特异诱导脐血CD8+抗白血病细胞毒淋巴细胞的研究

微小残留病灶(minimal residual disease,MRD)的存在是导致白血病复发的主要根源,特异性免疫治疗能有效地清除MRD,其中的策略之一就是诱导并回输白血病特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL).本实验的目的是研究脐血能否在体外诱导生成CD8+ CTL,所生成的CD8+ CTL能否特异性杀伤白血病细胞,从而确定脐血淋巴细胞的利用价值及用于特异性免疫治疗的可行性.通过联合细胞因子体外诱导脐血单个核细胞(mononuclear cells,MNC)分化为树突状细胞(dendritic cells,DC),同时负载U937冻融抗原;成熟DC刺激同源的脐血T淋巴细胞成为CTL,经MACS磁珠分选出CD8+ CTL.用倒置显微镜、扫描电镜及流式细胞仪等方法检测DC,四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法测定杀伤活性.结果显示,10份人脐血标本均可培养出形态典型、功能成熟的DC.3组效应细胞CD8+ CTL、CD8- CTL和T淋巴细胞(TL)组在相同效靶比(40∶1)对U937细胞株的杀伤率分别为(66.36±12.43)%、(34.47±8.19)%和(15.79±4.64)%,以CD8+ CTL组最高;效靶比为40∶1时,CD8+ CTL对U937细胞株的杀伤率(66.36%)高于对K562细胞株的杀伤率(41.97%)(P＜0.05);而CD8- CTL对U937细胞株和K562细胞株的杀伤率无显著差异(P＞0.05).结论用负载U937白血病细胞株抗原的成熟脐血DC可使脐血淋巴细胞诱导产生特异性的CD8+ CTL;CD8+ CTL对U937白血病细胞株的杀伤活性强于CD8- CTL的作用;CD8+ CTL对U937白血病细胞株杀伤具有特异性.     

特异诱导脐血CD8+抗白血病细胞毒淋巴细胞的研究

目的研究体外诱导脐血CD8+细胞毒性T淋巴细胞(CTL)特异性杀伤白血病细胞的作用,探讨脐血淋巴细胞用于特异性免疫治疗的可行性.方法联合细胞因子体外诱导10份脐血单个核细胞(MNC)分化为树突细胞(DC),同时负载U937冻融抗原;成熟DC刺激同源的脐血T淋巴细胞成为CTL,经MACS磁珠分选出CD8+CTL.倒置显微镜、扫描电子显微镜及流式细胞术等方法检测DC细胞特性.MTT法测定细胞杀伤活性.结果10份脐血标本均可培养出形态典型、功能成熟的DC.CD8+CTL、CD8-CTL和淋巴细胞(TL)组对U937细胞不同效靶比的杀伤率以CD8+CTL组最高;CD8+CTL在401效靶比时对靶细胞U937细胞的杀伤率高于对K562细胞的杀伤率[分别为(66.36±12.43)%和(41.97±14.24)%,(P《0.05)];而CD8-CTL在401效靶比时对U937细胞和K562细胞的杀伤率差异无统计学意义[分别为(34.47±8.19)%和(22.45±4.00)%(P》0.05)].结论用负载U937细胞抗原的成熟脐血DC,诱导出脐血淋巴细胞特异性的CD8+CTL;CD8+CTL对U937细胞的杀伤活性强于CD8-CTL;CD8+CTL对U937细胞的杀伤活性具有特异性.     

含癌胚抗原片段的重组腺相关病毒转染树突状细胞疫苗的体外杀伤作用

目的：观察研究人外周血单核细胞来源的树突状细胞（DC）转染含癌胚抗原（CEA）片段的重组腺相关病毒后所诱导的特异性T细胞对直肠癌细胞株LOVO和SW480的体外杀伤作用。方法：抽取HLA表型为A11的健康志愿者外周血，分离单核细胞，体外培养，使用含CEA片断的重组腺相关病毒转染未成熟DC，诱导特异性T细胞。检测体外培养的DC和CTL活性，并使用MTT法检测细胞毒性T细胞（CTL）对LOVO细胞的杀伤作用。结果：转染或未转染的体外培养的成熟DC高表达CD40、CD86、IL-12，诱导的细胞毒性T细胞高表达IFN-γ；转染后DC诱导特异性细胞毒性T细胞可有效识别并杀伤HLA-A11阳性的LOVO细胞。结论：重组腺相关病毒转染DC，不明显改变DC表型和刺激淋巴细胞增殖、分化功能，可诱导自体细胞毒性T细胞增殖，含CEA片断的腺相关病毒转染DC诱导自体细胞毒性T细胞对LOVO细胞有明显杀伤作用，DC疫苗可以作为直肠癌患者免疫治疗的有效补充。     

