树突状细胞疫苗联合胸腺肽α1对人源化免疫重建裸鼠结肠癌生长的抑制效应

目的观察胸腺肽αl（Ted）联合结肠癌细胞裂解物致敏树突状细胞（LyDCs）对人源化免疫重建裸鼠结肠癌的免疫治疗效应。方法常规DCs负载结肠癌细胞裂解物制备LyDCs疫苗，流式细胞仪（FCM）检测Tαl体外刺激前、后的LyDCs表型。HT-29结肠癌裸鼠模型成瘤后，经尾静脉注射人外周血T淋巴细胞6×10^6个／只，2d后，FCM检测裸鼠外周血人源性CD4^＋、CD8^＋T细胞；将人源化免疫重建裸鼠分为3组，分别用LyDCs＋Tctl、LyDCs和生理盐水皮下免疫注射治疗；治疗后7d，体外观察各组裸鼠脾脏淋巴细胞的肿瘤杀伤作用及IFN-γ、IL4分泌水平，实验结束时，观察LyDCs联合Tctl对荷瘤裸鼠的体内抑瘤作用。结果LyDCs的表型HLA．DR、CD80、CD86、CD83较刺激前明显上调；免疫重建裸鼠均检测到人源性CD4^＋、CD8^＋T细胞；LyDCs＋Tctl组裸鼠脾脏T淋巴细胞的肿瘤杀伤作用与LyDCs组比较差异有统计学意义（P〈0．01），LyDCs＋Ted组T细胞的IFN-γ分泌水平与LyDCs组比较差异有统计学意义（P〈0．01）；接种HT-29细胞58d后，LyDCs＋Tαl组、LyDCs组抑瘤率分别为60．41％、37．20％，两组之间抑瘤效应比较差异有统计学意义（P〈0．01），两组的抑瘤效应与对照组比较分别（P〈0．01）。结论Tαl能增强LyDCs诱导的CD4^＋，Th1细胞反应和CTLs杀伤效应，对DCs疫苗的抗癌免疫治疗功效具有明显的放大作用。     

单核细胞源性树突状细胞表达淋巴细胞趋化因子mRNA的初步研究

淋巴细胞趋化因子（Lptn）是趋化因子C族的惟一成员，具有趋化CD4^＋、CD8^＋T细胞和NK细胞的生物学特性，在肿瘤免疫治疗中具有重要意义。单核源性树突状细胞（DCs）分化过程中具有未成熟和成熟2个阶段，DCs具有不同的生物学特性与功能。我们采用体外实验研究了诱导培养的DCs（Mo-DCs）不同时期Lptn mRNA的表达情况。     

sCD40基因修饰树突状细胞抑制T细胞表型及细胞因子分泌

目的探讨sCD40基因修饰树突状细胞（DC）对T细胞表型及Th1和Th2类细胞因子分泌的影响及其体外诱导T细胞免疫耐受的机制。方法分别以sCD40基因及空载体修饰的13（2（实验组和对照组）作为刺激细胞，尼龙毛柱法收集Balb／c小鼠淋巴细胞作为反应细胞，初次进行混合淋巴细胞培养（MLC）7d。在培养的第1、3、4、5、7天时采用新型四唑氮盐（MTS）比色法检测淋巴细胞增殖率；酶联免疫吸附（EuSA）法测定培养液上清中的白细胞介素2（IL-2）、γ干扰素（IFN-γ）、白细胞介素4（IL-4）、白细胞介素10（IL-10）水平；第5天采用流式细胞仪检测CD4^＋、CD8^＋T细胞上CD25及CD69的表达。再次进行MLC5d，在培养的第1、3、5天时检测与初次MLC相同的项目，并比较初次及再次MLC后的各项检测指标。结果初次及再次MLC后，实验组（经sCD40基因修饰的DC）刺激细胞增殖反应明显低于对照组（空载体修饰的DC）。初次MLC中，实验组CD4^＋、CD8^＋T细胞比例及CD4^＋CD25^＋、CD8^＋CD25^＋、CD4^＋CD69^＋、CD8^＋CD69^＋T细胞比例明显低于对照组（P〈0．05）；实验组和对照组培养液上清中IL4和IL-10不能测出，而实验组培养液上清中IFN-γ和IL-2的水平均明显低于对照组（P〈0．01）。再次MLC后，培养液上清中IFN-γ、IL-2和IL-4、IL-10的分泌水平均明显低于对照组（P〈0．01）。结论体外MLC体系中，经sCD40基因修饰的DC可同时作用于CD4^＋和CD8^＋T细胞，使CD69和CD25表达降低，引起T细胞早期活化和成熟障碍，抑制T细胞增殖及Th1和Th2类细胞因子的分泌，诱导出T细胞免疫耐受状态。     

