肿瘤抗原联合CD40L致敏树突状细胞诱导CTLs杀伤K562细胞的实验研究

目的：以健康人外周血单核细胞为前体细胞，体外诱导为树突状细胞（DCs），负载K562细胞冻融抗原，并联合CD40L诱导产生特异性细胞毒性T淋巴细胞（CTLs）对K562细胞的杀伤作用。方法:密度梯度离心法、贴壁法分离健康人外周血单核细胞，应用rhGM-CSF、rhIL-4、rhTNF-α等细胞因子诱导扩增，培养DCs，用K562细胞冻融抗原联合rhsCD40L致敏DCs。实验分4组：K562细胞冻融抗原致敏DCs为实验组A，联合CD40L致敏DCs为实验组B，未致敏DCs为对照组A，单核细胞+异体淋巴细胞组为对照组B，观察CTLs对K562细胞的杀伤效应。结果:培养出具有典型特征的DCs，表达CD40最高达96%、CD86达97%、CD80为77%、CD1a为 69%，体外能诱导强烈的同种异体混合淋巴细胞增殖反应。在效靶比为 20∶1 时，实验组A对K562细胞的杀伤率为71.3%，实验组B为86.9%，对照组A为37.6%，对照组B为21.1%。实验组均显示高水平杀伤率，与对照组比较差异显著（P＜0.05），实验组B加CD40L杀伤率高于实验组A（P＜0.05） 。结论：K562细胞冻融抗原冲击致敏DCs能有效诱导T细胞特异性抗白血病作用，联合CD40L能增强其CTL的杀伤作用。     

SP2/0细胞膜抗原负载DC诱导体外特异性CTL效应

目的 研究SP2／0细胞膜抗原负载树突状细胞（DC）诱导的特异性细胞毒性T淋巴细胞（CTL）对肿瘤细胞的杀伤活性。方法 密度梯度离心法分离SP2／0细胞膜，负载经rmGM-CSF和rmIL-4诱导扩增的小鼠骨髓来源的DC，活化T淋巴细胞获得肿瘤特异性CTL，MTT法检测对SP2／0细胞的杀伤效果。结果 骨髓单个核细胞在rmGM-CSF和rmIL-4作用下获得了高表达CDS0、CD86和MHC-Ⅱ分子的DC，致敏的CTL对SP2／0骨髓瘤细胞具有高杀伤率，显著高于对I5178Y淋巴瘤细胞的杀伤活性（P〈0．01）。结论 SP2／0细胞膜抗原负载DC能诱导明显的特异性抗肿瘤免疫应答。     

自身骨髓瘤抗原致敏树突细胞介导特异性CTL反应的研究

目的：研究多发性骨髓瘤(MM)患者肿瘤冻融物致敏的树突细胞(DC)能否诱导特异性细胞毒T淋巴细胞(CTL)反应。方法：用MM患者骨髓CD34^ 细胞诱生DC。将MM患者骨髓瘤冻融物冲击致敏DC。MTT法检测骨髓瘤抗原致敏及非致敏DC诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞)的杀伤率。结果：骨髓瘤冻融物致敏DC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DC能有效诱导自身T细胞特异性抗瘤免疫。     