急性白血病RNA转染脐血源性树突状细胞介导的免疫作用

目的检测应用脐血诱导成熟树突状细胞(DC),并以急性白血病(AL)总RNA冲击该DC所诱导产生的特异性细胞毒T细胞(CTL)的杀伤活性。方法取AL患者外周血或骨髓,以淋巴细胞分离液获得单个核细胞(MNC),并提取RNA。分离脐血MNC,以2×106个细胞/孔接种于12孔培养板,培养体系中加入重组人粒单核细胞集落刺激因子(rhGM-CSF),重组人白细胞介素4(rhIL-4)和肿瘤坏死因子α(TNF-α),并在培养第5天,加入白血病总RNA,致敏脐血DC。培养过程中,观察树突状细胞形态,流式细胞仪检测表型。培养第12天收获成熟DC,与自体T淋巴细胞共培养,诱导产生白血病特异性CTL,乳酸脱氢酶法(LDH法)测定溶细胞活性。结果脐血来源DC表现出成熟DC典型形态和免疫表型特征,培养第5天,DC相关分化抗原CD1a、CD83、CD86、CD80的表达较培养前增高,差异有统计学意义(P<0.01);培养至第12天,DC相关分化抗原CD1a、CD83、CD86、CD80的表达较培养第5天增高,另外人类白细胞相关抗原(HLA-DR)的表达也较培养第5天增高,差异均具有统计学意义(P<0.01)。以白血病RNA转染的DC组所刺激的自体T淋巴细胞对自身白血病靶细胞显示出明显的杀伤活性,而未经抗原转染DC组无明显杀伤活性,按照各效靶比进行两者的比较,差异有统计学意义(P<0.01)。结论利用细胞因子体外诱导脐带血产生大量成熟DC,以急性白血病RNA转染此DC,并以此白血病特异性DC诱导产生抗白血病特异性CTL,不失为清除AL患者体内残留灶的积极措施。     

卵巢癌抗原冲击DC在体外对SKOV3细胞作用的研究

目的探讨卵巢癌患者外周血和腹水中培养的树突细胞(DC)在体外激活淋巴细胞产生的肿瘤特异性细胞毒T淋巴细胞(CTL)作用,及其对卵巢癌SKOV3细胞的杀伤作用。方法分别培养卵巢癌患者外周血及腹水来源DC,进行细胞形态学观察及细胞表面分子表达检测;以卵巢癌冻融抗原致敏DC,检测两种来源DC刺激T淋巴细胞增殖的能力及对IL-2水平的影响;检测抗原负载DC与T细胞诱导CTL对卵巢癌细胞的杀伤作用。结果镜下显示卵巢癌患者外周血及腹水来源的DC形态学无明显差异,腹水来源DC成熟时间较外周血来源DC晚,二者表面分子表达的阳性率除CD83外无明显差异;卵巢癌冻融抗原负载DC上调其表面相关分化抗原表达,统计学差异具有显著性(P0.05);经卵巢癌冻融抗原负载后的DC刺激T淋巴细胞的增殖,差异有显著性(P0.05),但两种来源DC间刺激指数无统计学差异;抗原负载DC与无抗原负载DC相比,其上清中IL-12浓度增高,差异具有显著性(P0.05);负载与不负载抗原的DC对卵巢癌细胞杀伤作用增强,与对照组相比具有显著性差异(P0.05),随着效应细胞数量的增高,杀伤作用增强,加入IL-2增强其杀伤作用,但外周血及腹水来源DC间相应情况下对卵巢癌细胞的杀伤作用无统计学差异(P0.05)。结论卵巢癌患者外周血及腹水来源的DC均具有诱导CTL作用,DC作为载体负载肿瘤抗原后可以激发T淋巴细胞形成CTL,在体外有效的杀伤卵巢癌SKOV3细胞。     