原发性肝癌患者外周血Th17与CD4^＋CD25^＋调节性T细胞表达的相关性研究

目的探讨原发性肝癌患者外周血Th17和CD4^＋CD25^＋调节性T细胞的表达水平及其相关性。方法选取2008年6月—2009年5月浙江大学医学院附属第一医院30例原发性肝癌患者和25名健康人群,采血并分离其外周血单个核细胞。利用流式细胞仪分别测定Th17和CD4^＋CD25^＋Foxp3^＋调节性T细胞的表达,采用t检验分析两组的表达差异。同时,采用Spearman检验对原发性肝癌患者外周血中Th17和CD4^＋CD25^＋调节性T细胞的表达进行相关性分析。结果健康对照组外周血中Th17细胞为（2．10±0．87）％,CD4^＋CD25^＋调节性T细胞为（7．10±2．32）％,原发性肝癌组外周血中Th17细胞为（3．38±1．68）％,CD4^＋CD25^＋调节性T细胞为（11．78±5．62）％,两组差异具有统计学意义（t=3．640和4．162,P值均〈0．01）。原发性肝癌患者组外周血Th17细胞与CD4^＋CD25^＋Fosp3^＋调节性T细胞表达呈正相关（r=0．821,P〈0．01）。结论原发性肝癌患者外周血Th17和CD4^＋CD25^＋Fosp3^＋调节性T细胞表达水平较高,二者呈正相关。CD4^＋CD25^＋Fosp3^＋调节性T细胞可能通过促进Th17细胞分化导致肿瘤的发生与发展。     

胃癌患者外周血调节性T细胞的多种细胞因子水平的检测及其意义

目的探讨细胞因子在CD4^＋CD25^＋调节性T细胞免疫抑制机制中的作用，观察胃癌患者外周血中的CD4^＋CD25^＋调节性T细胞及其效应性T细胞CD4^＋CD25^-T细胞产生具有不同生物活性的细胞因子的水平。方法采用免疫磁珠分选方法分离胃癌患者外周血中的CD4^＋CD25^＋T细胞与CD4^＋CD25^-T细胞后，将CD4^＋CD25^＋T细胞与效应性T细胞CD4^＋CD25^-T细胞在体外分别单独培养及按不同比例共同培养后，再用酶联免疫吸附试验（ELISA）法检测单独及混合培养时细胞因子干扰素（IFN）-γ、白细胞介素（IL）-10及转化生长因子（TGF）-β分泌的水平。结果健康对照及胃癌患者CD4^＋CD25^＋T细胞分泌抑制性细胞因子IL-10、TGF-β均显著高于CD4^＋CD25^-T细胞，而分泌细胞因子IFN-γ均显著低于CD4^＋CD25^-T细胞（P〈0．01）。CD4^＋CD25^＋T细胞在体外可明显抑制CD4^＋CD25^-T细胞产生IFN-γ的能力，且这种抑制能力呈效靶比关系（P〈0．01）；但CD4^＋CD25^＋T细胞与效应性T细胞CD4^＋CD25^-T细胞在体外共培养对IL-10及TGF-β的分泌无显著影响（P〉0．01）。结论CD4^＋CD25^＋调节性T在体外可能通过细胞因子的调节发挥对效应性T细胞的抑制作用。     

