4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

不同肝癌细胞抗原致敏树突状细胞体外诱导CTL活性的研究

目的比较不同方法提取的肝癌细胞(SMMC-7721)抗原致敏树突状细胞(Dendritic cell DC)后,对特异性细胞毒性T细胞(CTL)的诱导作用。方法分离脐带血单个核细胞,加入重组粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)诱导扩增出脐血DC;SMMC-7721细胞经反复冻融,直接超声破碎及热休克处理后再超声破碎细胞三种方法提取肿瘤抗原,然后分别致敏DC,以MTT法检测脐血DC诱导T细胞增殖及其特异性CTL杀伤活性。结果热休克处理后再超声破碎法提取的抗原致敏DC,能诱导更强的刺激T细胞增殖的能力,并且可以诱导更强的CTL活性。结论加热处理后再超声破碎细胞提取的肿瘤抗原致敏DC,在体外可诱导强大的抗肿瘤免疫保护效应。     

致敏DC诱导CTL特异性杀伤HL-60细胞的实验研究

目的 :探讨来自外周血单个核细胞的树突状细胞 (DC)负载冻融或弱酸洗脱的HL 6 0多肽抗原 ,体外诱导产生细胞毒性T淋巴细胞 (CTL)对HL 6 0细胞的杀伤作用。方法 :应用GM CSF、TNF α和IL 4自外周血单个核细胞诱导出DC ,使其负载两种HL 6 0多肽抗原 ,致敏同种T淋巴细胞 ,产生CTL。以胃癌细胞系SGC790 1为对照组 ,观察DC∶CTL∶HL 6 0 /SGC 790 1为 1∶10 0∶2 ,CTL对HL 6 0特异性免疫杀伤活性。结果 :弱酸洗涤法或冻融法所提取的抗原多肽致敏DC诱导的CTL对HL 6 0细胞的杀伤率分别为 6 8.2 9%± 11.37%和4 5 .95 %± 8.4 7% ,二者差异有极显著性意义 (P <0 .0 1)。对SGC 790 1细胞的杀伤率分别为 2 4 .6 0 %± 4 .4 0 %和 16 .0 9%± 4 .98% ,二者差异有极显著性意义 (P <0 .0 1)。结论 :相对于冻融HL 6 0细胞抗原多肽 ,DC可更有效地提呈酸洗HL 6 0抗原多肽 ,并通过特异性CTL产生对HL 6 0细胞高效而特异的杀伤作用     

树突状细胞负载甲胎蛋白、细胞毒性T细胞表位肽后的免疫应答

目的 用负载hAFP 218-226位LLNQHACAV九肽的树突状细胞（dendritic cells,DC)诱导自身淋巴细胞，使之活化为肝细胞癌（HCC）特异性细胞毒性T细胞（cytotoxic T lymphocyte,CTL)，并特异性杀伤HCC细胞。方法 在体外用GM-CSF和IL-4诱导HLA-A2^ 健康志愿者外周血单核细胞，使其分化为高纯度DC。用负载hAFP 218-226位LLNQHACAV九肽的DC诱导自身淋巴细胞，使之成为HCC特异性CTL。结果 诱导的DC分泌较高水平的IL-12（17.6-21.5pg/ml)，其中以第7天为最高（21.5pg/ml)，经DC和DC负载AFP表位肽后活化的淋巴细胞培养上清液中TNF水平均比在IL-2条件下培养的未活化淋巴细胞高。L929细胞死亡百分率分别为80.65和11.6%；经DC和DC负载AFP表位肽后活化的淋巴细胞培养上清液中IL-12水平分别为20.5pg/ml和46.9pg/ml，经DC活化后淋巴细胞增殖指数大于3。活化后的淋巴细胞不但特异性杀伤HLA-A2^ 的HCC细胞HepG2和负载AFP表位肽的T2细胞，而且还不同程度杀伤HLA-A3^ HCC细胞Alexander和其它表型HCC细胞，对NK细胞敏感的靶细胞慢性髓原白血病细胞K562也有较强的杀伤作用。结论 负载hAFPHLA-A2限制性CTL九肽的DC对表达AFP HCC的研究和治疗有潜在应用价值。     

