IL-12基因修饰树突状细胞体外诱导免疫杀伤肝癌细胞

目的探讨IL-12基因修饰树突状细胞(DC)体外诱导免疫杀伤肝癌细胞的效能及其机制。方法IL-12基因修饰肝癌病人外周血DC(DC-IL-12),ELISA法检测DC-IL-12培养上清中IL-12和IFN-γ水平,以冻融HepG2所获得的肿瘤相关抗原(TAA)刺激DC-IL-12,MTT法检测TAA负载的DC-IL-12刺激同源T淋巴细胞增殖分化能力,细胞毒试验检测DC-IL-12诱导的细胞毒T淋巴细胞(CTL)及其上清液对HepG2肝癌细胞株的杀伤作用,ELISA法检测CTL上清液IFN-γ水平。结果DC经IL-12基因修饰后48h分泌高水平IL-12(24·35±5·4)pg/ml及IFN-γ(725±30)pg/ml,均显著高于未转染DC组(P<0·01)。DC-IL-12诱导的CTL及其上清液对HepG2均有显著杀伤作用,杀伤率显著高于未转染DC组,分别为(82·43±8·7)%vs(57·4±4·3)%和(76·45±8·5)%vs(18·23±5·3)%(P<0·01),DC-IL-12诱导的CTL上清液IFN-γ水平显著高于未转染DC组,分别为(1125·0±32·7)pg/ml、(281·3±14·7)pg/ml(P<0·01)。结论IL-12基因修饰增强DC体外诱导自体T淋巴细胞产生特异性抗肝癌免疫,其机制与IL-12基因修饰促进DC分泌IL-12、增强T淋巴细胞活化及分泌IFN-γ密切相关。     

核因子-κB对小鼠树突状细胞分化成熟及其体外刺激T细胞免疫反应的影响

目的 探讨在小鼠树突状细胞(DC)成熟过程中核因子-κB(NF-κB)基因的作用。方法应用特异性NF-κB寡聚脱氧核苷酸诱骗剂(NF-κB ODN Decoys)阻断NF-κB活性，观察DC形态、细胞表面分子表达以及同种T淋巴细胞增殖反应的变化，了解NF-κB基因对DC成熟及免疫生物活性的影响。结果 NF-κB ODN Decoys的有效摄取，抑制了DC表面共刺激分子CD80、CD86、CD40的表达和白细胞介素-12(IL-12)的分泌，阻碍了DC的发育成熟，这种阻碍作用不可被脂多糖(LPS)所逆转。混合淋巴细胞反应显示，NF-κB ODN Decoys的应用可诱导DC刺激同种T淋巴细胞低反应活性，抑制了T细胞IL-2和γ-干扰素(IFN-γ)的分泌。结论 NF-κB是DC发育成熟过程中关键性调控基因。应用NF-κB ODN Decoys可抑制DC的成熟，从而为生成耐受原性未成熟DC、诱导移植免疫耐受提供了一种新的途径。     

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。     

转染IKK2dn的受者树突状细胞回输延长同种异体肾移植大鼠的存活时间

目的 探讨IKK2dn基因转染并负载供者抗原的受者未成熟树突状细胞(imDC)延长同种异体肾移植大鼠的存活时间及其机制.方法 获取和培养Lewis大鼠骨髓源性DC,转染IKK2dn并负载BN大鼠可溶性抗原进行体外实验,检测CD86和主要组织相容性复合物(MHC)Ⅱ的表达及DC刺激T淋巴细胞增殖的能力.肾移植受者为Lewis大鼠,用随即数字表法分DC组、空转染组、转染组、对照组,术前7d分别输注1×10~7个D、Adv-0-DC、负载BN抗原的Adv-IKK2dn-DC和等量生理盐水,供者均为BN大鼠.另设第三方供者组,术前处理同转染组,供者为Wistar大鼠.移植后检测各组受者T淋巴细胞的增殖能力及血清白细胞介素2(IL-2)和γ干扰素(IFN-γ)的表达水平,观察各组大鼠的存活时间和发生排斥反应情况.结果 DC的体外实验结果显示:与转染IKK2dn前相比,转染后DC仍能低水平表达CD86和MHC Ⅱ,负载供者抗原后CD86和MHCⅡ表达均增加,而转染IKK2dn后再负载供者抗原,CD86和MHC Ⅱ的表达未发生明显变化;DC负载供者抗原后,刺激T淋巴细胞增殖的能力明显增强(P<0.05),而转染IKK2dn并负载供者抗原后不能有效刺激T淋巴细胞增殖.肾移植术后的检测结果显示:转染组T淋巴细胞的增殖能力明显低于其他4组(P<0.05或P相似文献   