两种白血病细胞抗原负载DC的体外诱导特异性CTL应答的比较

目的: 比较树突状细胞 (DC)与白血病细胞融合及白血病细胞冻融抗原负载DC两种白血病DC疫苗诱导抗原特异性CTL应答的能力。方法: 采用羟乙基淀粉 Ficoll两步法分离外周血单个核细胞(PBMC), 通过贴壁 2h获得黏附的单核细胞, 用GM- CSF加IL- 4诱导培养 5d收获细胞。将细胞分为 4组: A组将K562细胞或原代慢性粒细胞白血病(CML)细胞在 500g/LPEG 100mL/LDMSO诱导下与DC融合; B组加入相应细胞数量的白血病细胞冻融抗原; C组将K562细胞或CML细胞与DC共培养; D组: 单独DC培养组。融合前以红色荧光染料PKH26标记K562细胞, 采用流式细胞仪(FCM)检测PKH26 /FITC 抗HLA ABC抗体双标细胞并评估融合率。培养的第 6天, 加入TNF -α诱导DC成熟, 然后分别与自体T细胞共培养, 用MTT比色法检测各组CTL对靶细胞的杀伤活性。结果: GM- CSF加IL -4和TNF- α依次诱导成熟的DC具有经典的DC的形态和表型特征, 在PEG -DMSO的介导下, DC与白血病细胞的融合率为 ( 17. 33 ~29. 94 )%。两种抗原负载方案激活的CTL均对表达K562抗原的细胞具有特异性的细胞毒作用。在效靶比相同时, 融合组诱导CTL的杀伤活性强于冻融抗原致敏DC组。结论: 与冻融抗原致敏的DC相比, DC与白血病细胞融合细胞递呈抗原的效率更高, 体外诱导的CTL特异性杀伤靶     

冻融抗原负载树突状细胞诱导细胞毒性T淋巴细胞特异性杀伤急性白血病细胞的实验研究

我们应用基因重组人白介素 4 (rhIL 4 ) ,基因重组人粒 /单细胞集落刺激因子 (rhGM CSF)联合培养体系自健康志愿者骨髓单个核细胞诱导产生DC ,使其负载急性髓系白血病 (AML)细胞冻融抗原 ,体外诱导细胞毒性T细胞 (CTL) ,观察负载AML细胞冻融抗原后DC的状态对所激发的T淋巴细胞表型及功能的影响及CTL对AML细胞特异性杀伤活性。我们发现负载AML冻融抗原后DC的CD1a、CD83、CD86、CD11C、HLA DR表达率为较培养前明显增高 (P <0 .0 1) ,且负载AML冻融抗原的DC诱导的CTL中CD3 CD8 T细胞比例 ,较诱导前明显增高 (P <0 .0 1) ,而负载AML细胞冻融抗原的DC诱导CTL对AML细胞有较强的杀伤作用 ,明显强于加或不加IL 2培养的T细胞对照组 (P <0 .0 1) ,而对K5 6 2细胞无明显的杀伤活性。通过以上实验我们认为经rhGM CSF ,rhIL 4培养产生的DC为CD14CD1a DC ,能诱导CTL对AML细胞产生明显的特异性杀伤作用 ,另外 ,负载AML细胞冻融抗原DC诱导的CTL中CD3 CD8 T细胞比例明显增高 ,提示CD8 T细胞在抗肿瘤免疫中具有极其重要的作用。     