MAGE-1多肽负载树突状细胞对人肝癌细胞的体外杀伤作用

目的:应用黑色素瘤基因(MAGE-1)多肽负载的树突状细胞(DC)在体外诱导出高度特异性的抗肝癌免疫应答。方法:分离肝细胞肝癌患者外周血单个核细胞;GM-CSF、IL-4、TNF-α联合培养获得DC;MAGE-1多肽负载DC激活自体T淋巴细胞,使之增殖、分化为细胞毒T细胞(CTL);用MTT法检测CTL对BEL-7402HCC细胞的杀伤作用。结果:MAGE-1多肽负载DC激活的T淋巴细胞对BEL-7402HCC细胞有明显的体外杀伤作用,而无关抗原和无抗原负载的DC活化的淋巴细胞无明显杀伤作用(P<0.01)。结论:MAGE-1多肽负载的HCC患者外周血DC在体外可诱导出高度特异性的抗肿瘤免疫应答,提示MAGE-1基因可作为免疫治疗HCC的攻击靶点,MAGE-1多肽负载的DC疫苗在HCC的治疗中起重要作用。     

抗原冲击致敏树突状细胞诱导特异性CTL杀伤Jurkat细胞实验研究

本研究以健康人外周血单核细胞为前体细胞，体外诱导其为树突状细胞（DC），分别负载Jurkat细胞冻融抗原及WT1多肽抗原，产生特异性细胞毒性T淋巴细胞（CTL），以探讨其对白血病Jurkat细胞的杀伤作用。联合应用rhGM—CSF、rhIL-4、rhTNF-α及rhsCD40L等细胞因子，密度梯度离心法、贴壁法从外周血获取单核细胞并进行诱导扩增，培养出DC，分别用冻融抗原及WT1多肽抗原冲击致敏DC。实验分4组：冻融抗原致敏DC组为实验组A，WT1多肽致敏DC组为实验组B，未致敏DC组为对照组A，单核细胞组为对照组B，观察CTL对Jurkat细胞的杀伤作用。结果表明：培养出了具有典型特征的DC，它高表达CD40、CD80、CD1a及CD86等表面标志，能体外诱导强烈的同种异基因混合淋巴细胞增殖反应。实验组显示高水平杀伤率，与对照组比较均具有统计学意义（P〈0.01），实验组A、B间比较无统计学意义。结论：Jurkat细胞冻融抗原及WT1多肽抗原冲击致敏DC能有效诱导T细胞抗白血病作用，为临床研制DC疫苗提供了实验依据。     

人脐血树突状细胞体外诱导抗白血病细胞毒效应

为了体外观察树突状细胞 (DC)诱导的抗白血病细胞毒效应 ,采用细胞因子组合体外扩增培养脐血单个核细胞 (CBMNC) ,对产生的DC进行形态学观察、免疫表型检测和功能鉴定 ;用DC制备细胞毒T淋巴细胞 (CTL)和51Cr释放法测定CTL的溶靶细胞毒性。结果表明 :CBMNC中T细胞亚群与成人外周血相似 ;CD1a阳性细胞含量极少 ,仅为 (0 .4 1± 0 .0 9) % ;在DC扩增培养过程中 ,DC形态发生明显变化 ,胞体变大 ,形态不规则 ,有的细胞出现毛刺状突起 ,有的细胞出现粗大树根状突起 ;在培养 15天时 ,细胞群中CD1a阳性细胞达 (2 8.4± 3.5 5 ) % ,有 (6 3.6 7± 2 3.33) %的细胞表达CD86 ,(8.7± 1.4 9) %的细胞表达CD83,(32 .5± 1.5 3) %的细胞表达HLA DR ;产生的DC对异基因淋巴细胞具有明显的增殖刺激作用 ;经冻融的HL 6 0全细胞抗原脉冲的DC致敏自体T淋巴细胞产生的CTL对HL 6 0细胞具有特异性细胞毒效应。结论 :本研究中的脐血DC培养体系能产生较高含量的DC ,脐血DC在体外能诱导产生抗白血病细胞毒效应。     

