肝素酶CTL表位负载的树突状细胞疫苗体外抗肿瘤免疫效应研究

目的研究肝素酶(Hpa)多肽疫苗体外能否诱发肝素酶特异性CTL反应,为肝素酶多肽疫苗的临床应用提供理论依据。方法5条HLA-A2限制性肝素酶抗原表位[Hpa(525-533)(PAFSYSFFV)、Hpa(353-361)(PLLSDTFAA)、Hpa(277-285)(KMLKSFLKA)、Hpa(400-408)(PLPDYWLSL)、Hpa(405-413)(WLSLLFKKL)]分别负载正常人外周血来源(PBMC)的树突状细胞(DC),诱导肝素酶特异性细胞毒T淋巴细胞(CTL),采用4 h标准51Cr释放实验检测上述CTL对不同肿瘤细胞的免疫杀伤效应,采用ELISPOT方法检测肝素酶表位肽负载的DC疫苗刺激的效应细胞IFN-γ的释放。结果5条肝素酶多肽表位中,只有Hpa(525-533)、Hpa(277-285)和Hpa(405-413)体外可以诱导肝素酶表位特异性CTL反应,对Hpa阳性且HLA-A2阳性的KATO-Ⅲ胃癌细胞、U2OS骨肉瘤细胞、SW480结肠癌细胞具有明显的杀伤效应,对HLA-A2阴性的HepG2肝癌细胞和Hpa阴性的MCF-7乳腺癌细胞不具有杀伤效应,但对转染了HLA-A2的HepG2细胞和转染了肝素酶质粒的MCF-7细胞恢复杀伤效应,对自体淋巴细胞不具有杀伤效应,进一步研究表明,Hpa(525-533)、Hpa(277-285)和Hpa(405-413)表位肽可以促进CTL细胞IFN-γ的释放。结论人肝素酶特异性抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)不仅能激发机体特异性的抗肿瘤效应,而且还能诱导一个非特异性抗肿瘤的良性循环,这种肝素酶多肽疫苗具有广谱、高效、特异、安全的优点,为肝素酶表位多肽疫苗的临床应用提供了理论依据。     

Eps8抗原HLA-A* 0201限制性CTL表位的预测和鉴定

目的:寻找并鉴定肿瘤相关抗原Eps8来源的HLA-A*0201限制性CTL表位,为临床开展基于Eps8表位的特异性免疫治疗奠定基础。方法:通过在线生物学软件从C-末端酶切位点、MHC-I类分子结合力及TAP转运效率3个方面初步预测Eps8抗原的HLA-A*0201限制性CTL表位,通过肽/MHC分子结合力及肽/MHC分子复合物稳定性实验初步验证预测结果,利用酶联免疫斑点检测(enzyme-linked immuospot assay,ELISPOT)方法检测候选表位刺激健康志愿者外周血单个核细胞(PBMNC)分泌IFN-γ的能力,以及LDH细胞毒实验验证其体外杀伤肿瘤细胞的功能,最后在HLA-A*0201/Kb转基因小鼠中检测候选表位的体内免疫功能。结果:通过在线生物学软件筛选得到4个候选表位。结合力实验结果显示,p360-368具有高结合力,p101-109、p276-284具有中等结合力。稳定性实验结果显示,该3个表位的DC50均大于8 h。LDH细胞毒实验中p101-109、p276-284、p360-368都能诱导产生特异性CTL,对靶细胞具有显著的杀伤效应,还能提高效应细胞分泌IFN-γ的水平。在HLA-A*0201/Kb转基因小鼠体内免疫功能检测中同样证实该Eps8抗原表位能产生强烈的免疫应答。结论:天然表位p101-109、p276-284、p360-368可能是Eps8抗原HLA-A*0201限制性CTL表位。     

