体内致敏的树突状细胞诱导特异性抗肿瘤免疫的基础研究

目的证实树突状细胞（dendritic cells,DC）可在体内通过吞噬凋亡肿瘤细胞获取抗原物质,探讨其在肿瘤免疫治疗中的意义.方法以615小鼠的前胃癌细胞株造模,在体外用rmGM-CSF和rmIL-4从荷瘤小鼠骨髓细胞分化、诱导未成熟树突状细胞.分为4组：小剂量化疗组、树突状细胞组、小剂量化疗＋树突状细胞组和对照组,以BAX试剂盒检测肿瘤细胞凋亡.在瘤体内注射树突状细胞,观察给药侧瘤体及对侧瘤体体积,生存期,和特异性细胞毒性T淋巴细胞（CTLs）对肿瘤细胞的特异性杀伤作用.结果小剂量化疗能诱导肿瘤细胞凋亡.小剂量化疗后瘤内应用树突状细胞,给药侧瘤体及对侧瘤体体积明显缩小（P＜0.05）,小鼠的生存率提高,体内凋亡肿瘤细胞致敏的DC诱导的CTL对MFC有显著的杀伤作用,在效靶比为40：1、20：1、10：1和5：1时72 h的杀伤率分别为87.64%、70.32%、34.63%和13.87%.并能特异性杀伤小鼠前胃癌细胞MFC（P＜0.01）.结论体外诱导分化的未成熟DC,能于体内捕获小剂量化疗诱导的凋亡肿瘤细胞所携带的肿瘤抗原,诱导机体特异性抗肿瘤免疫反应.     

p53转染黑色素瘤细胞修饰的树突状细胞疫苗体外抗肿瘤作用的研究

利用野生型p53质粒转染黑色素瘤B16细胞,反复冻融法提取p53修饰的肿瘤抗原(p53-Ag),将抗原体外冲击同基因小鼠骨髓来源的树突状细胞(dendritic cells,DC)制备特异性DC肿瘤疫苗;观察DC诱导的淋巴细胞增殖反应和细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)对黑色素瘤细胞的细胞毒效应,分析其诱导肿瘤抗原特异性免疫应答的机制。结果显示,p53-肿瘤抗原冲击的DC可显著刺激淋巴细胞增殖,其诱导的CTL效应对肿瘤细胞也有很好的杀伤效果。     

PEG10基因转染的树突状细胞疫苗对肝癌细胞的杀伤实验研究

目的:研究印记基因PEG10修饰的树突状细胞(DC)疫苗对肝癌细胞的杀伤效应,为肝癌的治疗提供新的策略。方法:将重组PEG10腺病毒rAd-PEG10感染HLA-A2阳性的人外周血来源的DC,制备PEG10基因修饰的DC疫苗,并在体外刺激HLA-A2阳性限制性的单个核细胞,酶联免疫斑点试验(Enzyme Linked Immunospot Assay, ELISPOT)和标准⁵¹Cr释放试验分别检测PEG10腺病毒感染的DC所诱导的特异性CTL活性,并检测对HLA-A2阳性的HepG2肝癌细胞的杀伤作用。结果:成功制备了PEG10基因修饰的树突状细胞(DC)疫苗,并在体外能有效诱导抗原特异性CTL效应,对HepG2肝癌细胞有明显的杀伤毒性。结论:PEG10基因修饰的树突状细胞能有效激发出特异性CTL应答,并对HepG2肝癌细胞有明显的杀伤毒性,为肝癌治疗提供了新思路。     

热处理肿瘤细胞抗原负载树突细胞抗小鼠结肠癌效应

研究了热处理肿瘤细胞抗原负载骨髓来源的树突细胞（dendritic cell,DC）对结肠癌小鼠的治疗作用.将小鼠结肠癌细胞CT26热处理后超声破膜,以其细胞裂解液负载BALB/c小鼠骨髓来源的DC,观察DC诱导肿瘤特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）杀伤活性;并将DC接种于荷瘤小鼠皮下,观察其对肿瘤生长的抑制作用及对荷瘤小鼠生存期的影响.发现致敏DC诱导的CTL对CT26肿瘤细胞具有显著的杀伤作用.用致敏DC免疫小鼠后,对小鼠肿瘤的生长具有显著的抑制作用,并能显著延长荷瘤小鼠的生存时间.热处理肿瘤细胞抗原负载的树突细胞对结肠癌小鼠具有显著的治疗效果.     