肿瘤细胞裂解物致敏树突状细胞对小鼠乳腺癌作用的研究

目的观察肿瘤细胞裂解物致敏树突状细胞 (DC)对小鼠乳腺癌的治疗作用。方法无菌取小鼠骨髓细胞 ,在体外培养条件下经细胞因子作用诱导为树突状细胞 (DC) ,用EMT6肿瘤抗原裂解物冲击致敏DC细胞 ,检测DC体外刺激活化淋巴细胞作用 ,以及经DC免疫产生的细胞毒T淋巴细胞 (CTL)体外杀伤肿瘤细胞的活性 ,观察致敏DC免疫对小鼠乳腺癌模型的治疗作用。结果镜下可见抗原致敏后的DC可吸引淋巴细胞聚集成团 ;致敏DC诱导生成的特异性CTL在体外对肿瘤细胞可产生杀伤作用 (与PBS对照组比较 ,P =0 .0 2 7) ,而未成熟DC细胞组和肿瘤抗原组与PBS对照组间无显著性差异 (P =0 .17,P =0 .0 72 ) ;经致敏DC注射免疫后 ,小鼠负荷肿瘤得到抑制 (与PBS对照组比较 ,P =0 .0 3 5 ) ,而单纯肿瘤抗原及未致敏DC免疫组与PBS对照组间无显著性差异 (P =0 .2 6,P =0 .11)。结论肿瘤抗原裂解物致敏的DC可有效递呈抗原并诱导淋巴细胞杀伤肿瘤细胞。     

体外诱导人脐血树突状细胞介导T细胞抗白血病的细胞毒性效应研究

为了研究脐血淋巴细胞能否在体外培养成为特异性杀伤白血病细胞的杀伤性T细胞(CTL),联合细胞因子体外诱导脐血单个核细胞分化为树突状细胞(DC),再吞噬凋亡白血病细胞并将其抗原呈递给相同脐血的T淋巴细胞,得到杀伤性T细胞。用形态学及流式细胞术检测DC。CTL细胞的杀伤功能用乳酸脱氢酶(LDH)释放法测定。结果表明:12份脐血标本均可培养出形态典型的DC,DC的表面标志CD1a 、HLA DR 、CD86 、CD83 表达水平显著升高(P<0.05)。CTL可以杀伤未经培养的白血病细胞(效∶靶=50∶1对AML细胞的平均杀伤率为44.76±17.42%,对ALL细胞的平均杀伤率为8.50±4.25%),对相同患者缓解期的骨髓细胞杀伤率极低。结论: 在外体应用多种细胞因子刺激可诱导脐血单个核细胞分化成典型的DC;负载有白血病抗原的DC可诱导同一脐血的淋巴细胞生成白血病特异的杀伤性T细胞(CTL),所得CTL可特异性杀伤未经培养的白血病细胞而不严重伤害相同患者缓解期的骨髓细胞。     

树突细胞介导的独特型瘤苗的体外抗骨髓瘤作用

目的研究树突细胞（DC）介导的独特型瘤苗诱导骨髓瘤抗原特异性细胞毒性T淋巴细胞（CTL）的抗肿瘤免疫反应。方法从多发性骨髓瘤（MM）患者外周血中分离获取DC前体细胞，使用GM-CSF、IL-4与TNF-α诱导分化促成熟。加入MM患者的M蛋白F（ab′），片段（Id），诱导MM肿瘤特异性CTL。光学显微镜下观察其培养过程中形态特征变化，电镜观察其超微结构，间接免疫荧光观察其表型特征，采用MTT法检测致敏DC促自体T淋巴细胞增殖的能力以及患者CTL对自体骨髓瘤细胞的特异性细胞毒杀伤作用。结果 GM-CSF、IL-4和TNF-α配伍可有效地从MM患者外周血单陔细胞中诱导出大量成熟的功能性DC。MM患者自体血清Id冲击致敏的成熟DC能够显著地提高T细胞的增殖能力，并且使幼稚T细胞活化成为肿瘤独特型CTL，各个剂量的CTLs均能够产牛针对自体MM细胞的抑制性杀伤反应。结论 在MM患者外周血中可获得典型的DC，使用负载了Id的DC疫苗能够诱导出有效的抗肿瘤免疫应答反应，以DC为基础的疫苗可能在MM免疫治疗中发挥重要的作用。     

IL-13 can substitute for IL-4 in the generation of dendritic cells for the induction of cytotoxic T lymphocytes and gene therapy.