树突状细胞激活的肿瘤浸润淋巴细胞对荷瘤小鼠免疫功能影响的研究

目的:探讨H22细胞全细胞性抗原致敏的DC激活的TIL体外抗小鼠肝癌活性;并将H22细胞全细胞性抗原致敏的DC激活的TIL(H22-DC-TIL)过继免疫荷瘤小鼠,研究其对荷瘤小鼠免疫功能的影响。方法:从小鼠四肢长骨骨髓中获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤全细胞性抗原致敏DC,然后用DC激活TIL,观察TIL在体外对H22细胞、Hepal-6细胞和B16细胞的杀伤活性;检测应用H22-DC-TIL后荷瘤小鼠的脾淋巴细胞的NK、LAK、CTL活性及血清TNF活性,并与对照组相比较。结果:(1)H22-DC-TIL具有很强的对H22细胞杀伤活性(杀伤率为71.31%±3.11%),明显高于其对Hepal-6和B16细胞的杀伤活性(杀伤率分别为50.11%±3.03%和30.31%±2.89%);也明显高于未经DC激活的TIL、H22-DC-小鼠脾淋巴细胞和未经DC激活的小鼠脾淋巴细胞对H22细胞杀伤活性(杀伤率分别为49.80%±3.21%、48.76%±3.60%和19.23%±2.71%)和对Hepal-6细胞杀伤活性(杀伤率分别为39.40%±3.21%、38.62%±2.87%和18.73%±2.40%)以及对B16细胞杀伤活性(杀伤率分别为26.38%±2.51%、25.82%±2.70%和18.34%±3.01%),同时B16-DC-TIL(TIL来源于H22瘤体)也可诱导相对较低的对B16细胞的特异性细胞杀伤活性。(2)H22-DC-TIL可明显诱导提高荷瘤小鼠脾淋巴细胞NK、LAK和CTL活性(活性分别为30.43%±1.35%、31.40%±1.80%和35.30%±1.20%),并可检测到血清TNF水平明显上升[血清TNF为(40.41±1.85)U/m l],它们均达正常对照组水平,与未经DC激活的TIL组、H22-DC-小鼠脾淋巴细胞组、未经DC激活的小鼠脾淋巴细胞组、生理盐水组分别对应比较,差异均有显著性(P<0.01)。结论:(1)H22-DC-TIL可产生很强的体外针对H22细胞的特异性杀伤活性。(2)H22-DC-TIL可明显诱导提高荷瘤小鼠特异性抗肿瘤免疫反应。     

不同大鼠胶质瘤抗原致敏的DC体外诱导CTL活性的研究

目的: 比较大鼠骨髓来源的树突状细胞(dendritic cells, DC)体外经由大鼠C6胶质瘤细胞由不同方式制备的不同抗原致敏后,对特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)的诱导作用。方法: 自大鼠骨髓分离DC前体细胞,经重组大鼠粒细胞巨噬细胞集落刺激因子(rrGM-CSF)+白细胞介素4(rrIL-4)诱导培养、扩增;由C6胶质瘤细胞经由反复冻融、煮沸灭活及超声破碎细胞抽提其总蛋白的方法制备各种不同抗原致敏DC,致敏的DC与T淋巴细胞进行共培养诱导CTL;以ELISA法检测CTL诱导过程中淋巴细胞趋化因子(lymphocyte chemoattractant factor)及细胞因子IFN-γ分泌水平:以 -TdR掺入法检测DC诱导T细胞增殖及其特异性CTL杀伤活性。 结果: 体外应用煮沸灭活瘤细胞制备的肿瘤抗原致敏DC,能诱导更强的刺激T细胞增殖的能力、并且可以诱导杀伤活性更强的CTL。结论: 应用煮沸灭活的瘤细胞制备瘤抗原负载DC获得瘤苗可获得更强的抗肿瘤保护作用。     

慢性粒细胞白血病患者DC对细胞因子诱导的杀伤细胞细胞毒作用的影响

目的 观察慢性粒细胞白血病（CGL）患者树突状细胞（DC）对自身细胞因子诱导的杀伤细胞（CIK）细胞毒的影响。方法 从CGL患者和正常人末梢血中分离单个核细胞（PBMC），用细胞因子（rhGM-CSF,rhIL-4和rHTNF-α)联合定向诱导培养DC。用CGL细胞抗原致敏DC，再将致敏的DC与CIK共同培养后，检测CIK对不同靶细胞的杀伤作用。结果 CGL患者和正常人的PBMC经细胞因子联合诱导培养后，DC占20．78％－56．0％。CGL患者的CIK和经CGL细胞抗原致敏的DC作用后的CIK，对自身白血病细胞的杀伤活性分别为56．0％和83．4％；正常人对照组则分别为30％和62％。经CGL患者CGL细胞抗原致敏的DC作用后的CIK, K562和SGC－7901的杀伤活性较未致敏的CIK活性低，而正常人对照组则无此现象。结论 从CGL患者的PBMC中能定向诱导扩增出DC。经CGL细胞抗原致敏的DC，能明显增强CIK对自身CGL细胞的杀伤活性。     