gp96多肽复合物对树突状细胞成熟的影响

目的研究热休克蛋白gp96多肽复合物与小鼠骨髓来源的树突状细胞（DC）成熟的关系。方法采用GM-CSF、IL-4刺激培养小鼠骨髓细胞，增殖产生大量DC；从小鼠肝癌细胞株H22细胞中提取gp96肽复合物，体外修饰DC；通过流式细胞仪检测上清液IL-12、TNF-α细胞因子含量和DC表面主要组织相容性Ⅱ类抗原、CD40、CD80等分子表达；DC与小鼠脾淋巴细胞共同培育后，检测CD4、CD8和IFN-γ、IL-10双阳性细胞比例；以^51Cr释放法测定细胞毒性T淋巴细胞（CTL）杀伤活性。结果gp96多肽复合物修饰的DC大量分泌IL-12、TNF-α等细胞因子，高表达MHCⅡ类抗原、CD40、CD80等分子；并且能激活小鼠脾淋巴细胞，CD8^＋-IFN-γ^＋和CD4^＋-IFN-γ^＋双阳性细胞比例显著升高；能够特异性的杀伤H22癌细胞。结论gp96多肽复合物能够诱导DC成熟，体外产生特异性CTL反应。     

趋化因子巨噬细胞炎症蛋白-1α动员的树突状细胞经基因修饰后抗胃癌效应

目的观察趋化因子巨噬细胞炎症蛋白-1α（MIP-1α）体外动员的树突状细胞（DC）经基因修饰后在荷瘤小鼠体内的抗胃癌效应。方法615小鼠通过尾静脉注射MIP-1α，流式细胞仪分选出B220^- CD11c^＋细胞，加入鼠粒-巨噬细胞集落刺激因子（mGM-CSF）、白细胞介素-4（IL-4）和鼠肿瘤坏死因子-1α（mTNF-1α）贯续培养进行诱导分化。通过细胞表型和混合淋巴细胞反应，检测细胞因子培养前后B220^- CD11c^＋细胞的异同。收集培养后的B220^- CD11c^＋细胞，加入编码黑色素瘤抗原基因-3（MAGE-3）的重组腺病毒进行转染，制备表达肿瘤抗原的DC疫苗。制备小鼠前胃癌（MFC）实体瘤模型，DC疫苗于MFC细胞接种后经皮下注射，观察小鼠瘤体生长和存活情况，研究DC疫苗的免疫治疗作用。结果MIP-1α注射8h后外周血中B220^- CD11c^＋细胞数量即升高，48h达到高峰，占外周血单个核细胞（MNCs）（13．68±0．95）％。新鲜分离的B220^- CD11c^＋细胞不具有成熟DC的特征，而经体外细胞因子诱导分化后则具有典型的DC表型，并具有极强的刺激T细胞增殖的能力。小鼠皮下接种MFC细胞后注射基因修饰的DC疫苗，小鼠瘤体生长缓慢，存活时间明显延长，与对照组之间差异有统计学意义（P〈0．01）。结论注射趋化因子MIP-1α可快速动员B220^- CD11c^＋细胞进入小鼠外周血，并经细胞因子可诱导分化为成熟DC。MIP-1α动员的DC经基因转染制备的DC疫苗，在体内对荷瘤小鼠有明显的免疫治疗作用，且较荷载全肿瘤细胞抗原的DC疫苗作用明显增强。     

负载NDV-ATV的树突状细胞诱导的抗胃癌效应研究

目的研究负载新城鸡瘟病毒修饰的自体肿瘤细胞瘤苗(NDV-ATV)抗原的树突状细胞(DCs)诱导的抗胃癌效应。方法新城鸡瘟病毒感染MNK45细胞并灭活,GMCSF、IL-4和TNF-α体外诱导扩增DCs并负载NDV修饰后的胃癌抗原,制备胃癌抗原特异性CTL;用CytoTox96TM检测其对MNK45体外杀伤效应。并以冻融胃癌细胞抗原为对照。结果负载新城鸡瘟病毒胃癌抗原DCs诱导的特异性CTL对MNK45的杀伤率达90.15%,显著高于冻融抗原的杀伤率(P<0.05)。且其对同种不同分化类型的胃癌细胞株MGC803、SGC7901也有较高的杀伤效应,而对LOVO及HepG2肿瘤细胞无显著杀伤作用(P<0.01)。结论新城鸡瘟病毒修饰的胃癌细胞,联合树突状细胞可诱导产生高效和特异的抗胃癌效应。提示NDV-ATV联合DCs可作为胃癌免疫治疗的一种有效的新方法。     