多发性骨髓瘤患者间充质干细胞的免疫原性及其对免疫功能的调控作用

目的 探讨多发性骨髓瘤(MM)患者的骨髓间充质干细胞(MSC)的免疫原性及其调控免疫的功能和机制.方法 获取MM患者的骨髓MSC,在低血清培养液中培养和扩增.流式细胞仪检测免疫表型;酶联免疫吸附试验检测MSC培养上清液中细胞因子的分泌水平;混合淋巴细胞反应检测骨髓MSC抑制异体T淋巴细胞增殖的能力;流式细胞仪检测MSC对树突状细胞(DC)吞噬功能的影响;混合淋巴细胞反应检测MM骨髓MSC抑制DC介导的异体T淋巴细胞增殖能力.结果 MM患者的MSC不表达HLA-DR和共刺激分子CD80、CD83、CD86和CD40.MM患者的MSC通过分泌转化生长因子β1 (TGF-β1)和肝细胞生长因子(HGF)抑制异体T淋巴细胞的增殖.MM患者的MSC具有抑制DC吞噬作用的功能;MM骨髓MSC抑制DC分泌白细胞介素12(IL-12),并可以抑制DC介导的异体T淋巴细胞增殖.结论 MM骨髓MSC具有低免疫原性及体外调节免疫的功能,该免疫调节功能与其分泌细胞因子有关.     

Rel-A siRNA对大鼠树突状细胞的影响

目的 探讨siRNA沉默Rel-A基因表达对大鼠树突状细胞(DC)生物学效应的影响.方法 采用细胞因子诱导培养法体外扩增大鼠骨髓DC,经免疫磁珠纯化获得高纯度DC后, 针对Rel-A基因,采用半定量RT-PCR及Western blot分别从mRNA和蛋白质水平检测Rel-A的表达情况,筛选一对高效RNA干扰(RNAi)行慢病毒载体包装并转染DC(RNA干扰组), 以未转染RNAi的DC作为对照组.分别给予1mg/L的脂多糖(LPS)刺激,透射电镜观察RNAi对LPS促DC成熟的影响,并利用流式细胞术(FCM)检测DC表面CD80,CD86及MHC Ⅱ分子的变化;混合淋巴细胞反应(MLR)法检测T细胞增殖能力;ELISA法检测DC分泌肿瘤坏死因子α(TNF-α)、干扰素γ( IFN-γ)和白介素12( IL-12)的浓度.结果 与空白对照组相比, 序列2组降低最明显,抑制率达90%,差异有统计学意义;透射电镜下可见RNAi Rel-A DC内多吞噬泡样结构;RNA干扰组CD80,CD86,MHC Ⅱ分子的表达、T细胞增殖能力及TNF-α,IFN-γ,IL-12的浓度与对照组相比均显著下降. 结论 RNA干扰技术能显著下调大鼠Rel-A基因的表达, Rel-A基因沉默DC的生物学效应表现为未成熟DC的特点.这种RNAi Rel-A DC可望作为一种新型的致耐受DC,而应用于免疫耐受的诱导研究.     

移植免疫耐受中Th1/Th2细胞因子对CD4~+T细胞功能的影响

目的:探讨移植免疫耐受中干扰素γ(IFN-γ)、白细胞介素10(IL-10)、白细胞介素4(IL-4)等Th1/Th2细胞因子对CD4+T细胞功能的影响。方法:取行心脏移植后的同种异系受体小鼠的脾脏,从中提取CD4+T细胞,测定其细胞增殖活性,并测定其IFN-γ、IL-10、IL-4等细胞因子的分泌水平。提取移植免疫耐受脾脏的CD4+T细胞进行体外细胞培养,使用抗IFN-γ单克隆抗体、抗IL-10受体单克隆抗、抗IL-4单克隆抗中和相应细胞因子以测定上述细胞因子对CD4+T细胞功能的影响。结果:移植免疫耐受受体的CD4+T细胞较发生排斥反应的CD4+T细胞具有更低的细胞增殖活性。移植免疫耐受受体的CD4+T细胞分泌更低水平的IFN-γ,并分泌更高水平的IL-10、IL-4。使用相应抗体中和IFN-γ、IL-10后,移植免疫耐受CD4+T细胞的细胞增殖活性明显升高;而中和IL-4后,移植免疫耐受CD4+T细胞的增殖活性反而降低。结论:移植免疫耐受中IFN-γ、IL-10对于维持CD4+T细胞的低增殖活性具有重要作用,而IL-4不但不能维持CD4+T细胞的低增殖活性,反而升高了增殖活性。     

