SLC基因修饰对肿瘤细胞疫苗免疫效果的初步研究

目的：构建次级淋巴组织趋化因子（secondary lymphoid—tissue chemokine,SLC）基因修饰的趋化型恶性黑色素瘤疫苗,并初步研究其免疫效果。方法：SLC基因转染小鼠黑色素瘤细胞B16,并用适当条件的丝裂霉素处理,制备趋化型肿瘤细胞疫苗。在体外,利用趋化小室法检测趋化型疫苗培养上清对小鼠脾淋巴细胞的趋化活性。在体内,用趋化型疫苗免疫小鼠,检测脾淋巴细胞的CTL活性,并观察疫苗免疫对B16移植瘤生长的影响。结果：筛选获得了SLC基因转染的小鼠黑色素瘤B16细胞,以100μg／mL丝裂霉素处理1.5h,制备趋化型肿瘤细胞疫苗。趋化型疫苗在体外培养48h后,其培养上清对淋巴细胞具有明显的趋化活性。与野生型疫苗相比,用趋化型疫苗免疫后,小鼠脾淋巴细胞的CTL活性更强,小鼠免疫后再次接种B16细胞,肿瘤的生长受到更明显的抑制。结论：成功构建了SLC基因修饰的趋化型黑色素瘤疫苗,并在体内外初步检测到该疫苗对抗肿瘤免疫的影响和对移植瘤生长的抑制,该疫苗有可能被应用于恶性黑色素瘤的免疫治疗。     

腺病毒介导IL-2基因转染的瘤苗体内抗肿瘤作用

目的 :探讨重组腺病毒介导的 IL- 2基因转染的瘤苗的体内抗肿瘤作用及其免疫学机制。方法 :应用腺病毒介导的鼠 IL- 2基因转染 CT2 6小鼠结肠癌细胞 ,灭活后用作瘤苗治疗荷瘤小鼠 ,观察皮下肿瘤生长及其存活期。采用乳酸脱氢酶释放法检测荷瘤小鼠脾细胞 CTL、L AK、NK细胞的杀伤活性。结果 :鼠 IL- 2基因转染瘤苗治疗能显著抑制荷瘤小鼠皮下肿瘤生长并明显延长其存活期 (P<0 .0 1)。体内免疫功能检测表明 ,鼠 IL- 2基因转染疫苗治疗组小鼠脾细胞 CTL 活性、L AK活性和 NK活性显著高于对照组 (P<0 .0 1)。结论 :腺病毒介导鼠 IL- 2基因转染的瘤苗体内具有较强的抗肿瘤效应 ,其机制可能是提高了荷瘤小鼠特异性和非特异性抗肿瘤免疫反应     

基因枪介导的neu基因DNA疫苗抗肿瘤作用的实验研究

目的 探讨DNA疫苗pWRG-neu的皮内免疫,对高表达neu基因的小鼠移植瘤生长和转移的抑制作用。方法 向小鼠黑色素瘤B16F10细胞系转染pcDNA-neu,用有限稀释法筛选一株高表达neu基因的细胞株B16F10-neu。在基因枪介导下,向C57BL／6小鼠导入DNA疫苗pWRG-neu,通过观察免疫动物的生存期,评价DNA疫苗的抗肿瘤作用。分离免疫动物脾细胞,经自体淋巴细胞混合培养实验,分析DNA疫苗体内免疫后机体的CTL应答。结果 筛选到一株高表达neu基因的B16F10-neu细胞株,转基因过程和外源基因的表达没有改变细胞系的增殖特性。用基因枪轰击,进行DNA疫苗pWRG-neu皮内免疫,对小鼠黑色素瘤B16F10-neu进行预防、治疗和抗转移的实验研究,结果表明,DNA疫苗的免疫能够明显推迟移植瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。DNA疫苗免疫后可诱导小鼠脾淋巴细胞CTL活性。结论 基因枪介导的DNA疫苗pWRG-neu经皮内免疫,能够有效的诱导机体的细胞免疫应答,预防和治疗小鼠移植瘤的发生,并有一定的预防肿瘤肺转移的作用。     