Immunization with tumor-associated antigen pulsed dendritic cells (DC) has been shown to elicit both protective and therapeutic antitumor immunity in a variety of animal models and is currently being investigated for the treatment of cancer patients in clinical trials. In this study we show that DC can be generated from peripheral blood mononuclear cells of healthy donors as well as breast and melanoma cancer patients using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13) and that these DC have many of the same characteristics as DC differentiated using GM-CSF and IL-4. The DC generated in GM-CSF and IL-13 are CD14- and express high levels of the cell surface markers CD86, HLA-DR, and CD58, as do DC generated in GM-CSF and IL-4. The purity and yield of both DC populations are not significantly different. Furthermore, both populations of DC are effective at presentation of alloantigen as determined in a mixed lymphocyte response, and both are able to process and present soluble tetanus toxoid antigen to CD4+ T cells. Because we are interested in the generation of DC for antigen-specific cytotoxic T lymphocyte (CTL) generation, we compared the ability of peptide-pulsed DC differentiated in GM-CSF and IL-4 versus GM-CSF and IL-13 for the generation of influenza and MART-1 specific CTL. Both populations of DC induced CD3+ CD8+ CD4- and CD56- CTL, which could lyse the appropriate targets in an antigen-specific manner. Finally, both GM-CSF and IL-4 DC and GM-CSF and IL-13 DC yielded similar beta galactosidase expression levels after transduction with recombinant adenovirus containing the LacZ gene. These results suggest that DC generated in GM-CSF and IL-13 may be useful for immunotherapy and gene therapy protocols.     

白血病细胞系来源的树突状细胞诱导及其抗肿瘤免疫功能研究

树突状细胞（DC）在抗肿瘤免疫反应中起关键作用，但大多数白血病病病人有DC功能缺陷，在外体扩增DC并增强其抗肿瘤免疫功能及以DC为基础的肿瘤疫苗是对白血病有效的免疫治疗方法，为了探讨由不同的髓性白血病细胞诱生DC的条件及其抗白血病反应，选用HL－60，K562和THP－1细胞与不同的细胞因子组合诱生DC，以光学和电子显微镜术观察形态特征，用流式细胞术和单克隆抗体检测细胞表型，以同种混合淋巴细胞反应观察刺激淋巴细胞增殖，用^51Cr释放法检测诱生细胞的细胞术和单克隆抗体检测细胞表型，以同种混合淋巴细胞反应观察刺激淋巴细胞增殖，用^51Cr释放法检测诱生细胞的细胞毒作用，用ELISA法测定DC培养及DC＋血单个核细胞培养的血清中IL－12及INF－γ的量，结果表明，由K562，HL－60和THP－1细胞诱生的DC具有村突状细胞的形太学特征，细胞表达DC的表面分化抗原，其中GM－CSF＋IL－4＋TNF-γ刺激HL－60－DC和THP－DC和GMCSF＋IL－4＋IL－12刺激的K562－DC中有抗原表达。3种细胞诱生的DC对混合淋巴细胞反应CTL反应有强刺激细胞因子能诱导髓性白细胞产生DC，不同细胞需要不同的细胞因子和培养条件，这些DC表达抗原呈递细胞的表型具有刺激T淋巴细胞增殖和诱导CTL反应以及分泌IL－2和促进T细胞分泌INF－γ的作用。     

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.     

Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage–colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product.
STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-γ (IFN-γ). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated.
RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-γ and soluble CD40L/IFN-γ were used.
CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.     

Reciprocal activating interaction between natural killer cells and dendritic cells

We analyzed the interaction between human peripheral blood natural killer (NK) cells and monocyte-derived immature dendritic cells (DC). Fresh NK cells were activated, as indicated by the induced expression of the CD69 antigen, and their cytolytic activity was strongly augmented by contact with lipopolysaccharide (LPS)-treated mature DC, or with immature DC in the presence of the maturation stimuli LPS, Mycobacterium tuberculosis or interferon (IFN)-alpha. Reciprocally, fresh NK cells cultured with immature DC in the presence of the maturation stimuli strongly enhanced DC maturation and interleukin (IL)-12 production. IL-2--activated NK cells directly induced maturation of DC and enhanced their ability to stimulate allogeneic naive CD4(+) T cells. The effects of NK cells were cell contact dependent, although the secretion of IFN-gamma and TNF also contributed to DC maturation. Within peripheral blood lymphocytes the reciprocal activating interaction with DC was restricted to NK cells, because the other lymphocyte subsets were neither induced to express CD69, nor induced to mature in contact with DC. These data demonstrated for the first time a bidirectional cross talk between NK cells and DC, in which NK cells activated by IL-2 or by mature DC induce DC maturation.     