雷帕霉素对大鼠髓源性树突状细胞表型和功能的影响

目的 观察雷帕霉素(RAPA)对树突状细胞(DC)表型和功能的影响.方法 诱导分化Lewis大鼠骨髓源性DC,第6天同时加入脂多糖(LPS)和10μg／L或100 μg/L RAPA,48 h后检测共刺激分子、细胞凋亡、上清液白细胞介素(IL) -10和IL-12水平;混合淋巴细胞培养观察DC刺激Brown Norway大鼠T淋巴细胞增殖能力.结果 RAPA能抑制DC成熟,共刺激分子的上调显著受抑,细胞凋亡增加,100 μg/L RAPA组DC凋亡率达53％.DC在脂多糖作用下分泌IL-10、IL-12上升至( 1533±117)、(2575 ±58) ng/L,但同时加入100μg/L RAPA后,两者下降至(330±159)、( 245±63) ng/L.RAPA处理的DC对T淋巴细胞增殖有抑制作用.结论 雷帕霉素能抑制DC成熟并诱导其凋亡,RAPA处理的DC分泌细胞因子降低,且抑制T淋巴细胞增殖.     

CD40配体刺激的树突细胞增强CEA转基因小鼠细胞免疫的实验研究

目的探讨CD40配体（CD40L）对CEA转基因小鼠中的树突细胞（Dc）的激活作用,并进一步评价其对DC诱导的细胞免疫的效能。方法使用CEA转基因小鼠的骨髓细胞,通过粒-巨噬细胞集落刺激因子（GM—CSF）和IL-4在体外的刺激,贴壁法定向诱导分化出未成熟DC,利用CD40L刺激DC成熟后．体外荷载CEA526-533抗原肽．使DC具备诱导特异性细胞免疫的能力。通过定量测定DC表面分子、DC分泌的细胞因子、淋巴细胞增殖试验等来研究DC诱导细胞免疫的有效性。结果实验组DC的IL-12分泌量为（937．81±51．99）pg／10^6 DC,明显高于对照组[（83．06±8．58）pg／10^6DC,P〈0．01]。实验组DC的促CD8＋T细胞及脾细胞增殖活性明显强于对照组（P〈0．05）。刺激CD8T细胞增殖时,实验组和对照组淋巴细胞的IFN-γ分泌量分别为（33．900±4．550）和（5．226±0．460）ng／L．差异具有统计学意义（P〈0．01）;刺激脾细胞增殖时。实验组和对照组淋巴细胞的IFN-1分泌量为（69．802±11．407）和（2．912±0．562）ng／L,差异同样具有统计学意义（P〈0．01）。结论在CEA转基因小鼠的动物模型中．体外使用CD40L刺激骨髓来源DC可以促进其分化成熟,该成熟DC能有效增强CEA转基因小鼠的细胞免疫。     

RNA干扰抑制大鼠T淋巴细胞CD28基因表达的实验研究

目的：探讨RNA干扰（RNAi）后对大鼠T淋巴细胞共刺激分子CD28基因的表达及其对细胞功能的影响。方法：设计针对目标基因的小干扰RNA（siRNA）,转染大鼠T淋巴细胞。以FCAS检测CD28水平,MTT检测转染后T淋巴细胞对异体淋巴细胞增殖能力的影响。Reahime-PCR及ELISA法检测细胞因子水平。结果：siRNA转染大鼠T淋巴细胞后可抑制CD28的表达,T细胞增殖能力、IL-2、IFN-γ平明显低于空白对照组（P〈O．05）。结论：siRNA可特异性抑制大鼠T淋巴细胞共刺激分子CD28的表达,降低大鼠T细胞的增殖能力,抑制IL-2、IFN-γ细胞因子的基因水平表达和分泌水平．从而产生免疫耐受效应,为器官移植免疫研究提供实验依据。     