热休克蛋白70激发树突状细胞抗肝癌作用的实验研究

目的探讨以树突状细胞（DC）为载体，用肝癌细胞提取的热休克蛋白70-肿瘤肽复合物（HSP-PC）刺激DC，观察经活化后的DC对肝癌细胞SMMC-7721的杀伤作用。方法用脐带血在体外培养诱导DC，用从SMMC-7721提取的HSP70—PC与DC共同培养，检测其对肝癌细胞的杀伤活性。结果经HSP70-Pc刺激后的DC对SMMC-7721的杀伤能力明显高于单纯DC对肿瘤细胞的杀伤能力。结论用HSP70—PC刺激的DC经体外实验证实具有明显的抗肿瘤效应，为临床肿瘤免疫治疗的开展提供了有效的数据。     

负载酸洗脱食管癌抗原肽的脐血树突状细胞对CTL杀伤活性的影响

分离脐血中的单个核细胞,以酸洗脱食管癌抗原肽负载获得分化成熟树突状细胞（DCs）,检测其对特异性细胞毒T淋巴细胞（CTL）的诱导和体外杀伤效应的影响。结果发现,负载食管癌抗原肽的脐血DCs诱导的CTL对酸洗前食管癌细胞株T.Tn的杀伤率显著高于酸洗后组和各对照组（P均〈0．05）,而对无关肿瘤细胞株Hep2无显著杀伤作用。认为负载食管癌抗原肽的脐血DCs体外可诱导CTL产生特异性杀伤效应。     

负载胰腺癌抗原的树突状细胞疫苗诱导T淋巴细胞抗肿瘤效应的研究

培养树突状细胞(DC),反复冻融法裂解培养的负载胰腺癌细胞(PC-3),提取细胞抗原,致敏DC,获得负载胰腺癌抗原的DC疫苗后诱导特异性细胞毒性T淋巴细胞(CTL)的生成,MTT法检测CTL对不同肿瘤细胞的杀伤作用.发现负载PC-3细胞抗原的DC疫苗能诱导产生肿瘤特异性的CTL,其对PC-3细胞具有明显地杀伤效应,而对人乳腺癌细胞MCF-7、人肝癌细胞7721细胞杀伤作用弱.认为负载胰腺癌抗原的DC疫苗能够诱导高效而特异地CTT杀瘤活性,为将来DC疫苗在胰腺癌的免疫治疗中提供了实验依据.     

人脐血来源树突细胞抗乙型肝炎病毒细胞免疫的体外实验研究

目的借助DC的强大抗原呈递能力,使其负载HBsAg后以激活HBsAg特异性的CTL,从而探索一种可有效活化机体抗HBV细胞免疫的免疫方式。方法从健康产妇分娩的胎儿脐血中分离出单个核细胞,在细胞因子组合的作用下诱导出DC,于体外负载HBsAg后,激活自体T细胞成CTL,于体外培养环境下检测其对表达HBsAg的靶细胞HepG2-S的杀伤效应。结果人脐血来源的单个核细胞可在多种细胞因子的作用下,被诱导为DC。DC在负载HBsAg后可于体外活化HBsAg特异性的CTL。效应细胞为负载HBsAg的DC活化脐血单个核细胞,靶细胞分别为不表达和表达HBsAg的HepG2,当效应细胞和靶细胞比为1:1时出现最大的杀伤效率,CTL对靶细胞的杀伤率分别为(16.36±6.42)%和(78.09±9.66)%,差别有统计学意义(P<0.01)。结论健康人脐血来源的DC,具有较强的抗原呈递功能,可在体外激活产生HBsAg特异性CTL,在表达HBsAg的靶细胞模型上可观察到一定杀伤效果,为人脐血来源DC用于抗HBV治疗进行了有益的探索。     