CTP-BCR-ABL抗原肽负载树突状细胞体外致敏T淋巴细胞靶向杀伤CML细胞

BCR-ABL为慢性髓细胞白血病特异胞质抗原,为良好的免疫治疗靶标。该研究选择BCR-ABL融合位点的两段抗原肽SSKALQRPV(SS)、GFKQSSKAL(GF)为靶点,与胞质转导肽融合表达,负载小鼠骨髓源性树突状细胞。在胞质转导肽介导下,SS、GF短肽进入树突状细胞并定位于内质网,具备了被树突状细胞识别为内源性抗原并以MHC I类分子递呈的条件。在体外培养中,用致敏的树突状细胞刺激脾脏CD8⁺T淋巴细胞,获得针对CML的细胞毒性T淋巴细胞,同时检测该细胞毒性T淋巴细胞体外抗CML的效应。结果证实,胞质转导肽介导的GF抗原短肽负载的树突状细胞能够诱导CD8⁺T淋巴细胞增殖活化并产生针对CML的细胞毒性杀伤效应。因此,GF抗原肽有望作为CML免疫治疗的靶点。该研究为鉴定出靶向CML细胞的T淋巴细胞表面的特异TCR序列准备了条件,进而为后续制备靶向CML的TCR-T细胞奠定了基础。     

阿霉素处理的肿瘤抗原致敏树突状细胞的抗肿瘤免疫研究

研究表明化疗药物作用于肿瘤细胞后可有效激发免疫应答,这与肿瘤细胞的性质和化疗药物有关。该研究主要探讨阿霉素（adriamycin,ADM）处理小鼠宫颈癌u14细胞获得的肿瘤抗原致敏树突状细胞（dendriticceils,DCs）的免疫应答及对肿瘤的杀伤效应。分别应用ADM和反复冻融法处理小鼠宫颈癌U14细胞,取其离心上清液,致敏小鼠骨髓来源的DC,观察DC诱导的淋巴细胞增殖反应和细胞毒性T淋巴细胞（cytotoxicTlymphocyte,CTL）对宫颈癌细胞的细胞毒效应。结果显示：ADM处理的U14细胞抗原致敏后的DC组所激发和扩增的T细胞数及对宫颈癌细胞的杀伤效果显著高于对照组（P〈0．05）。因而提示ADM处理的肿瘤抗原能有效地致敏DC并产生抗肿瘤免疫效应。     

Hs-exosomes致敏树突状细胞介导的抗卵巢癌免疫治疗的实验研究

目的检测卵巢癌细胞的外排小体Hs-exosomes致敏树突状细胞DC诱导T淋巴细胞CTL特异性杀伤卵巢癌细胞的能力。方法:分离卵巢癌细胞株2780释放的Hs-exosomes,将其致敏DC并与T淋巴细胞共培养,检测CTL体外细胞毒活性。结果:Hs-exosomes致敏DC激活CTL的抗肿瘤能力显著高于未致敏DC组。结论:2780细胞释放的Hs-exosomes致敏DC激活T淋巴细胞对肿瘤具有很强的杀伤作用。     

重组人p53腺病毒转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应

目的：研究重组人p53腺病毒转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应。方法：采集本科室初诊的弥漫性大B细胞淋巴瘤（DLBCL）肿大淋巴结分离单个核细胞（MNC）进行体外DC的诱导培养,分为实验组A（rAd-p53-DC）、对照组A（rAd-DC）、空白对照组A（N-DC）,同时采集患者的外周血分离单个核细胞（MNC）,进行体外DC的诱导培养,分为实验组B（rAd-p53-DC）、对照组B（rAd-DC）、空白对照组B（N-DC）,用重组人p53腺病毒（rAd-p53）转染2种来源的DCS,流式检测DCS免疫表型,用Western-blotting鉴定P53蛋白的表达,ELISA法检测上清中的细胞因子IL-12的含量,混合淋巴细胞反应（mixed lymphocyte reac-tion,MLR）测定DCS刺激同种异体淋巴细胞增殖能力,用乳酸脱氢酶（lactate dehydrogenase,LDH）释放法检测经rAd-p53转染的两种来源DCS的细胞毒性T淋巴细胞反应（CTL）。结果：DC的表型（CD1a除外）CD83、CD80、CD86和HLA-DR实验组均较对照组及空白对照组明显增高（p〈0.05）。Western-blotting可检测到实验组P53蛋白的表达。上清液中IL-12分泌水平实验组均较对照组及空白对照组明显增高（p〈0.05）。实验组具有明显的刺激自体淋巴细胞增殖的能力,且刺激能力随rAd-p53-DC与淋巴细胞比例的增加而升高。对照组及空白对照组也能刺激同种自体淋巴细胞增殖的能力,但较实验组差（p〈0.05）。对靶细胞的细胞毒作用（CTL效应）结果显示,实验组介导同种异体的淋巴细胞杀伤率显著高于对照组及空白对照组（P〈0.05）。且实验组A的CTL效应明显高于实验组B,两者之间有显著性差异（P〈0.05）。结论：rAd-p53-DC为基础的肿瘤疫苗有可能在解决淋巴瘤的MRD、DC免疫耐受等问题上发挥强大的治疗作用。     