急性髓系白血病患者树突状细胞诱导抗自身白血病T细胞免疫的实验研究

目的　探讨自体白血病细胞裂解物 (ACL)冲击的完全缓解期的急性髓系白血病 (AML CR)患者骨髓细胞衍生的树突状细胞 (DC)体外能否刺激自体T细胞产生特异性抗AML细胞的细胞毒活性。方法　应用羊红细胞玫瑰花结程序从AML CR患者的骨髓中分离出去T细胞的骨髓单个核细胞 (TD BMNC) ,并培养在含联合细胞因子 (GM CSF、IL 4、SCF或TNF α)的条件下以产生DC ,并在培养的第 5天用ACL进行冲击。培养 7d后收获细胞 ,用流式细胞仪测定其成熟DC表型。同时 ,这些细胞与经抗CD3抗体激活过的自体T细胞在低浓度IL 2条件下共培养 7d ,以产生细胞毒性T淋巴细胞(CTL)。用乳酸脱氢酶释放实验测定溶细胞活性。结果　 1 2例AML CR患者培养的骨髓单个核细胞均分化为成熟DC。其中 6例完成CTL活性实验。当效∶靶 =2 0∶1时 ,ACL冲击的DC致敏的自体T细胞与单纯IL 2或IL 2加无抗原冲击的DC组比较对自体AML细胞有明显的杀伤活性 ,而对K562细胞均无明显影响 (P <0 .0 1 )。结论　用AML CR患者白细胞裂解物冲击的骨髓细胞衍生的DC体外致敏自体T细胞可以产生AML细胞相关抗原特异性的CTL。     

Proinflammatory cytokines and CD40 ligand enhance cross-presentation and cross-priming capability of human dendritic cells internalizing apoptotic cancer cells

Human monocyte-derived dendritic cells (DC) can ingest apoptotic tumor cells (ATC) and present tumor-associated antigens (TAA) to T cells, leading to the generation of tumor-specific cytotoxic effector cells (Cancer Res 2000;60:3542-9). To further augment antitumor effector cell responses, attempts were made to modify antigen presentation and cross-priming of T cells by DC fed with ATC. Proinflammatory cytokines (PC), CD40 ligand (CD40L) and/or interferon-gamma (IFN-gamma) were found to markedly enhance the immunogenicity of TAA presented by DC. While PC upregulated expression of major histocompatibility complex class I/II and costimulatory molecules on the surface of DC, CD40L +/- IFN-gamma increased interleukin (IL)- 12 and to a lesser extent, IL-15 production by DC. Additionally, lactacystin, a specific proteasome inhibitor, significantly abrogated the effects of IFN-gamma and, in part, also those of CD40L or PC. The ability of DC + ATC to cross-prime TAA-inexperienced ("naive") T cells was significantly enhanced by PC and CD40L or CD40L + IFN-gamma, but not by IFN-gamma alone. These results indicate that future vaccines for patients with cancer incorporating DC fed with ATC could be made more effective by the addition of proinflammatory cytokines or CD40L +/- IFN-gamma to improve the DC function.     

Generation of tumor-associated cytotoxic T lymphocytes requires interleukin 4 from CD8(+) T cells.

Activation of tumor-associated CD8(+) cytotoxic T lymphocytes (CTLs) often requires antigen representation, e.g., by dendritic cells (DCs), and CD4(+) T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin 4 (IL-4) during the immunization but not effector phase. To determine the source and target cells of IL-4, we performed adoptive T cell transfers using CD4(+) and CD8(+) T cells from IL-4(-/-) and IL-4R(-/-) mice and analyzed CTL generation. Even though necessary for CTL generation, CD4(+) T cells did not need to express IL-4 or IL-4R. Surprisingly, CTL generation required IL-4 but not IL-4R expression by CD8(+) T cells. As IL-4 (a) was expressed by naive CD8(+) T cells within 24 h after antigen encounter, (b) IL-4 induced DC maturation, and (c) CTL development was impaired in T cell-reconstituted IL-4R(-/-) mice, CD8(+) T cell-derived IL-4 appears to act on DCs. We conclude that CD4(+) and CD8(+) T cells provide different signals for DC activation during CTL generation.