树突状细胞HBsAg疫苗抗乙型肝炎病毒免疫的体外研究

目的 研究人单核细胞来源的树突状细胞（DC）激活的HBsAg特异性细胞毒性T淋巴细胞（CTL）对表达HBsAg的HepG2/S靶细胞的杀伤效应，以探索DC-HBsAg疫苗在抗乙型肝炎病毒（HBV）中的作用。方法 从健康外周血中分离邮单核细胞，在粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素4（IL-4）的作用下培养7d诱导出DC，然后以DC为刺激细胞在体外诱导出HBsAg特异性CTL；将携有HBV-S基因的pLXSN/S重组质粒电击导入肝癌细胞系（HepG2)，建立表达HBsAg的靶细胞模型HepG2/S；用染色法检测HBsAg特异性CTL对HepG2/S靶细胞的杀伤效应。结果 DC激活的HBsAg特异性CTL对HepG2/S靶细胞具有较强的杀伤效应，不同浓度HBsAg(0μg/L，50μg/L和100μg/L）诱导的CTL的杀伤率分别为3.8%,69.5%和85.1%，而CTL对HepG2细胞无明显杀伤效应。结论 由DC激活的HBsAg特异性CTL具有较强制 特异性抗HBV作用。     

Activation of HIV type 1 specific cytotoxic T lymphocytes from semen by HIV type 1 antigen-presenting dendritic cells and IL-12

Seminal HIV-1-specific cytotoxic T lymphocytes (CTLs) could provide an important immune defense against local HIV-1 infection, and be important in impeding the spread of HIV-1 infection. In this study, we demonstrate that autologous blood-derived dendritic cells (DCs) loaded in vitro with synthetic HIV-1 peptides representing known CTL epitopes activated HLA class I restricted, anti-HIV-1 CTLs and interferon gamma responses in seminal CD8+ T cells from subjects with chronic HIV-1 infection on antiretroviral therapy. CTLs specific for the same HLA-restricted epitopes were detected in semen and blood of the same individuals by stimulation with peptide-loaded DCs. Anti-HIV-1 CTL responses from semen were enhanced by stimulation with DCs loaded with HIV-1 peptides and interleukin 12. Our results suggest that blood-derived DCs have HIV-1 antigen-presenting capacity for seminal CTL in HIV-1-infected subjects. The DC-T cell system can serve as a model for immunotherapy of HIV-1 infection in the local genital tract as well as systemic blood circulation.     

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.     

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM To evaluate antihepatoma effect ofantisense phosphorothioate oligodeo-xyribonucleotides(S-ODNs)targeted to alpha-fetoprotein(AFP)genes in vitro and in nudemice.METHODS AFP gene expression was examinedby immunocytochemical method or enzyme-linked immunosorbent assay.Effect of S-ODNson SMMC-7721 human hepatoma cell growth invitro was determined using microculturetetrazolium assay.In vivo antitumor activitiesof S-ODNs were monitored by measuring tumorweight differences in treated and control micebearing SMMC-7721 xenografts.Induction of cellapoptosis was evaluated by fluorescence-activated cell sorter(FACS)analysis.RESULTS Antisense S-ODN treatment led toreduced AFP gene expression.Specificantisense S-ODNs,but not control S-ODNs,inhibited the growth of heaptoma cells in vitro.In vivo,only antisense S-ODNs exhibitedobvious antitumor activities.FACS analysisrevealed that the growth inhibition by antisenseS-ODNs was associated with their cell apoptosisinduction.CONCLUSION Antisense S-ODNs targeted toAFP genes inhibit the growth of human hepatomacells and solid hepatoma,which is related totheir cell apoptosis induction.     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

BACKGROUND/AIMS: The aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia. METHODS: HLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24+ patients with acute hepatitis B. RESULTS: An HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117-125 and P756-764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24+ patients with acute hepatitis B, respectively. CONCLUSIONS: We identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.     