慢性特发性荨麻疹患者外周血T及Th淋巴细胞亚群的表达

目的：探讨慢性特发性荨麻疹患者外周血T及辅助性T淋巴细胞（Th）亚群的表达及其在慢性特发性荨麻疹发病机制中的作用。方法：采用流式细胞术检测经四色荧光抗体染色的慢性特发性荨麻疹患者及正常对照外周血CD3^＋、CD4^＋、CD8^＋T淋巴细胞数及CD4^＋／IFN-γ’（Th1）、CD4^＋／IL-4^＋（Th2）细胞含量。结果：慢性特发性荨麻疹组外周血CD3^＋T淋巴细胞数无明显变化、CD4^＋T淋巴细胞数、CD8^＋T淋巴细胞数均降低;CD4^＋／CD8^＋比值增高,差异有统计学意义（P〈0．01）。慢性特发性荨麻疹患者外周血Th1细胞含量、Th1／Th2比值均明显低于正常对照组（P〈0．01,P〈0．05）,Th2细胞含量高于正常对照组（P〈0．01）。结论：慢性特发性荨麻疹患者外周血存在着T及Th淋巴细胞亚群分化失衡,这可能为慢性特发性荨麻疹发病的机制之一。     

肝癌mRNA致敏的DC对SCID荷瘤鼠的抑瘤作用

目的通过重建人肝癌PBL-SCID嵌合模型来观察mRNA致敏的树突状细胞疫苗（mRNA DC）在体内的抑瘤效应并探讨机制。方法采用人外周血淋巴细胞腹腔注射法建立Hu-PBL-SCID鼠模型,尾静脉分别注射mRNA DC疫苗、抗CD4^＋、CD8^＋＋mRNA DC、未致敏的树突状细胞（DC）。每周1次,共两次,然后接种2×10^6HepG-2cells,观察鼠成瘤率、成瘤潜伏期、肿瘤体积以及测定特异性CTL活性。结果ELISA法可检测到鼠血清中人IgG水平,Hu-PBL-SCID嵌合模型重建成功,各组小鼠间成瘤率无明显差异,但mRNA DC组成瘤潜伏期延长,肿瘤生长缓慢,2周后肿瘤体积明显小于抗CD4^＋、CD8^＋＋mRNA DC组、DC组和PBS组,差异有统计学意义（P〈0.05）,实验组脾淋巴细胞对HepG-2细胞有特异性杀伤效应,而对胃癌SGC-7901细胞则无杀伤活性。结论mRNA致敏的树突状细胞疫苗体内能诱导产生明显的抑瘤作用。     

In vitro induction of tumor-specific HLA class I-restricted CD8+ cytotoxic T lymphocytes from patients with locally advanced breast cancer by tumor antigen-pulsed autologous dendritic cells