黄芪多糖对分泌白细胞介素12树突细胞亚群的免疫调控效应与机制

目的 观察黄芪多糖(APS)对分泌IL-12树突细胞(DC)亚群CD11chighCD45RBlowDC功能的影响.方法磁珠分选技术获得BALB/c小鼠脾脏CD11chighCD45RBlowDC和CD4+T淋巴细胞.在CD11 chighCD45RBlowDC中加入不同浓度APS(50、100、200μg/mL)处理,以不加APS的细胞作为对照,应用ELISA法检测细胞培养上清液中IL-12水平,流式细胞仪检测细胞表面分子CD40、CD80、CD86、I-A/E及Toll样受体4(TLR4)的表达.将CD4+T淋巴细胞分为正常对照组(未行任何处理)、未刺激组(加入未经APS处理的CD11chighCD45RBlowDC与CD4+T淋巴细胞混合培养)、高浓度APS刺激组(加入经200μg/mL APS处理后的CD11chighCD45RBlowDC与CD4+T淋巴细胞混合培养)、高浓度APS刺激+抗体1组(加入经200μg/mL APS处理后的CD11chighCD45RBlowDC、IL-12抗体与CD4+T淋巴细胞混合培养)和高浓度APS刺激+抗体2组(加入经200 μg/mL APS处理后的CD11chighCD45RBlowDC、IL-12同型对照抗体与CD4+T淋巴细胞混合培养).采用噻唑蓝法测定CD4+T淋巴细胞增殖能力,流式细胞仪检测细胞培养液中IL-4和γ干扰素水平.对数据行多组间单因素方差分析.结果与未加APS刺激相比,3种浓度APS均显著增强CD11chighCD45RBlowDC表面分子CD40、CD80、I-A/E及TLR4表达及IL-12分泌,其中IL-12分泌呈APS浓度依赖性;CD86表达无明显变化.高浓度APS刺激组CD4+T淋巴细胞增殖能力高于未刺激组(F=13.438,P＜0.05);高浓度APS刺激组细胞γ干扰素水平为(2784±137)pg/mL,高于未刺激组[(1952±101)pg/mL,F=12.177,P＜0.05];高浓度APS刺激组细胞IL-4水平为(172±20)pg/mL,明显低于未刺激组[(193±19)pg/mL,F=11.963,P＜0.05].高浓度APS刺激+抗体1组前述3项指标表达水平较未刺激组明显改善,高浓度APS刺激+抗体2组前述3项指标表达水平与高浓度APS刺激组接近.结论 APS能够通过促进CD11chighCD45RBlowDC中IL-12的表达,诱导CD4+T淋巴细胞向Th1型反应分化,通过激活CD11chighCD45RBlowDC增强免疫活性.