HLA-B7 beta-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses

Chronic rejection of transplanted allografts is the major cause of graft loss after clinical solid organ transplantation. Recent data link the indirect presentation of allopeptides to chronic graft loss; thus, identification of immunodominant epitopes on major histocompatibility complex (MHC) antigens could significantly contribute to establishing novel ways for monitoring and managing chronic rejection. Here, we show that synthetic allo-MHC-derived peptides covering the polymorphic region 56 to 120 of HLA-B7 modulate alloresponses. In particular, the 2 beta-pleated sheet-derived peptides covering residues 91 to 105 and 96 to 120, respectively, but not sequences from the alpha1 helix, were presented by autologous peripheral blood lymphocytes to induce T-cell proliferation. In addition, the 2 beta-pleated sheet-derived peptides and the alpha1-derived peptide residues 60 to 75 abrogated lysis of HLA-B7 target cells by anti-HLA-B7 cytotoxic T lymphocytes (CTLs). Although most residues between 91 and 120 are normally not directly accessible to T cells, our results indicate that peptides derived from the lower surface of the peptide-binding groove of HLA-B7 are immunodominant in HLA-B7 alloresponses. To characterize the binding and stability of allopeptides to T cells, the 62-70 peptide-derived from the 60-75 allopeptide that blocked cytotoxicity of anti-HLA-B7 CTL-was synthesized and coupled with fluorescein isothiocyanate. The peptide specifically labeled anti-B7 CTL, but not anti-HLA-A2 CTL as measured by flow cytometry. Peptide binding to CTL was specific at 4 degrees C and remained stable for 12 hours, whereas it remained stable for less than 2 hours at 37 degrees C. These studies allow the identification of HLA-B7 T-cell epitopes and reveal for the first time a novel, previously unrecognized application of synthetic HLA-derived allopeptides to visualize alloreactive T cells.     

Induction of cytotoxic T-cell responses against the oncofetal antigen-immature laminin receptor for the treatment of hematologic malignancies

The oncofetal antigen immature laminin receptor protein (OFA-iLRP) is a highly conserved protein that is preferentially expressed in fetal tissues and in many types of cancer, including hematopoietic malignancies, whereas OFA-iLRP is not detectable on healthy differentiated adult cells. To investigate whether OFA-iLRP-specific cytotoxic T lymphocytes (CTLs) are capable of killing OFA-iLRP-expressing hematologic targets, CTLs were generated from healthy HLA-A*0201-positive volunteers by incubating T cells with autologous dendritic cells (DCs) transfected with OFA-iLRP RNA. OFA-iLRP-specific CTLs lysed HLA-A2+ OFA-iLRP+ tumor cells, including several lymphoma and leukemia cell lines, as well as fresh leukemic targets from patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), indicating that OFA-iLRP-derived peptides are naturally processed and presented by hematologic tumors. Healthy OFA-iLRP-negative target cells (CD14+ monocytes, activated B cells, DCs, bone marrow cells) were not attacked by OFA-iLRP-specific CTLs. Furthermore, in an established murine B-cell lymphoma model (A20), treatment with syngeneic DCs transfected with OFA-iLRP-coding RNA resulted in powerful antitumor effects in a significant portion of mice. For the first time, these data show that OFA-iLRP can be used as a target for T-cell-based immunotherapeutic strategies against hematologic malignancies.     

Patient-derived dendritic cells transduced with an a-fetoprotein-encoding adenovirus and co-cultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against hepatocellular carcinoma cells.

BACKGROUND/AIMS: Breaking immunologic tolerance towards the hepatocellular carcinoma (HCC)-associated alpha-fetoprotein (AFP) antigen is possible. The use of this potential for the treatment of immunocompromised HCC patients is limited. In this study, we analyzed whether dendritic cells (DCs) from HCC patients transduced with a human AFP (hAFP)-expressing adenovirus and co-cultured with cytokine-induced killer (CIK) cells can induce a strong specific immune response against HCC-cells. METHODS: An hAFP-encoding adenovirus (Ad-hAFP) was generated. DCs from healthy donors or patients were transduced at a very high efficacy. Afterwards, DCs were co-cultured with autologous CIK-cells, and their ability to lyse HCC-cells was analyzed. RESULTS: AFP-transduced DCs stimulated CIK cells strongly to lyse about 70% of AFP-expressing HCC cells. Cytotoxicity was significantly higher when lymphocytes were co-cultured with Ad-hAFP-transduced DCs than with Ad-mock-transduced DCs, indicating an AFP-specific immune response. More interestingly, CIK cells from patients with AFP-positive HCC could be stimulated to lyse AFP-expressing HCC cells as effectively as CIK cells from healthy individuals and stronger than CIK cells from patients without AFP-expressing HCC. CONCLUSIONS: The data demonstrate that patient-derived DCs that were transduced with an AFP-expressing adenovirus and co-cultured with autologous CIK cells induce an AFP-specific, strong immune response against HCC cells. Therefore, this approach may have a potential for an adoptive and/or DC-based immunotherapy for HCC patients.