树突状细胞体外诱导抗肿瘤血管内皮细胞特异性免疫的研究

目的:探讨肿瘤内皮细胞抗原负载的树突状细胞(DC)诱导的细胞毒性T淋巴细胞(CTL)的特异性杀伤效应。方法:采用肿瘤细胞的培养上清诱导人脐静脉血管内皮细胞(HUVEC)增殖,制备肿瘤血管衍生的内皮细胞(TdEC)。用RTPCR检测肿瘤内皮标志物(TEM)的表达。制备TdEC的冻融抗原,负载从外周血中扩增的DC,用MTS比色法检测DC刺激自体淋巴细胞增殖的效应;用LDH法检测DC诱导的CTL的特异性杀伤效应。结果:TdEC可表达TEM1和TEM8。负载TdEC抗原的DC,可显著刺激自体淋巴细胞增殖。由其诱导的CTL对TdEC具有特异性的杀伤作用。在效靶比为20∶1和10∶1时,杀伤率分别为33%和27%,高于对照组的14%和10%。结论:TdEC抗原负载的DC,在体外可有效地诱导CTL产生,并特异性地杀伤TdEC。     

树突状细胞负载肝癌抗原肽疫苗体外诱导特异性免疫学反应的研究

目的：研究树突状细胞(dendritic cell，DC)负载肝癌抗原肽EPVTKAEML体外诱导特异性CTL的能力及其抑癌效果。方法：用顺序特异引物聚合酶链反应技术(PCR—SSP)选择HLA—B7表型供者，从脾组织中分离、培养DC-EPVTKAEML特异性CTL。用^51Cr释放法检测CTL的杀伤活性，并用抗HLA-1分子单抗(mAb)进行杀伤抑制实验。结果：找到4例HLA-B7杂合子供者，用DC负载HLA-B7限制的抗原肽EPVTKAEML可诱导特异性CTL反应，对肝癌细胞HHCC有较强的杀伤作用。结论：DC负载抗原肽EPVTKAEML在体外可诱发较强的特异性免疫反应。     

凋亡肿瘤细胞负载的树突状细胞对抗原特异性CTL的激活

为探讨凋亡Daudi细胞负载的树突状细胞 (DC )激发诱导肿瘤抗原特异性CTL及其生物学特性 ,采用常规方法从人外周血单个核细胞 (PBMC )诱导DC ,激发型CD40mAb联合 50Gyγ射线辐照诱导Daudi凋亡后负载DC ,然后与自体T细胞共育 ,并联合IL 2激发诱导肿瘤特异性CTL ;采用免疫荧光标记和流式细胞仪测定分析膜分子的表达 ;ELISA检测细胞因子的产生 ;Dextran FITC内吞实验分析DC的抗原摄取能力 ;3H掺入试验和51 Cr释放试验分别测定CTL的增殖和细胞毒效应。结果 :(1 )细胞因子序贯体外诱导 7d的DC ,对Dextran吞噬能力最强 ;(2 )CD40mAb诱导联合γ射线辐照 ,能有效介导Daudi细胞的凋亡 ;(3 )抗原负载联合CD40mAb激发可使DC上调表达CD1a、CD80、CD86和HLA DR ,并能促进IL 1 2的分泌 ;(4)凋亡Daudi负载后的DC在激发型CD40mAb作用下 ,能激发和扩增对Daudi细胞具有高效和特异杀伤作用的细胞毒性T细胞。因而认为凋亡肿瘤细胞负载联合CD40mAb刺激的DC可有效激活和扩增肿瘤特异性CTL。     