生物肽-蛋氨酸脑啡肽增强树突状细胞抗肿瘤活性研究

采用反复冻融法制备Rac-1淋巴瘤细胞总抗原,与树突状细胞（DC2.4）共培养制备DC瘤苗;设置不同实验组,M组及RM组加入蛋氨酸脑啡肽（MENK）,研究MENK对DC瘤苗抗肿瘤细胞的杀伤活性影响。结果表明：MENK作用于DC瘤苗后,DC2.4形态上更趋向于成熟,且CD86、MHC-Ⅱ类分子表达水平与对照组相比较明显升高;DC2.4酸性磷酸酶（ACP）含量与对照组相比较明显减少而IL-12的分泌水平升高;同时,DC2.4诱导淋巴细胞增殖能力增强,且诱导活化的淋巴细胞对Rac-1淋巴瘤细胞的杀伤活性与对照组相比较明显增强。结果可见,MENK可明显促进特异性负载Rac-1淋巴瘤抗原的DC2.4的成熟,并增强特异性诱导活化的淋巴细胞对Rac-1淋巴瘤细胞的杀伤活性。     

045树突细胞前体可受登革病毒和人类免疫缺陷病毒感染

黏膜表面、皮肤和血液中的树突细胞（dendritic cells，DC）由于能在这些部位摄取抗原，易成为病毒感染的初始靶细胞。含有Birbeek颗粒的CDl4^＋细胞亚群能分化成朗格汉斯型DC，表明了这些亚型是DC的前体。这些CDl4^＋细胞具有髓样DC前体的特性，能表达凝集因子XⅢa或C型凝集素（lectin）DC，SIGN等特异性表面标志。通过加入单核细胞集落刺激因子（M-CSF）对CD34^＋前体细胞进行培养可以获得CDl4^＋亚群，     

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

烟曲霉抗原Asp f16HLA-A*0201限制性的CD8+CTL抗原表位生物信息学预测与实验室鉴定

目的 预测与鉴定烟曲霉抗原Asp f16的HLA-A *0201限制性CD8+细胞毒性T细胞(CTL)抗原表位.方法 以国人常见的HLA-A*0201位点为靶点,依据生物信息学软件扫描烟曲霉特异性抗原Asp f16的全部427个氨基酸序列.使用HLA-A *0201转基因小鼠制备骨髓来源的树突状细胞(DC)和CTL.流式细胞仪技术检测DC表面MHC Ⅱ类抗原,CD80,CD86和CD11c的表达来验证其是否成熟.ELISPOT试验检测烟曲霉抗原多肽特异性CTL产生的细胞因子IFN-γ.四聚体(Tetramer)试验证实烟曲霉特异性CTL与抗原肽,HLA-A*0201分子复合体的亲和性.结果 根据与MHC I类分子结合的半衰期评分,选择了3个HLA-A*0201限制性抗原表位.流式细胞仪分析示成熟DC高表达HLA Ⅱ类抗原,CD80,CD86和CD11c.Tetramer试验证实烟曲霉特异性T细胞受体与抗原肽,HLA-A*0201分子复合体的高亲和性.ELISPOT实验结果 表明烟曲霉抗原肽体外可以活化CD8+CTL,被负载了抗原肽的DC刺激活化后可以产生IFN-γ.结论 本研究成功鉴定烟曲霉抗原Asp f16的HLA-A*0201限制性CD8+CTL表位,可作为疫苗设计的候选表位,为进一步研发新型抗烟曲霉疫苗提供参考.     