BACKGROUND: Early dissemination of treatment-resistant tumor cells remains the major cause of metastatic recurrence and death in breast cancer patients. Dendritic cells (DCs) are the most powerful antigen-presenting cells, and recently DC-based vaccination has shown great promise for the treatment of human malignancies by immunological intervention. MATERIALS AND METHODS: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous breast tumor antigen-pulsed DCs were tested for their ability to induce a HLA class I restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. To correlate cytotoxic activity by CTL with T cell phenotype, two-color flow cytometric analysis of surface markers and intracellular cytokine expression was performed. RESULTS: DC pulsed with breast tumor extracts consistently elicited a tumor-specific HLA class I restricted CTL response in vitro in three consecutive patients harboring locally advanced breast cancer. CTL expressed strong cytolytic activity against autologous tumor cells but did not lyse autologous Epstein Barr virus-transformed lymphoblastoid cell lines and showed variable cytotoxicity against the natural killer-sensitive cell line K-562. In all patients, two color flow cytometric analysis of surface markers and intracellular cytokine expression demonstrated that tumor-specific CTL exhibited an heterogeneous CD8+/CD56+ expression and a striking Th1 cytokine bias (IFNgamma(high)/IL-4 (low)). CONCLUSIONS: Tumor lysate-pulsed DCs can consistently stimulate specific CD8+ CTLs able to kill autologous tumor cells in patients with locally advanced breast cancer in vitro. Tumor antigen-pulsed DC-based vaccinations may be appropriate for the treatment of residual and/or chemotherapy-resistant breast cancer refractory to standard salvage treatment modalities.     

五聚体辅助胰腺癌黏蛋白4抗原HLA限制性CTL表位筛选

目的 筛选和验证黏蛋白4(MUC4)的有效抗原表位并证实MUC4抗原表位在体内的自然递呈作用.方法 采用生物信息学方法预测、T2细胞体外实验初步筛选后,通过人类组织相容性抗原(HLA)表位肽负载树突状细胞(DC)、酶联免疫斑点试验、细胞毒T淋巴细胞(CTL)试验验证相应表位的体外HLA限制性免疫效应;合成MUC4 HLA-A2限制性表位五聚体;采用该五聚体检测临床胰腺癌患者体内自然递呈的MUC4表位.结果 通过多参数表位预测得到了5个HLA-A*0201限制性表位并合成抗原肽,并经T2细胞稳定性试验初步验证;体外实验中,肽P1126诱导的CTL在不同的效靶比可特异性杀伤P1126负载的T2细胞和MUC4+、HLA-A2+的癌细胞株HCT-116;能够产生IFN-γ的细胞数(130.3±6.6)明显高于阴性对照组.五聚体检测发现,MUC4(+)患者外周血检出特异性CTL的频率显著高于MUC4(-)患者;但MUC4(+)患者中,HLA-A2(+)患者外周血检出特异性CTL的频率与HLA-A2(-)患者的差异无统计学意义.结论 肿瘤相关抗原MUC4的HLA-A*0201限制性CTL表位P1126,能诱导人外周血单核细胞的CTL反应,该活化的CTL能分泌免疫活性物质诱导特异性靶细胞的凋亡.P1126能在胰腺癌患者外周血中自然递呈,但HLA限制性不完全.     

A novel dendritic cell-based cancer vaccine produces promising results in a syngenic CC-36 murine colon adenocarcinoma model

BACKGROUND: This study was conducted to test the efficacy of a new cancer vaccine, composed of dendritic cells (DCs) pulsed with an interleukin-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) in a CC-36 murine colon adenocarcinoma model. MATERIALS AND METHODS: CC-36 tumor cells were injected subcutaneously into the left flank of four- to six-week old male BALB/c mice. The mice were divided into three groups, each of which received one of the following treatments: (1) DCs pulsed with the IL-2 gene-encoded vaccinia oncolysate (DC-IL-2VCO), (2) DCs pulsed with the tumor oncolysate alone (DC-CO), or (3) no treatment (control). Tumor incidence was measured, and survival rates were compared using a paired Student's t-test. Cytolytic T cell activity was measured in peripheral blood lymphocytes (PBL) and splenic lymphocytes using a (51)Cr-release assay. Lastly, mice were depleted of either CD4+ or CD8+ lymphocytes prior to receiving the vaccine to test the mechanism of tumor immunity in these mice. RESULTS: Mice treated with DC-IL-2VCO demonstrated decreased tumor burden, increased survival, and greater cytolytic activity compared with control mice and mice receiving DC-CO. In addition, mice depleted of CD8+ T cells prior to immunization with IL-2VV + DC-IL-2VCO had a significant increase in the incidence of tumor, similar to the untreated control mice. CONCLUSIONS: DCs pulsed with an IL-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) produced safe and effective immune responses in a murine CC-36 colon adenocarcinoma model. This vaccine (DC-MelVac; Patent no. 11221/5) has the potential to treat humans with cancer, and has received FDA approval for use in Phase I clinical trials.     