重组人MUC1-MBP融合蛋白的抗肿瘤作用

目的： 研究重组人MUC1MBP的抗肿瘤作用。方法：用重组人MUC1MBP皮下注射途径免疫健康鼠，采用MTT法测定小鼠抗MUC1特异的CTL活性；通过免疫鼠成瘤及荷瘤鼠治疗实验检测其对肿瘤的预防和治疗作用。结果：MUC1MBP免疫鼠CTL对MCF7及Lewis肺癌细胞的杀伤率分别为(47.7±4.3) %和(67.5±6.5) %；免疫鼠成瘤结果免疫组小鼠共长5个肿瘤，存活时间明显延长，对照组共长51个，平均生存时间23 d；荷瘤鼠治疗结果显示治疗组小鼠肿瘤平均体积386 mm3，对照组小鼠肿瘤平均体积4 000 mm3。结论：重组人MUC1MBP具有明显的治疗、预防肿瘤和抑制肿瘤转移的作用,有希望发展成人类抗肿瘤疫苗。     

表达VEGFR-2的减毒鼠伤寒沙门氏菌疫苗诱导特异性抗胶质瘤血管免疫应答

背景与目的:血管内皮生长因子(vascular endothelialgrowth factor,VEGF)及其主要受体血管内皮生长因子受体-2(vascular endothelialgrowth factorreceptor-2,VEGFR-2)在肿瘤新生血管和肿瘤基质形成过程中起着重要作用。本研究的目的是观察表达鼠VEGFR-2的重组减毒沙门氏疫苗菌诱导的抗血管特异性免疫应答及抗胶质瘤作用。方法:构建真核表达载体pcDNA3.1-VEGFR2,通过电转化法将pcDNA3.1-VEGFR2导入减毒鼠伤寒沙门氏菌SL7207中,经由胃管饲予C57BL/6J小鼠,对小鼠进行基因免疫。采用ELISA法检测免疫小鼠血清中特异性抗VEGFR2-IgG抗体,分离免疫小鼠的脾细胞,分析重组疫苗菌免疫后小鼠体内的特异性细胞毒性T细胞(cytotoxic T lym phocyte,CTL)应答。用携带pcDNA3.1-VEGFR2的重组沙门氏菌免疫治疗胶质瘤荷瘤小鼠,通过测量荷瘤小鼠肿瘤大小,检测肿瘤微血管密度及肿瘤细胞凋亡,评价重组疫苗菌的抗血管及肿瘤生长抑制作用。结果:重组疫苗菌免疫后小鼠产生了高水平的抗VEGFR2-IgG抗体,诱导小鼠脾淋巴细胞产生针对VEGFR2的特异性CTL活性。重组疫苗菌的免疫能够明显抑制胶质瘤的生长。NaH CO3对照组、载体对照组、重组疫苗菌组的平均微血管密度分别为26.5±5.8、27.2±4.5、8.8±1.9,平均凋亡细胞数分别为4.41.2、3.     

树突状细胞与肿瘤细胞融合后诱导T细胞介导的抗肿瘤效应

目的：研究树突状细胞（DC）与肿瘤细胞（SP2／0）融合作为肿瘤疫苗免疫小鼠后抑制肿瘤生长的作用及其机理。方法：BALB／C小鼠DC与SP2／0细胞体外融合,形成的杂交瘤分2次免疫接种同基因小鼠皮下,然后用SP2／0细胞攻击免疫的小鼠,观察肿瘤的生长情况及免疫小鼠脾细胞特异性CTL功能。结果：DC－SP2／0杂交瘤接种小鼠能产生明显的抗肿瘤效应,其拮抗SP2／0细胞攻击的能力明显强于用灭活的死SP2／0细胞与DC混合,和单用死SP2／0细胞免疫的小鼠及用生理盐水的对照组小鼠。DC－SP2／0杂交瘤免疫接种小鼠的脾细胞特异性CTL杀伤活性强于其它各组小鼠。结论：DC与肿瘤细胞融合后作为肿瘤疫苗接种可在体内产生明显的抗肿瘤免疫,其机理主要是特异性CTL的作用。     