DC与CIK细胞治疗难治复发急性髓细胞白血病的近期临床观察

目的：评估自体DC与CIK细胞治疗难治复发急性髓细胞白血病的近期疗效与安全性。方法：给予20例难治复发急性髓细胞白血病患者树突状细胞（DC）与细胞因子诱导的杀伤细胞（CIK）治疗,20例难治复发的应用同样化疗方案的急性髓细胞白血病患者做为对照组;治疗后4周观察两组患者临床疗效和生存质量（KVS）评分,DC与CIK细胞治疗前和治疗后1周检测T细胞亚群（CD3＋、CD3＋CD4＋、CD3＋CD8＋、CD3＋CD56＋）和细胞因子（IL-12、IL-2、IL-7、IFN-γ及TNF—α）水平的变化。结果：（1）DC与CIK细胞治疗组有效率和KPS评分明显高于对照组（P〈0．05）,所有患者的不良反应轻微,均可耐受。（2）DC与CIK细胞治疗后1周,患者T细胞亚群百分比和细胞因子含量较治疗前均明显升高,其中CD3＋、CD3＋CD56＋及IL-12、IL-7明显升高（P〈0．05）。结论：DC与CIK细胞免疫治疗难治复发急性髓细胞白血病安全有效。     

Identification of novel MAGE-A6- and MAGE-A12-derived HLA-A24-restricted cytotoxic T lymphocyte epitopes using an in silico peptide-docking assay

Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called ??-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24⁺ melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.     

FKBP-RAP-FRB系统转运BCR-ABL入核对K562细胞的增殖抑制效应

BCR-ABL融合蛋白是慢性粒细胞白血病（chronic myeloid leukemia,CML）发病的基础。其中,BCR-ABL只能定位于细胞浆、不能易位至细胞核是其致病的关键因素。因此,转运BCR—ABL入核可能是治疗CML的潜在方法。该研究利用基因重组技术,构建HA-2FKBP-ABD（HF2A）和FLAG-3NLS—FRB＊（FN3R）重组腺病毒,与雷帕霉素类似物（Rapamycin analog）同组成FKBP-RAP-FRB系统,转运K562细胞胞浆中的BCR—ABL癌蛋白至细胞核,并探究其对K562细胞增殖的影响。结果显示,成功构建了高滴度的重组腺病毒,Westernblot证实目的蛋白在K562细胞内成功表达。FKBP—RAP—FRB系统可通过转运BCR—ABLA入核。抑制K562细胞生长和克隆形成的能力。结果揭示,FKBP-RAP—FRB系统转运BCR—ABL入核有望为CML提供新的治疗手段。     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

Induction of Tc2 cells with specificity for prostate-specific antigen from patients with hormone-refractory prostate cancer

Prostate-specific antigen (PSA) is a potentially useful antigen for targeted T-cell immunotherapy of prostate cancer (CaP). Our laboratory has identified a synthetic nonamer peptide (PSA 146-154) homologue of PSA, which binds to the prevalent human leukocyte antigen, HLA-A2, and elicits specific cytotoxic T-lymphocyte (CTL) responses from normal individuals of the HLA-A2 phenotype. In the present study, we report on the induction of CTL from peripheral blood mononuclear cells (PBMC) of patients with hormone-refractory CaP, which exhibit the same specificity. T-cell lines were established from two patients by stimulation of PBMC with PSA 146-154 peptide in vitro. The T-cell lines exhibited specific cytolytic activity against T2 cells pulsed with PSA 146-154 peptide, but not a control HLA-A2 binding peptide (HIV-RT 476-484) via chromium release assay (CRA). The T-cell lines also showed PSA 146-154 peptide-specific IL-4 responses, but no detectable interferon-gamma (IFN-gamma) responses via enzyme-linked immuno-spot assays. Magnetic immuno-selection studies of one of the T-cell lines demonstrated that both cytolytic and interleukin-4 (IL-4) responses were mediated by CD8(+), but not by CD4(+) T cells. This Tc2 line was further characterized for the ability to recognize endogenously processed PSA epitopes. The line specifically secreted IL-4 in response to HLA-A2(+) target cells transfected to express PSA and specifically lysed the PSA(+) target cells, but not control transfected cells. The results indicate that the PSA 146-154 peptide emulates a naturally processed and presented peptide epitope of PSA that is within the T-cell repertoire of HLA-A2(+)patients with CaP.