Regulatory function of human CD4+ cytolytic T lymphocytes.

Allograft rejection is mediated by both CD4+ and CD8+ T cells. The lytic function of the classic CD8+ cytolytic T lymphocytes (CTL) occurs through recognition of allogeneic major histocompatibility complex (MHC) class I on the surface of the graft. CD4+ CTL recognize MHC class II through a direct recognition pathway or an indirect pathway where MHC peptides are presented in the context of self MHC class II. Lytic CD4+ cells may destroy graft tissue or, we hypothesize, the indirect CD4+ T cell may down regulate CD8+ CTL by recognition of donor MHC peptides presented by self MHC class II expressed on CD8+ T cells. To define the role of CD4+ CTL in allograft outcome we used a CD4+ CTL that is MHC class II restricted, recognizing human leucocyte antigen (HLA)-A1 and HLA-B8 peptides in the context of HLA-DR4. This line (MDSxA1/B8) will lyse DR4+ B lymphoblastoid cells (LCL) pulsed with HLA-A1/B8 peptides (amino acids 60-84 of the alpha1 domain of the MHC class I molecule). These T cells will also lyse peptide-pulsed antigen-specific T cell clones, both CD4+ and CD8+, that express HLA-DR4. These clones must process and present the MHC class I peptides for recognition and lysis to occur. These results suggest a possible mechanism to explain allograft tolerance. Lytic CD4+ T cells, that recognize donor HLA peptides through an indirect antigen presentation pathway, down-regulate donor-specific CTL through peptide-specific lysis resulting in graft tolerance.     

MAGE-3抗原肽体外诱导肝癌患者免疫应答的研究

目的：利用载有MAGE－3抗原肽的树突状细胞（DC）活化原发性肝细胞癌（HCC）患者T淋巴细胞，探讨是否可以体外诱导特异性细胞毒T细胞细胞（CTL）应答。方法：通过逆转录多聚酶链反应和测序分析MAGE－3多肽表位密集区的核苷酸变异状况，体外培养HLA－A2表型的HCC患者及正常献血员外周血来源DC，并经孵育携带MAGE－3／HLA－A2抗原肽FLWG－PRALV，用以活化T淋巴细胞，利用特异性杀伤实验检测CTL应答。结果：中国HCC患者表达的MAGE－3序列高度保守。用特定细胞因子和无血清培养基可成功培养HCC患者外周血精源的DC。经多肽冲击的DC诱导，3例HCC例者和3例正常献血员可成功培养HCC患者外周血来源的DC。经多肽冲击的DC诱导，3例HCC患者的3例正常献血员中各有2例产生CTL免疫应答。结论：载有MAGE－3抗原肽的DC可以体外诱导特异性的CTL免疫应答。提示HLA－A2限制性的MAGE－3抗原肽经DC递呈可以作为有潜力的肝癌免疫治疗疫苗。     