树突细胞联合β-榄香烯对小鼠胰腺癌治疗的实验研究

背景与目的：树突细胞（Dendritic cells，DC）是目前发现的功能最强大的肿瘤抗原专职免疫递呈细胞，DC疫苗是目前最具临床应用潜能的治疗性疫苗。肿瘤细胞凋亡小体被DC的MHC—1分子提呈并诱导的特异性CTL免疫反应抗癌作用显著。本研究探讨了DC负载凋亡癌细胞抗原后诱导的免疫应答联合β-榄香烯对小鼠胰腺癌治疗作用。方法：制备C57BL／6小鼠骨髓源性DC并鉴定，负载肿瘤抗原后制备成疫苗。分别观察DC诱导的CTL和β-榄香烯对胰腺癌细胞的杀伤作用。体内观察DC疫苗联合β-榄香烯对小鼠胰腺癌的抑制作用。结果1经电镜及流式细胞鉴定可获得典型的具有抗原加工及递呈作用的DC，利用TNF-α可使成熟的树突状细胞比例增加，MHC-Ⅱ、CD83、CD86等表面分子的表达较单纯DC组显著增强。负载凋亡抗原后DC诱导的特异性CTL体外对胰腺癌细胞有显著杀伤作用，效靶比30：1时，诱导的CTL对胰腺癌细胞抑制率达93％。β-榄香烯对癌细胞增殖抑制明显，DC疫苗与β-榄香烯联合治疗可明显抑制荷瘤小鼠肿瘤的生长，使其生存期延长，联合治疗组生存期为49．4天，与单纯β-榄香烯组（24．8天）、DC疫苗（38．5天）、DC组（20．7天）及对照组（17．5天）相比有显著差异（P〈0．05）。结论：经凋亡肿瘤细胞致敏的DC回输荷瘤小鼠可激发宿主针对特异肿瘤的Th1及CTL免疫应答，以DC、介导的免疫反应联合中药的治疗方法可成为抗肿瘤的崭新且有效手段。     

重组EGFR噬菌体疫苗抗肿瘤效果的研究

目的构建重组EGFR噬菌体疫苗并且观察其抗肿瘤效果。方法将鸡源EGFR膜外部分的5个基因片段插入到T7噬菌体展示系统中,EGFR蛋白以融合蛋白的形式展示在噬菌体的外壳蛋白10B上,从而构建成噬菌体疫苗。Western blot方法检测噬菌体壳蛋白上是否有EGFR表达。利用ELISA方法检测免疫小鼠血清中抗EGFR抗体。分离免疫小鼠脾细胞,检测淋巴细胞对靶细胞A431的细胞毒作用。C57小鼠经过4次免疫,接种Lewis肺癌,记录肿瘤的生长情况,评价疫苗的抗肿瘤效果。结果本实验成功构建5种鸡源EGFR噬菌体疫苗。Western blot。显示噬菌体壳蛋白上有EGFR表达。免疫后的小鼠血清中检测到抗EGFR抗体。体外分离脾细胞证明免疫后的小鼠脾细胞对EGFR受体高表达的A431细胞具有杀伤作用。靶细胞和效应细胞的比例在1:10时,最大杀伤率可达(45．74±7．21)％(P<0．001)。肿瘤生长曲线显示免疫后的C57小鼠Lewis肺癌生长受到明显的抑制。结论利用噬菌体展示技术构建的噬菌体疫苗是一种有效的抗肿瘤方法。     