Abstract：

Objective To investigate immunomodulatory effect of Astragalus polysaccharides (APS) on IL-12-secreting dendritic cell (DC) subset CD11chigh CD45RBlow DC. Methods Spleen CD11chighCD45RBlow DC and CD4 +T lymphocytes in BALB/c mice were purified by magnetic beads sorting,and were treated with 0 (as control), 50, 100, 200 μg/mL APS. Immunofluorescence staining and flow cytometry were used to determine expressions of CD11chighCD45RBlow DC surface molecules, including CD40,CD80, CD86, I-A/E, and Toll-like receptor (TLR) 4. IL-12 level in CD11chighCD45RBlow DC culture supernatant was determined by ELISA. The CD4+ T lymphocytes were divided into: normal control group,non-stimulation group ( CD4 + T lymphocytes cocultured with APS-unstimulated CD11 chigh CD45RBlow DC ) ,high-dose APS stimulation group (CD4+T lymphocytes cocultured with 200 μg/mL APS-stimulated CD11ch'ghCD45RBlow DC) , high-dose APS stimulation + antibody 1 group ( CD4 + T lymphocytes cocultured with 200 μg/mL APS-stimulated CD11chighCD45RBlow DC and IL-12 antibody), high-dose APS stimulation +antibody 2 group (CD4 +T lymphocytes cocultured with 200 μg/mL APS-stimulated CD11chigh CD45RBlow DC and IL-12 antibody isotype). Proliferation ability of CD4 + T lymphocytes was determined with MTT method.IL-4 level as well as IFN-γ level in CD4 + T lymphocyte culture supernatant was determined by flow cytometry. Data were processed with one-way analysis of variance. Results Compared with those in control, the expressions of CD 11 chigh CD45 RBlow DC surface molecules ( except for CD86 ) on CD 11 chigh CD45RBlow DC surface, as well as IL-12-secreting level with dose-dependence were increased in cells stimulated with 50,100, 200 μg/mL APS. Proliferation ability of CD4 +T lymphocytes in high-dose APS stimulation group was higher as compared with that in non-stimulation group ( F = 13. 438, P ＜0.05). IFN-γlevel in high-dose APS stimulation group [(2784 ± 137 ) pg/mL] was higher than that in non-stimulation group [(1952 ±101 ) pg/mL, F = 12. 177, P ＜0.05]. IL-4 level in high-dose APS stimulation group was (172 t 20) pg/mL,which was lower than that in non-stimulation group [( 193 ± 19) pg/mL, F = 11.963, P ＜0.05]. Proliferation ability of CD4+ T lymphocytes, IFN-γ level, and IL-4 level in high-dose APS stimulation + antibody 1 group were all ameliorated when compared with those in non-stimulation group; while levels of the 3 indexes in high-dose APS stimulation + antibody 2 group were similar to those in high-dose APS stimulation group.Conclusions APS can activate IL-12-producing CD11 chighCD45RBlowDC, and further induce the activation of immune function of T lymphocyte with shifting of Th2 to Th1 in vitro. APS can enhance the immune response via promoting the phenotypic and functional maturation of CD11 chighCD45RBlow DC.     

转存活素基因树突状细胞抗消化道肿瘤的免疫效应

目的研究转染存活素基因对树突状细胞(DC)免疫功能的影响,观察修饰后的DC在体外诱导的抗消化道肿瘤免疫效应。方法脂质体介导存活素基因转染入DC,用蛋白印迹法检测培养上清存活素的表达,检测转存活素基因DC分泌细胞因子白细胞介素12(IL12)、肿瘤坏死因子α(TNFα)的功能,以及经流式细胞仪检测DC表面CD1a、CD83、MHCⅡ、CD80、CD86表达的高低,用噻唑蓝(MTT)法诱导人特异的细胞毒性T淋巴细胞(CTL)的能力。结果培养上清中均可以检测到存活素表达;转存活素基因DC的上清IL12、TNFα含量分别为(2652±327)pg/ml和(4371±835)pg/ml,比单纯DC组高(P<005);CD1a、CD83、MHCⅡ、CD80、CD86等在单纯DC表面低表达,在转基因DC表面高表达;MTT法检测,经转染存活素基因的DC提呈的细胞对胃癌细胞、结肠癌细胞、胆管癌细胞杀伤率分别为65%、77%、85%,而单纯DC杀伤作用较低。结论存活素基因转染修饰的DC能诱导细胞毒性T淋巴细胞的特异性,显著地提高DC的抗原提呈功能,体外能诱导高效而特异的抗癌免疫效应。     