K562细胞总RNA转染对人树突状细胞抗原提呈、成熟及功能影响的初步研究

探讨人的树突状细胞(dendriticcell,DC)体外经K562细胞株总RNA转染后,能否诱导出p210蛋白表达,并诱导特异性CTL,为DC疫苗的临床应用提供理论基础。自健康人浓缩白细胞制剂分离有黏附特性的单核细胞,经GM-CSF、IL-4培养5d后,获得未成熟的DC(imDC);体外以脂质体DOTAP或直接电穿孔转染K562细胞总RNA。抽提后即时以及转染24h后的DC内RNA进行RT-PCR检测,Westernblot分析p210蛋白表达,流式细胞仪检测,以及诱导CTL杀伤K562细胞功能检测等。结果显示,RT-PCR检测表明:DC经K562细胞总RNA转染后,细胞内出现Bcr-Abl的cDNA条带,24h后消失。Westernblot实验表明:转染24h后,开始表达p210特征性蛋白。与对照组相比,转染K562细胞总RNA的DC,在24h后CD80、CD83、CD86、HLA-DR等表面标志均不同程度表达增高,并可促进T细胞对K562的杀伤活性。该研究从多方面角度说明应用肿瘤总RNA负载DC来制备DC疫苗的可行性,提示改良的DC疫苗抗肿瘤可能的应用前景。     

Immunological characterization of respiratory syncytial virus N protein epitopes recognized by human cytotoxic T lymphocytes

Virus-specific cytotoxic T lymphocytes (CTLs) are crucial for the control of respiratory syncytial virus (RSV) infection. This study has identified CTL epitopes of the RSV N protein in healthy subjects. We screened the primary structure of the N protein for HLA-A 0201-binding amino acid consensus motifs, identifying three peptides designated as N-RSV1, N-RSV2, and N-RSV3. These peptides were used to generate CTL lines by stimulating human HLA-A 02.01 peripheral blood mononuclear cells (PBMCs) in vitro. These CTL lines were then characterized by performing CTL chromium release assays and IFN-gamma secretion detection by intracellular cytokine staining. N-RSV1 and N-RSV3 peptides elicited the strongest cytolytic activity against RSV-infected cells and they could be useful epitopes for the analysis of CTL responses to RSV and for understanding immune-induced disease pathogenesis.     

Monocyte inflammatory protein-1 alpha facilitates priming of CD8(+) T cell responses to exogenous viral antigen

Dendritic cells (DC) derived from bone marrow precursors of BALB/c or C57BL/6 mice in low-serum cultures supplemented with granulocyte macrophage colony stimulating factor and Flt(3) ligand were pulsed in vitro with hepatitis B surface antigen (HBsAg) particles. DC processed exogenous HBsAg and presented its MHC class I-binding epitopes to cytotoxic T lymphocytes (CTL). This specific and restricted interaction of DC with CTL stimulated release of IFN-gamma and macrophage inflammatory protein (MIP)-1 alpha from the responding CTL. MIP-1 alpha enhanced the survival of DC in vitro but did not induce proliferation. Furthermore, co-delivery of MIP-1 alpha facilitated CTL priming in vivo to exogenous HBsAg in low responder C57BL/6 (H-2(b)) mice: a single injection of a low dose of HBsAg particles (without further adjuvants) successfully primed K(b)-restricted CTL responses to HBsAg only when the exogenous antigen was co-delivered with 100 ng MIP-1 alpha. These in vitro and in vivo data point to an important role of MIP-1 alpha in the DC-dependent priming of CTL to exogenous viral antigens.     

Bovine lymphocyte antigen-A11--specific peptide motif as a means to identify cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1.

Major histocompatibility complex (MHC) class I molecules present 8- to 10-mer viral peptides to antiviral cytotoxic T lymphocytes (CTLs). Identification of the allele-specific peptide motifs (ASPMs) of class I molecules enables the prediction of potential CTL epitopes of a virus from its protein sequences. Based on the bovine herpesvirus 1 (BHV-1) protein sequences that conform to the BoLA-A11 ASPM that we identified previously, potential CTL epitopes of BHV-1 were synthesized for use in cytotoxicity assays with CTLs from BHV-1-immunized calves. A peptide binding assay used to select the peptides that are most likely to be CTL epitopes categorized the peptides into groups of high, intermediate, and low binding capacity. Synthetic peptides stimulated lymphocytes from BHV-1-immunized calves to secrete interferon-gamma. Groups of peptides from the major glycoproteins of BHV-1 restimulated CTLs in vitro and sensitized targets for lysis by means of restimulated bulk CTLs.     