肿瘤干细胞-DC疫苗联合白介素-2治疗小鼠恶性黑色素瘤的研究

摘 要：［目的］ 研究荷载肿瘤干细胞（CSCs）抗原的树突状细胞疫苗联合白介素-2（IL-2）治疗小鼠恶性黑色素瘤的研究。［方法］ 利用Aldefluor染料从小鼠恶性黑色素瘤细胞系D5中分选出肿瘤干细胞,将CSCs制备成细胞裂解液加载在DC细胞上制备成CSCs-DC疫苗。将D5细胞接种至C57BL/6小鼠皮下建立小鼠恶性黑色素瘤模型,随机分成磷酸盐缓冲液组 (PBS)、白介素-2组（IL-2）、CSCs-DC疫苗组（CSCs-DC）、CSCs-DC疫苗联合白介素-2组（CSCs-DC+ IL-2）,并给予相应的治疗。测量并记录各组肿瘤大小变化和小鼠生存期,实验终点取小鼠脾脏,体外培养激活后利用ELISA法测定B细胞分泌IgG水平,LDH细胞毒性实验测定细胞毒性淋巴细胞(CTL)活性。 ［结果］ CSCs-DC+IL-2组肿瘤生长明显慢于PBS组,IL-2组及CSCs-DC组（P<0.05）。小鼠生存期CSCs-DC+IL-2组最长为58.33±1.67天,CSCs-DC组为51.67±0.33天,较IL-2组43.33±2.40天长,PBS组平均生存时间最短,为34.00±1.53天。CSCs-DC+IL-2组小鼠脾脏中B细胞分泌IgG水平和CTL活性最高,CSCs-DC组次之,IL-2组较CSCs-DC组和CSCs-DC+IL-2组低,但较PBS组高,差异有统计学意义（P<0.01）。［结论］CSCs-DC疫苗联合IL-2方案在治疗恶性黑色素瘤的小鼠模型中效果较CSCs-DC疫苗组和IL-2组好,与IL-2提高T/B细胞活性有关。     

异种EGFR口服DNA疫苗抑制小鼠Lewis肺癌的生长

目的：观察表达异种鸡EGFR(chicren EGFR, cEGFR)与 IgGγFc融合基因的口服减毒鼠伤寒沙门菌疫苗对高表达EGFR 的肺癌Lewis细胞小鼠移植瘤生长的抑制作用。方法：将pVAX1-cEGFR-γFc质粒转化减毒沙门菌SL7207,重组菌SL7207/pVAX1-cEGFR-γFc体外感染小鼠腹腔巨噬细胞,免疫荧光法检测cEGFR-γFc融合蛋白的表达。SL7207/pVAX1-cEGFR-γFc重组菌口服免疫小鼠3次后接种Lewis细胞,Western blotting检测小鼠体内融合蛋白的表达,ELISA法检测免疫小鼠血清抗EGFR抗体的水平。接种Lewis细胞14 d后处死小鼠,瘤体称质量,检测SL7207/pVAX1-cEGFR-γFc疫苗对Lewis肺癌生长的抑制作用,测定荷瘤小鼠的生存时间。结果：成功构建减毒沙门菌疫苗SL7207/pVAX1-cEGFR-γFc,SL7207/pVAX1-cEGFR-γFc感染后,在小鼠后体内外都能检测到cEGFR-γFc融合蛋白的表达;SL7207/pVAX1-cEGFR-γFc疫苗口服免疫后小鼠能够产生高水平的抗EGFR抗体,口服SL7207/pVAX1-cEGFR-γFc疫苗能够有效抑制小鼠Lewis移植瘤的生长,延长荷瘤小鼠的生存时间。结论：异种EGFR口服DNA疫苗能够有效地抑制高表达EGFR肺癌的生长,是EGFR分子靶向治疗的一条新途径。     