不同浓度γ干扰素诱导大鼠脾脏来源树突状细胞表达吲哚胺2,3-双加氧酶的变化及对T淋巴细胞增殖的影响

目的 观察不同浓度γ干扰素(IFN-γ)作用下大鼠脾脏来源的树突状细胞(DC)中吲哚胺2,3-双加氧酶(IDO) mRNA和蛋白表达的变化及IDO对同种异体T淋巴细胞增殖的影响.方法 应用细胞因子体外诱导培养大鼠脾脏来源DC,应用流式细胞仪测定大鼠DC特异性分子OX62 和表面分子CD80、CD86的表达.分别用不同浓度(0、100、300、500 U/ml)的IFN-γ诱导作用DC后,实时聚合酶链反应( Real-time PCR)测定DC中IDO mRNA的相对表达水平,Westemblot检测DC中IDO蛋白在DC中的表达水平.同种异体混合淋巴细胞反应(MLR)检测不同浓度IFN-γ诱导后的DC对同种异体T淋巴细胞增殖的影响.结果 体外诱导培养的DC光学显微镜下观察,具有典型的树枝状突起.0X62表达率达到80％以上,CD80、CD86的阳性表达也在80％左右;DC的IDOmRNA(2 -△△Ct值分别为:1.010 ±0.094、1.340±0.114、1.700±0.087、2.080±0.150)和蛋白的相对表达量(分别为:0.861 ±0.612、1.155±0.059、1.308±0.068、1.755±0.118)随着IFN-γ作用浓度的增大逐渐增大,分别为:不同浓度组间差异有统计学意义(P＜0.05);各组IFN-γ作用后DC与对照组比较,T淋巴细胞的增殖率显著降低(P＜0.05);且随着IFN-γ作用浓度的增大T淋巴细胞的增殖率逐渐降低,A值分别为:0.458±0.041、0.423±0.030、0.349±0.019、0.312±0.036,各组间差异有统计学意义(P＜0.05).结论 采用改良的培养黏附法体外获得纯度较高的大鼠脾脏来源的DC;IFN-γ可以诱导大鼠脾脏来源DC表面活性IDO的表达增加,减弱脾脏DC对同种T细胞增殖的刺激能力.     

树突状细胞在肾小管间质炎症病变中的作用及抗黏附干预调节的研究

目的 探讨树突状细胞(DC)在肾小管间质炎症损伤中的作用，以及抗P-选择素功能域单抗(PsL-EGFmAb)对DC浸润及体外成熟与功能的干预调节。 方法 (1)建立大鼠单侧输尿管梗阻(UUO)模型。分别采用免疫组化和免疫双染与图像分析，观察P-选择素及CD1a+CD80+DC在肾组织表达和分布变化。(2)从脐血CD34+造血干细胞中诱导扩增DC，并于成熟过程中采用流式细胞仪分析细胞表面分子表达；RT-PCR检测细胞NF-κB P50、P65 mRNA表达；混合淋巴细胞反应(MLR)检测DC对T细胞刺激能力；以及ELISA测定MLR上清液IL-12 p70分泌含量。 结果 (1)与假手术组比较，UUO大鼠从第1天起，随着P-选择素以肾小管上皮细胞为主的小管间质表达，CD1a+CD80+DC以肾间质为主浸润；至第7天P-选择素上调且CD1a+CD80+DC显著聚集，两者明显相关且与肾小管间质病变程度显著相关。经PsL-EGFmAb处理后，大鼠肾组织P-选择素表达下调，CD1a+CD80+DC浸润减少，且肾小管间质损害程度减轻。(2)经TNF-α刺激炎性状态下，培养人DC成熟过程中基本不表达或低表达P-选择素，但持续高表达与P-选择素同属C型凝集素的DC-SIGN。经PsL-EGFmAb处理后，可明显抑制DC-SIGN及细胞内NF-κB基因表达，并相应抑制DC黏附共刺激分子表达、IL-12分泌及刺激T细胞增殖能力。 结论 DC也是肾小管间质炎症病变启动因素，针对P-选择素功能域的单抗对其浸润具抑制作用。此外，该单抗对人DC成熟与功能有调节效应，提示与抑制作为DC模式识别及黏附受体的DC-SIGN有关，并可能通过影响NF-κB途径起作用。     

Sequential delivery of maturation stimuli increases human dendritic cell IL-12 production and enhances tumor antigen-specific immunogenicity

BACKGROUND: Despite the increasing use of dendritic cells (DCs) in clinical trials, questions regarding the optimal means of DC preparation, in particular how to achieve optimal maturation, remain unanswered. We hypothesized that delivering two separate sequential maturation signals to DC in vitro, mimicking the process of DC maturation that occurs in vivo, would enhance the ability of DCs to generate antigen-specific effector T cells in an experimental in vitro antimelanoma model. MATERIALS AND METHODS: Human monocyte-derived DCs were transfected with mRNA encoding melanoma-associated antigen Mart-1 (MART) or influenza M1 matrix protein (M1). After mRNA transfection, DCs were left untreated or exposed to different maturation stimuli either added simultaneously or delivered sequentially 18 h after first stimulation. Phenotypic DC cell-surface marker changes and IL-12 secretion were analyzed. Specific antigen presentation by DCs was measured by IFN-gamma release Elispot assay using a CD8(+) MART peptide-specific T cell clone. RNA-transfected and treated DCs were cultured with autologous naive T cells and the induction of antigen-specific effector T cells were assessed by IFN-gamma release Elispot assay. RESULTS: DCs transfected and matured had increased cell-surface expression of CD40 and costimulatory molecules CD80, and CD86. DCs matured and further treated by soluble CD40 ligand (sCD40L) had a 10- and 2-fold increase in MART antigen presentation compared to untreated (immature) DCs and DCs treated only with a first maturation signal, respectively (Elispot P = 0.02). Delivery of sequential maturation stimuli resulted in maximal DC IL-12 secretion compared to simultaneous stimuli. Last, generation of antigen-specific effector T cells more than doubled with the sequential addition of sCD40L to mature DC stimulators (Elispot P = 0.009). CONCLUSIONS: Maturation of DCs following mRNA transfection increases expression of cell-surface costimulatory molecules. Delivery of a second sequential maturation stimulus enhances antigen presentation, increases IL-12 secretion, and augments immunogenicity as evidenced by generation of tumor antigen-specific effector T cells. This strategy should be considered in the future development of RNA-based DC vaccine strategies for the treatment of cancer.     