高三尖杉酯碱联合IFNα-2b可促进白血病源性树突状细胞成熟

目的： 研究高三尖杉酯碱（HHT）、干扰素α-2b（IFNα-2b）和两者联合（HHT+IFNα-2b）对白血病源性树突状细胞（DCs）的促成熟作用。方法: 在白血病多药耐药细胞K562/A02中加入细胞因子rhGM-CSF和rhIL-4,7 d后Wright-Gimsa染色观察细胞形态的变化,流式细胞术检测细胞免疫表型的变化,同种混合淋巴细胞反应、CTL杀伤效应、细胞因子IL-12的分泌来评价此白血病源性DC的成熟程度,然后在上述白血病源性DC中分别加入HHT、IFNα-2b和HHT+ IFNα-2b,3 d后以上述同样方法检测它们对此白血病源性DC的促成熟作用。结果: K562/A02细胞在rhGM-CSF和rhIL-4作用7 d后,CD83、HLA-DR和CD86分子的表达分别为（65.50±8.40）％、（32.00±4.32）％和（18.65±3.20）％,经HHT作用后这3种分子的表达升高到（85.36±8.42）％、（39.58±7.68）％和（35.53±4.35）％;经IFNα-2b作用后升高到（87.15±7.59）％、（40.53±6.30）％和（38.45±6.42）％;经HHT+ IFNα-2b联合作用后升高到（94.38±6.59）％、（52.75±8.51）％和（42.98±9.87）％,与作用前相比差异均显著。经HHT、IFNα-2b和HHT+ IFNα-2b作用后具有典型DC形态的细胞增多。经HHT和 IFNα-2b作用的DC均可刺激T细胞产生较强的增殖效应,诱导产生的CTL对靶细胞K562/A02的杀伤率明显增强且当效靶比为20∶1时杀伤效率最强,IL-12分泌明显增高。结论: HHT和IFNα-2b对白血病源性DC有促成熟作用,且两者联用时作用最强。     

A simple method for the quantitation of specific cytotoxic precursors to synrgeneic tumors

Cytotoxic T lymphocytes (CTLs) to EL4, a syngeneic lymphocytic leukemia of C57BL/6 (B6) (H-2^b) mice, were obtained by culturing normal B6 spleen cells with irradiated EL4 tumor cells for 4 days in conical bottom mitrotiter trays. A much lower, though significant, amount of cytotoxicity towards EL4 was observed in B6 spleen cell cultures not stimulated with EL4. The cytotoxic effector cells were shown to be Thy-1⁺ and their cytolytic activity to EL4 tumor cells was inhibited by an anti-H-2^b serum. Cold target competition studies suggest that the B6 anti-EL4 CTLs could discriminate between EL4 tumor cells and a second H-2^b lymphoma, C1498. The converse experiment yielded similar results. Culture conditions limiting for CTL precursors (CLPs) to EL4 were attained by including interleukin 2 (IL2), a lymphokine obtained by stimulating murine or rat spleen cells with concanavalin A, in the culture medium. In the presence of IL2, the CLP frequencies in B6 spleen cells to EL4 and C1498 in antigen-stimulated cultures were 113 and 231 per 10⁶ responder cells, respectively. In non-antigen stimulated B6 spleen cell cultures, the apparent CLP frequencies to EL4 and C1498 were 6 and 33 per 10⁶ responder cells, respectively. In agreement with the cold target competition studies using CTLs generated in the absence of IL2, we found that the CTL clones produced in cultures stimulated with EL4 and C1498 in the presence of IL2 were specific for the stimulating antigen. The specificity of the CTL clones obtained from cultures stimulated with IL2 in the absence of antigen appears to be different from those derived from antigen-stimulated cultures. The potential applications and limitations of this culture system are discussed.