KDEL修饰的HSV-2CD8~+T细胞表位促进CTL效应的研究

目的:研究赖氨酸天冬氨酸谷氨酸亮氨酸(Lys-Asp-GluLeu,KDEL)修饰的2型单纯疱疹病毒(herpessimplexvirustype2,HSV2)CD8+T细胞表位促进细胞毒T淋巴细胞(cytotoxicityTlymphocyte,CTL)效应。方法:将雄性C57BL/6小鼠随机分为5组,每组5只,用各抗原肽免疫C57BL/6小鼠,采用3HTdR掺入法检测淋巴细胞增殖反应、标准4h51Cr释放试验检测CD8+T细胞特异性CTL效应,观察HSV2CD8+T细胞表位(SSIEFARL,S1)、KDEL修饰的HSV-2CD8+T细胞表位(SSIEFARL-KDEL,S1-KDEL)、4拷贝串联的CTL表位[(SSIEFARL)4,S4]及KDEL修饰的串联CTL表位[(SSIEFARL)4KDEL,S4-KDEL]的特异性细胞免疫应答。结果:3HTdR掺入试验中,S4组和S4KDEL组cpm值显著高于对照组和S1组、S1KDEL组(P<0.05);S4KDEL组的cpm值显著高于S4组(P<0.05);S1组和S1KDEL组与对照组比较,差异无显著性(P>0.05);S1组与S1KDEL组比较,cmp值亦无显著性差异(P>0.05)。以S1致敏的EL4细胞为靶细胞的杀伤实验中,S4和S4KDEL组诱导的CTL活性明显高于对照组、S1组及S1KDEL组(P<0.05);S4KDEL组诱导的CTL活性明显高于S4组(P<0.05),S1组和S1KDEL组与对照组比较,差异无显著性(P>0.05);S1组与S1KDEL组比较,差异亦无显著性(P>0.05);以EL4细胞为靶细胞的实验中,实验组的杀伤率均在10%以下,与对照组?     

Induction of cytolytic T lymphocytes against pediatric solid tumors in vitro using autologous dendritic cells pulsed with necrotic primary tumor

Purpose

Effective and generally applicable methods for generating cancer vaccines in children have not been defined. Dendritic cells (DCs) are the most potent professional antigen-presenting cells capable of activating primary cytolytic T cells. We tested the ability of DCs generated from pediatric patients' peripheral blood monocytes and pulsed with a necrotic tumor to activate autologous tumor-specific cytolytic T cells.

Methods

Tumor and peripheral blood cells were obtained from pediatric patients undergoing biopsy or resection for advanced solid tumors according to an institutional research board-approved protocol and after acquiring informed consent from them. To generate DCs, we treated peripheral blood monocytes with granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Maturation was induced with a cytokine cocktail (CC) containing tumor necrosis factor-α, IL-6, IL-1β, and prostaglandin E₂. The DC phenotype was assayed using flow cytometry. Tumor necrosis was induced by exposure to UV-B irradiation (1000 mJ). Dendritic cells pulsed with a UV-B-treated primary tumor and matured with CC were used to stimulate autologous peripheral blood lymphocytes weekly. Tumor-specific cytolytic activity was assayed using 4-hour ⁵¹Cr release.

Results

Peripheral blood monocytes isolated from pediatric patients differentiated into immature DCs (CD14⁻, MHCII⁺ [major histocompatibility complex], CD80^low, CD86^low) in the presence of granulocyte-macrophage colony stimulating factor and IL-4. Cytokine cocktail induced maturation of DCs, as characterized by increased expressions of MHCII, CD83, CD80, and CD86. Patients' peripheral blood lymphocytes stimulated in vitro with DCs loaded with a necrotic primary tumor and matured with CC specifically lysed autologous neuroblastoma in 7 of 9 patients.

Conclusion

Dendritic cells generated from the peripheral blood of children with advanced solid tumors and pulsed with a necrotic primary tumor undergo maturation and effectively stimulate autologous tumor-specific cytolytic T cells in vitro. We describe a simple method for generating a vaccine capable of activating cytotoxic T cells against pediatric solid tumors that does not require the genetic identification of tumor-associated antigens.     

Human accessory cells activate fresh, normal, tumor-distant T lymphocytes but not tumor-infiltrating T lymphocytes to lyse autologous tumor cells in a primary cytotoxic T lymphocyte assay in renal cell carcinoma.