血管内皮生长因子受体-2胞外区基因修饰树突状细胞的抗实验性肺转移作用

目的研究血管内皮生长因子受体-2胞外区基因修饰树突状细胞(DC)的抗肿瘤转移作用及其机制。方法电穿孔法基因转染DC,ELISA检测基因转染DC表达sVEGFR-2水平;经尾静脉给C57BL／6小鼠分别注射PBS、DC、pcDNA3．1修饰的DC(DC-vector)、pcDNA3．1／sVEGFR-2修饰的DC(DC-sVEGFR-2),连续3次,每次间隔1周,最后一次免疫注射后10 d,以51Cr释放法检测小鼠脾细胞VEGFR-2特异性CTL活性,藻酸钠小珠法检测肿瘤细胞诱导的新生血管形成,以B16黑色素瘤肺转移模型观察DC-sVEGFR-2免疫的抗肿瘤转移作用。以抗-CD4单抗或抗-CD8单抗分别剔除体内CD4+T细胞及CD8+T细胞,研究DC-sVEGFR-2免疫抗肿瘤转移作用的免疫学机制。结果DC- sVEGFR-2能有效表达sVEGFR-2,而DC-vector和DC不表达sVEGFR-2。DC-sVEGFR-2免疫小鼠脾细胞能有效杀伤VEGFR-2+的靶细胞H5V和3LL-sVEGFR,与对照组相比,差异有统计学意义(P<0．01),但对VEGFR-2-的同系EL4和3LL细胞无杀伤作用。DC-sVEGFR-2免疫能显著抑制肿瘤诱导的新生血管形成,DC-sVEGFR-2免疫小鼠藻酸钠小珠中FITC-dextran的摄取量仅为对照组的50％,与对照组相比,差异有统计学意义(P<0．01)。DC-sVEGFR-2免疫组小鼠B16黑色素瘤肺转移灶数目显著减少,瘤灶体积显著小于对照组,与DC-vector免疫组相比,肺表面转移灶数目下降81．9％(49．7±12．7:9．0±3．2,P<0．01)。体内T细胞亚群剔除试验显示,DC-sVEGFR-2免疫的抗肿瘤转移作用由CD8+T细胞介导。结论DC-sVEGFR-2免疫能打破自身免疫耐受,诱导针对VEGFR-2的CTL应答,抑制肿瘤诱导的新生血管形成,显著抑制肿瘤的转移,其抗肿瘤转移作用由CD8+T细胞介导。     

Syngeneic monoclonal antibodies to B16 melanoma viral antigens

Four stable IgM monoclonal antibody-producing hybridomas were generated by fusing mouse myeloma cells with spleen lymphocytes from C57BL/6 mice hyperimmunized against the syngeneic B16 melanoma. All four monoclonal antibodies (R31/15, R37/4, R37/6, and R37/7), in common with polyclonal antiserum from immunized mice, recognized antigens on the same complex of related cell surface molecules specified by endogenous AKR-type murine leukemia virus, designated the B16-gp/70/80/85 antigen complex. Reactivity with this antigen complex was demonstrated by radioimmunoprecipitation. Specificity for viral Mr 70,000 glycoprotein-related antigens was indicated by absorption of antibody activity by endogenous AKR virus and by inhibition of antibody binding to B16 melanoma cells by monospecific antiserum to murine leukemia virus Mr 70,000 glycoprotein. Neither polyclonal nor monoclonal antibodies recognized antigens on fish, guinea pig, swine, or human melanoma cell lines. Polyclonal antiserum reacted with several other mouse melanomas and with certain mouse lymphoma lines induced by, or harboring, endogenous murine leukemia viruses, but the monoclonal antibodies were unreactive except for recognition of antigens on Harding-Passey mouse melanoma cells by antibody R37/4 and on RL male 1 mouse lymphoma cells by antibody R37/7. Only monoclonal R37/7 was cytotoxic for cultured B16 melanoma cells in an antibody- and complement-dependent assay with guinea pig complement, although all antibodies were cytotoxic with rabbit complement. In reflecting the predominant humoral immune response to the B16 melanoma detected in syngeneic mice during tumor growth, these monoclonal antibodies will permit experimental amplification of that response to help determine how that immunity influences tumor growth and metastatic dissemination.     

