeneic HCC cell line to induced cytotoxic T‐lymphocyte (CTL) response against autologous HCC cells compare with autologous tumor cells. Results: These DC/ BEL7402 fusion cells co‐expressed tumor‐associated antigens and DC‐derived costimulatory and major histocompatibility complex molecules. Both CD4+ and CD8 T+ cells were activated by the fusion cells as demonstrated by the proliferation of T‐cells, the production of cytokines and the simultaneous induction of specific CTL responses. Significantly, CTL induced by dendritic cell/allogeneic BEL7402 fusion cells were able to kill autologous HCC cells by human leukocyte antigen‐A2 restricted mechanisms. The results did not show significant difference between DC fusion with autologous hepatocellular carcinoma cells and DC fusion with allogeneic hepatocellular carcinoma cell line. Conclusions: The fusion of allogeneic HCC cell line and autologous DCs may have applications in antitumor immunotherapy through cross‐priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

树突状细胞瘤苗对老年非小细胞肺癌自体癌细胞的杀伤作用

目的研究老年非小细胞肺癌（NSCLC）患者的树突状细胞（dendritic cell,DC）瘤苗对细胞因子诱导的杀伤细胞（cytokines-induced killers,CIK）的促生长作用,及CIK在体外对患者自身肿瘤细胞的杀伤作用。方法提取老年NSCLC患者外周血单个核细胞（peripheral blood mononuclear cells,PBMC）,分别培养成DC与CIK;将患者自身的肿瘤组织分离,培养出肿瘤细胞,制备肿瘤细胞冻融物作为抗原,用于负载DC,制备成Ag-DC,同时以单纯DC作为对照,各组DC经OK-432激活后,流式细胞仪检测DC表面分子的表达;将DC与CIK共培养,制备成DC-CIK、Ag-DC-CIK,同时以单纯CIK作为对照,四氮唑盐（MTT）法测定各组CIK对自身肿瘤细胞的杀伤力。结果负载肿瘤细胞冻融抗原的Ag-DC其表面CD1a、CD80、CD86、CD83、人类主要组织相容性抗原（HLA-DR）的表达上调,明显高于未负载肿瘤抗原的DC（P〈0.05）。各组DC与CIK共培养后,Ag-DC-CIK的增殖速度明显加快,其中CD3＋CD56＋细胞的含量增加,对自身肿瘤细胞的杀伤力亦明显加强,其作用均强于其他各组DC-CIK及CIK（P〈0.05）。结论老年NSCLC患者PBMC来源的DC在负载自身肿瘤细胞冻融抗原后,可分化为成熟DC,该DC可促进CIK的增殖,提高CIK对自身肿瘤细胞的杀伤力。     

Immunotherapy by autologous dendritic cell vaccine in patients with advanced HCC

Background

Dendritic cells (DCs) could be used as potential cellular adjuvant for the production of specific tumor vaccines.

Objectives

Our study was aimed to evaluate the safety and efficacy of autologous pulsed DC vaccine in advanced hepatocellular carcinoma (HCC) patients in comparison with supportive treatment.

Methods

Thirty patients with advanced HCC not suitable for radical or loco-regional therapies were enrolled. Patients were divided into 2 groups, group I consisted of 15 patients received I.D vaccination with mature autologous DCs pulsed ex vivo with a liver tumor cell line lysate. Group II (control group, no. 15) received supportive treatment. One hundred and 4 ml of venous blood were obtained from each patient to generate DCs. DCs were identified by CD80, CD83, CD86 and HLA-DR expressions using flow cytometry. Follow up at 3, and 6 months post injection by clinical, radiological and laboratory assessment was done.

Results

Improvement in overall survival was observed. Partial radiological response was obtained in 2 patients (13.3 %), stable course in 9 patients (60 %) and 4 patients (26.7 %) showed progressive disease (died at 4 months post-injection). Both CD8⁺ T cells and serum interferon gamma were elevated after DCs injection.

Conclusion

Autologous DC vaccination in advanced HCC patients is safe and well tolerate.     

Generation of Aspergillus-specific T lymphocytes with cytotoxic activity

Dendritic cells (DC) are highly specialized antigen presenting cells with the unique capacity to initiate and direct immune responses. The superior ability of DC to present antigens to T cells has led to the development of DC-based strategies for the purpose of enhancing the immune response against tumors and infectious agents. In this study Aspergillus (Asp)-pulsed DC were used to generate Asp-specific cytotoxic T-lymphocytes (CTL). Two different Asp-antigen preparations were used here. Asp-specific CTL were generated by four stimulations with autologous, mature, monocyte (Mo)-derived DC that are pulsed with either Aspergillus crude extract (CE) or culture filtrate (CF) antigens. The cytolytic activity of the generated CTL was assessed one week after the 4th stimulation by chromium release assay. No significant difference (p > 0.05) was found between the proliferative responses induced by either CF or CE Asp-pulsed DC. Both types of Asp-pulsed mature DC were capable of priming Asp-specific CTL responses. Analysis of the Asp-CTL effectors revealed that they are mixed of CD3+/CD4+ and CD3+/CD8+ populations and that they secrete IFN-gamma in response to Asp-pulsed mature DC and specifically kill autologous DC pulsed with the same Aspantigen. The killing was higher in bulk-cultures generated using Asp-CF pulsed DC. The percentage of CD3+/CD8+ cytotoxic T cells was significantly increased (p < 0.001) in Asp-CF bulk-culture when compared with Asp-CE bulk-culture (31.55 +/- 1.96% versus 9.70 +/- 1.84%, respectively). In conclusion, Asp-specific T cell lines with cytotoxic activity can be generated from healthy donors. These cells may be used as prophylaxis in high-risk immunocompromised patients.