OBJECTIVES: Search for an ideal responder T-lymphocyte source for adoptive T-lymphocyte therapy in renal cell carcinoma (RCC). METHODS: Cytotoxic T-lymphocyte (CTL) activity of (a) normal, tumor-distant, renal T lymphocytes, (b) tumor-infiltrating T lymphocytes and (c) peripheral blood T lymphocytes against autologous tumor epithelial cells (EC) of 10 patients with organ-confined, primary RCC was analyzed in a primary CTL assay. Freshly enriched T lymphocytes were cultured with or without autologous, mitomycin-C-treated normal or tumor EC in the presence or absence of antigen-presenting cells (APC) for 7 days. RESULTS: Both tissue T-lymphocyte populations displayed a similar CD4:CD8 ratio (1:1). Elevated CD62L coexpression of CD4+ T lymphocytes in normal, tumor-distant, renal tissue resulted in a significantly higher transient T-cell activation than that seen in renal tumor tissue (46 vs. 27%; p = 0.002). All trials to induce significant lysis of autologous, renal tumor EC in tumor-infiltrating and peripheral blood T lymphocytes failed. Only when normal, tumor-distant, renal T lymphocytes were stimulated by autologous APC and tumor EC was significant autologous tumor EC lysis obtained (mean 14%; p<0.05). Costimulation by anti-CD3 (mean 21%; p<0.05) or interleukin-2 (mean 31%; p<0.05) further increased tumor EC lysis significantly. CONCLUSIONS: Increased turnover of T lymphocytes in normal, tumor-distant, renal tissue was associated with a higher yield of pre-CTL which can be transformed into a functionally active effector T-cell pool by stimulation via antigen plus APC. Thus, tumor-distant renal tissue has to be included in the tissue-sampling procedure for adoptive immunotherapy.     

Presence and specificity of tumor associated lymphocytes from ascites fluid in prostate cancer

BACKGROUND: In the present study, we had the rare opportunity to study immunological responses of TAL from ascites fluid in a patient with hormone-refractory prostate cancer. METHODS: We evaluated tumor antigen-specific T-cell responses, induced by either prostate specific antigen (PSA) pulsed dendritic cells (DCs) or PSA peptides, in TAL and peripheral blood lymphocytes. RESULTS: DC stimulation with PSA protein induced recognition of naturally processed PSA epitopes by both blood and ascites T cells. In contrast, only ascites T cells recognized the PSA-3 peptide, after stimulation with PSA-pulsed DCs or peptides. Finally, although IFNgamma secreting T cells were detectable in both blood and ascites by ELISPOT, multiplex cytokine assay detected the presence of predominantly Th2 cytokines. CONCLUSIONS: Although tumor antigen-specific TAL were detected in ascites fluid, these cells were producing immunosuppressive cytokines which may contribute to tumor escape from recognition and/or destruction by the immune system.     

Cytokine expression in circulating T lymphocytes from patients undergoing carotid endarterectomy

AIM: We investigated a possible relationship of cytokine expression and phenotype features of circulating T lymphocytes with the histological type of atherosclerotic plaque removed during carotid endarterectomy. METHODS: Peripheral blood samples were taken from 20 patients with carotid atherosclerosis and from 8 healthy blood donors. Patients were divided into 2 groups according to the histological type of their atherosclerotic plaques (types V and VI). Expression of intracellular tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin-4 (IL-4), and of surface antigens (CD4, CD8, CD45RA, CD45RO, T cell receptor (TCR) alpha/beta, TCR gamma/delta) in circulating T lymphocytes was determined by 3-colour cytofluorimetric analysis. RESULTS: The percentage of T lymphocytes primed for TNF-alpha, IFN-gamma and IL-4 was higher in blood samples from patients than from healthy subjects; the difference was statistically significant for TNF-alpha-producing cells (p=0.01). In patients, the percentage of TNF-alpha-producing cells was significantly higher in the CD4+ subset than in the CD8+ subset (p=10(-4)). The percentage of TNF-alpha-, IFN-gamma- and IL-4-primed cells was higher in patients with type VI plaques (complicated lesions) than in patients with type V plaques (less complicated lesions). The difference was statistically significant for TNF-alpha-primed cells (p=0.05). No statistically significant differences were found in T cell phenotype features among patients or between patients and healthy subjects. CONCLUSION: Our results suggest a relationship between the percentage of circulating T lymphocytes expressing TNF-alpha and possibly IFN-gamma and IL-4 and the histological type of atherosclerotic plaque in patients with carotid artery disease.