乳腺癌细胞抗原负载的自体树突状细胞体内诱导特异性T细胞应答

目的 以乳腺癌细胞抗原体外负载自体树突状细胞(DCs)对24例乳腺癌患者进行自身免疫,探讨其体内诱导特异性T细胞免疫应答的能力.方法 新鲜癌组织制备成热休克凋亡细胞抗原负载外周单核细胞来源DC,分别在采血后第1、2、4、6周于患者腹股沟淋巴结富集区进行皮内注射,细胞剂量为4×10+～6×106/次.结果 DC免疫治疗后患者血清抗瘤免疫因子水平明显上升:白细胞介素(IL)-2治疗前为(33.8±7.2)ng/L,治疗后为(68.5±12.4)ng/L;IL-12治疗前为(48.5±10.9)ng/L,治疗后为(118.2±31.5)ng/L;肿瘤坏死因子(TNF)-α治疗前为(18.7±5.3)ng/L,治疗后为(78.3±11.5)ng/L;干扰素(IFN)-γ治疗前为(20.5±6.3)ng/L,治疗后为(92.6±14.9)ng/L,治疗前后比较差异有统计学意义(P<0.01);DTH试验阳性率为7/10;4/10例IFN-γ+CD8+T细胞频率较治疗前明显增加.随访发现:除1例患者在治疗后3个月内疾病进展(PD),其余患者病情稳定无复发与转移症状(SD).结论 以乳腺癌细胞为抗原负载自体DC免疫患者,能够有效提高患者非特异免疫水平,激发体内特异性T细胞免疫应答,是一种安全、副反应较小、有效的乳腺癌辅助治疗手段.     

慢性粒细胞白血病患者的骨髓间充质干细胞对树突状细胞功能的调控

目的 探讨慢性粒细胞白血病(CML)患者骨髓间充质干细胞(MSC)对单个核细胞来源的树突状细胞(DC)的功能的调控及其机制.方法 采用细胞贴壁法获取CML患者骨髓MSC,在低血清培养液中培养和扩增.获取正常成人志愿者外周血单个核细胞,直接在体外诱导生成DC(正常DC组),另加入正常志愿者MSC(正常诱导组)或CML患者骨髓MSC(CML诱导组)以诱导生成DC.用流式细胞仪检测各组收获DC的免疫表型及MSC对DC吞噬功能的影响;用酶联免疫吸附试验检测各组DC白细胞介素12(IL-12)的分泌;混合淋巴细胞反应检测MSC对DC介导的异体T淋巴细胞增殖能力的抑制作用.结果与正常DC组相比较,正常诱导组和MSC诱导组培养7 d后未成熟DC的共刺激分子CD1a、CD80、CD83、CD86、CD40和HLA-DR的表达量较低(P＜0.05);与正常DC组相比较,诱导9 d后,正常诱导组和MSC诱导组成熟DC的CD40、CD86和CD83的表达量较低(P＜0.05).与正常DC组相比较,CML诱导组的不成熟DC的吞噬功能显著降低(P＜0.05),CML诱导组DC IL-12的分泌量显著下降(P＜0.05).CML诱导组的DC对T淋巴细胞增殖的刺激作用有所下降(P＜0.05).结论 CML患者骨髓MSC对DC的分化成熟以及其免疫调控能力有一定的抑制作用.