Genetic immunization of mice with human tyrosinase-related protein 2: implications for the immunotherapy of melanoma

The melanosomal protein TRP2 expressed by melanocytes and most melanoma cells is an attractive, clinically relevant model antigen for the experimental development of melanoma immunotherapy in mice. A peptide shared by murine and human TRP2 can be recognized by melanoma-reactive CTL in C57BL/6 mice, as well as in human melanoma patients. Previous experiments demonstrated that gene gun immunization of mice with plasmid DNA encoding autologous murine TRP2 was unable to induce protective immunity against B16 melanoma cells naturally expressing TRP2. In the present study, we investigated whether the use of cDNA encoding xenogeneic human TRP2, which is highly homologous to murine TRP2, would be more effective. Genetic immunization of mice with human TRP2 resulted in coat depigmentation as a sign of autoimmune-mediated destruction of melanocytes and provided significant protection against metastatic growth of B16 melanoma Induction of protective immunity was associated with TRP2-reactive antibodies and CD8+ T cells. Furthermore, immunization with recombinant adenovirus was more effective than immunization with plasmid DNA using the gene gun. Our results provide new insights for the development of antigen-specific immunotherapy of melanoma.     

Activation in vitro of mouse macrophages by syngeneic, allogeneic, or xenogeneic lymphocyte supernatants.

Macrophages from normal C57BL/6 mice, those with a subcutaneous B16 melanoma, and mice immunized against the tumor were examined for in vitro cytotoxicity to B16 tumor cells. Macrophages were treated by incubation with supernatants from B16 cells grown either in unmixed cultures or in cultures containing syngeneic, normal, or sensitized allogeneic (A mouse), or xenogeneic (rat) lymphocytes. The various treated and untreated macrophages were then cultured for 5 days with viable B16 cells prelabeled with 125I-5-iodo-2'-deoxyuridine; the cultures were terminated, and the extent of destruction of the B16 target cells was determined from the amounts of radioactivity remaining in adherent tumor cells. Of the untreated macrophages, only those from immunized mice were cytotoxic to the tumor cells; macrophages from normal and tumor-bearing mice became cytotoxic by incubation with supernatants from cultures containing lymphocytes from immunized syngeneic mice, sensitized allogeneic mice, or sensitized rats; and macrophages incubated with supernatants from cultures containing normal nonsensitized allogeneic or xenogeneic lymphocytes showed no cytotoxicity. Thes results suggested that macrophages from tumor-bearing animals are potentially cytotoxic to their syngeneic tumors and can be activated by mediators released from sensitized syngeneic, allogeneic, and/or xenogeneic lymphocytes in vitro.     

应用细胞因子缓释微球的肿瘤疫苗治疗肝癌的研究

目的 探讨采用细胞因子缓释微球的肿瘤疫苗预防和治疗肝癌的疗效及抗癌机制。方法 我们研制开发了一种肿瘤疫苗,其组成是固定的肿瘤细胞或组织碎片、细胞因子缓释微球和免疫辅助药。采用多聚甲醛固定的小鼠Hepal-6细胞或肿瘤碎片、微球包装的GM—CSF和／IL—2和合成TiterMax Gold等不同成份的瘤苗皮内接种C57BL／6J小鼠,随后肝内接种活体Hepal-6细胞。结果 对照组15只小鼠全部发展成肝肿瘤;含有固定Hepal-6细胞和IL-2及GM—CSF微球的肿瘤疫苗,80％小鼠获得保护。再加入免疫辅助剂TiterMax Gold的肿瘤疫苗,则87％小鼠获得保护。将Hepal-6细胞接种于左躯干皮下。肿瘤长至直径5mm时,皮内接种肿瘤疫苗2次。结果显示,对照组肿瘤继续生长。疫苗组在第2次接种后7—10天,10只小鼠中9只肿瘤生长受到抑制,随后明显缩小。60％小鼠的肿瘤完全消散。细胞毒性实验结果显示,未接种疫苗的小鼠脾细胞不能杀灭Hepal-6细胞和其他肿瘤细胞;而接种疫苗的小鼠脾细胞对Hepal-6细胞杀瘤活性达41％,但对B16—Fl,Lewis肺癌细胞(LLC),肾癌细胞(Renca),膀胱癌细胞(MBT-2)则无效。疫苗的Ⅰ期临床实验结果显示肝癌疫苗能有效地预防肝癌术后复发,诱导DTH反应。结论 肝癌疫苗能有效预防和治疗原发性肝癌,其抗瘤机制是诱导内源性抗原特异性CTL反应,其杀瘤特性是由典型的：MHC—Ⅰ限制的CD8^ T细胞所介导的。     

Dendritic cells break tolerance and induce protective immunity against a melanocyte differentiation antigen in an autologous melanoma model

Tyrosinase-related protein (TRP) 2 belongs to the melanocyte differentiation antigens and has been implicated as a target for immunotherapy of human as well as murine melanoma. In the current report, we explored the efficacy of nonmutated epitopes with differential binding affinity for MHC class I, derived from mouse TRP2 to induce CTL-mediated, tumor-reactive immunity in vivo within the established B16 melanoma model of C57BL/6 mice. The use of nonmutated TRP2-derived epitopes for vaccination provides a mouse model that closely mimics human melanoma without introduction of xenogeneic or otherwise foreign antigen. The results demonstrate that vaccination with TRP2 peptide-loaded bone marrow-derived dendritic cells (DCs) results in activation of high avidity TRP2-specific CTLs, displaying lytic activity against both B16 melanoma cells and normal melanocytes in vitro. In vivo, protective antitumor immunity against a lethal s.c. B16 challenge was observed upon DC-based vaccination in this fully autologous tumor model. The level of protective immunity positively correlated with the MHC class I binding capacity of the peptides used for vaccination. In contrast, within this autologous model, vaccination with TRP2 peptide in Freund's adjuvant or TRP2-encoding plasmid DNA did not result in protective immunity against B16. Strikingly, despite the observed CTL-mediated melanocyte destruction in vitro, melanocyte destruction in vivo was sporadic and primarily restricted to minor depigmentation of the vaccination site. These results emphasize the potency of DC-based vaccines to induce immunity against autologous tumor-associated antigen and indicate that CTL-mediated antitumor immunity can proceed without development of adverse autoimmunity against normal tissue.     

Induction of interleukin-2, natural killer cell activity and anti-melanoma antibodies resulting from immunization of mice with formalinized extracellular antigens (FECA) of murine melanoma.

Extracellular products from melanoma cells may play an important role in the pathogenesis of metastatic melanoma. Studies were designed to evaluate the effect of vaccination with formalinized extracellular antigens (FECA) of murine melanoma cells (MMM B16-F10) on survival and immune response of C57BL/6 mice. The cellular immune response was evaluated by assessing interleukin-2 (IL-2) production and natural killer cell activity, whereas the humoral immune response was examined by measuring the production of specific antibodies to extracellular antigens (ECA). IL-2 production by the splenocytes from immunized animals was significantly higher (4.7 U/ml and 3.7 U/ml) than that of controls (1.38 U/ml). The splenocytes from immunized mice revealed significantly higher natural killer cell activity. Similarly, immunized animals responded by producing specific antibodies against the extracellular melanoma antigens as detected by ELISA. The peak production of antibodies against ECA was observed on the 21st day post-immunization. These results suggest that FECA are immunogenic and may enhance active cellular and humoral anti-melanoma immunity.     

MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor

Summary Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n=23), untransduced DCs (n=17), or sterile saline (n=22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challengein vivo.