Abstract：

Objective To study the effects and mechanisms of mesenthymal stem cells (MSCs)derived bone marrow of patients with chronic myelogenous leukemia (CML) on function of monocytederived dendritic cells in vitro. Methods Bone marrow mononuclear cells from CML patients were obtained and cultured. Peripheral blood mononuclear cells (PBMCs) derived from normal volunteers were isolated and cultured in DC differentiational condition. Moreover, PBMCs were co-cultured with CML bone marrow-derived MSCs (CML-MSC) or normal volunteers' bone marrow-derived MSC (normal-MSC) in DC differentiational condition. Immunophenotype and the endocytosis of monocytederived DCs were investigated by FACS. The level of IL-12 was evaluated by enzyme linked immunosorbant assay (ELISA). The immunoregulatory ability was detected by mixed lymphocyte culture assay. Results CML-MSCs or normal-MSC inhibited the up-regulation of CD1a,CD40,CD80,CD86,and HLA-DR during DC differentiation and reduced CD40,CD86,and CD83 expression during DC maturation. CML-MSCs inhibited the endocytosis of DCs and decreased their capacity to secret IL-12. CML-MSC could significantly suppress the function of DCs stimulating proliferation of T lymphocytes. Conclusion CML-derived MSCs harbored effect on the differentiation and maturation of DCs in vitro ; CML-MSC could inhibit the immunregulation of DCs.     

Generation of dendritic cells with regulatory properties

Dendritic cells (DCs) are professional antigen presenting cells with the ability to induce and regulate an immune response. DCs that capture and present antigen under noninflammatory conditions maintain an immature phenotype and acquire tolerogenic properties. These DCs generate regulatory T lymphocytes that potentiate tolerogenic responses. Here we developed a method for the generation of immature murine DCs able to process and present a specific antigen in a tolerogenic context. Immature DCs were prepared from bone marrow precursors after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of vitamin D₃ and characterized by their low expression of major histocompatibility complex class (MHC) II and CD86 molecules. Purified phagosomes containing either MHC II molecules or ovalbumin were used to deliver antigens to immature DCs. More than 80% of the DCs captured the phagosomes, while maintaining a low expression of maturation markers and showing basal levels of secretion of activating cytokines such as interleukin (IL)-2 and IL-12. Treatment of the immature DCs with lipopolysaccharides (LPS) increased IL-10 secretion, in agreement with their anti-inflammatory and immune regulatory properties. Cocultures of transgenic OT-II T lymphocytes with the immature DCs carrying OVA-phagosomes succeeded in generating a subpopulation of regulatory T lymphocytes characterized by the expression of CD4, CD25, CD62L, and Foxp3. Taken together, our results suggest that vitamin D₃ generates immune tolerance through the modulation of DC phenotype and could be useful to induce tolerance to allotransplants.     

High-avidity antitumor T-cell generation by toll receptor 8-primed, myeloid- derived dendritic cells is mediated by IL-12 production

BACKGROUND: High-level production of heterodimeric p70 interleukin (IL)-12 by myeloid-derived dendritic cells (DCs) requires 2 signals: interferon gamma (IFN-gamma) and a maturation signal provided by CD40 ligation (CD40L) or lipopolysaccharide (LPS). METHODS: In the current study we demonstrate that signaling through toll-like receptor (TLR) 8, but not TLR3, TLR2, or TLR4, provides a priming signal to myeloid-derived DC for high IL-12 p70 heterodimer production. RESULTS: All the TLR agonists induced maturation of DC as evidenced by increased expression of CD83, CD80, and CD86. Both IFN-gamma and TLR7/8 agonist R848 increased expression of TLR8 in immature monocyte-derived DCs. The combination of TLR7/8 agonist R848 and maturation signals LPS or CD40L induced high-level expression of IL-12p35 and p40 similar to that induced by IFN-gamma plus LPS. In contrast, receptor agonists specific for TLR7 did not prime for IL-12 production. The p70 IL-12 produced by the TLR8-primed DC polarized CD4+ T for Th1 cytokine production and induced CD8+ T cells, displaying high functional avidity with enhanced tumor cell recognition. CONCLUSIONS: The data suggest that toll 8 receptor agonists are useful for inducing type-1 polarized DCs for vaccine design in treating cancer and infectious disease.