结核杆菌抗原Rv0173的HLA-A*0201限制性CTL表位的预测及鉴定

目的 鉴定结核杆菌(Mycobacterium tuberculosis,Mtb)抗原Rv0173中的HLA-A*0201限制性CTL表位.方法 应用数据库SYFPETTHI预测结核杆菌Rv0173中可能存在的HLA-A*0201限制性CTL表位,经流式细胞术分析各抗原肽与HLA-A*0201的亲合力,经时间分辨荧光法检测外周血单个核细胞(PBMCs)对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定Rv0173的HLA-A*0201限制性CTL表位.结果 位于Rv0173氨基酸序列肽3(21-30aa)和抗原肽7(161-170aa)与HLA-A*0201分子具有较高的亲合力,并都能刺激HLA-A*0201阳性个体PBMCs增殖,并诱导产生特异性杀伤活性.结论 肽3(RLSQSADQYL)(21-30aa)和肽7(LLRGGGLVNL)(161-170aa)是抗原Rv0173上HLA-A*0201限制性CTL的优势表位,可作为结核疫苗设计的候选表位.     

隐球菌抗原MP98的HLA-A*0201限制性CD8+CTL表位的预测及鉴定

目的鉴定隐球菌抗原MP98中的HLA-A＊0201限制性CD8＋CTL表住。方法应用数据库SYFPEITHI预测隐球菌MP98中可能存在的HLA-A＊0201限制性CD8＋CTL表位，经流式细胞术分析各抗原肽与HLA-A＊0201的亲合力，经时间分辨荧光法检测外周血单个核细胞（PBMCs）对各抗原肽产生的增殖反应，经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性，逐步鉴定MP98的HLA-A＊0201限制性CD8＋CTL表位。结果位于MP98氨基酸序列肽5（436-444aa）与HLA-A＊0201分子具有较高的亲合力，并都能刺激HLA-A＊0201阳性个体者PBMC增殖，并诱导产生具有特异性杀伤活性的CTL。结论肽5GMFDGLSGV（436-444aa）是MP98上HLA-A＊0201限制性CD8＋CTL的优势表位，可作为隐球菌疫苗设计的候选表位。     

bcr-abl融合基因真核表达载体诱导小鼠特异性免疫应答

目的在小鼠体内外研究bcr-abl融合基因真核表达载体诱导的特异性免疫应答,探索肿瘤免疫治疗的新途径。方法构建表达bcr-abl融合基因cDNA片段的真核细胞质粒pVbcr-abl,将 pVbcr-abl质粒用纳米颗粒聚乙烯亚胺包裹后给BALB／c小鼠肌内注射,检测小鼠血清中bcr-abl特异性抗体水平。小鼠免疫后20 d皮下接种具有相同遗传背景的SP2／0／ber-abl细胞(H-2~d),观察小鼠生存情况、移植肿瘤的生长情况和肿瘤细胞浸润情况。利用免疫组织化学方法观察肿瘤组织中T淋巴细胞浸润情况;流式细胞术分析免疫小鼠脾脏T细胞亚群的变化;LDH释放法检测脾脏细胞毒性T淋巴细胞(CTL)活性。结果成功构建真核表达载体pVbcr-abl,bcr-abl融合基因cDNA片段在真核细胞中可得到高效表达。pVbcr-abl免疫BALB／c小鼠能激活机体免疫系统,诱导产生特异性抗体和特异性CTL活性,并形成特异性免疫保护力。免疫小鼠的移植肿瘤形成时间、表面出现破溃时间、生长速度明显降低,荷瘤生存时间明显延长。免疫小鼠移植肿瘤组织中有大量CD3~+T细胞浸润。免疫小鼠的脾细胞杀伤SP2／0／bcr-abl细胞的细胞毒活性明显升高。小鼠脾脏T细胞亚群发生改变,CIM~+／ CD8~+细胞比值升高到1．54±0．29。结论pVbcr-abl真核表达载体除能在小鼠体内诱导产生特异性抗体以外,还能诱导高水平特异性CTL活性,直接杀伤肿瘤细胞,抑制肿瘤细胞的生长速度。     

氧化型甘露聚糖修饰的LL/2c肿瘤细胞疫苗诱导Th1免疫反应

目的：观察氧化型甘露聚糖修饰的LL／2c肿瘤细胞疫苗(Oxidative mannan-conugated LL/2c,ox-ML)能否诱导Th1抗瘤免疫反应。方法：乙醇固定的LL/2c细胞(fixed LL／2c,f—L)、ox—M—L腹腔免疫小鼠,取脾脏制备T淋巴细胞悬液,采用ELISA检测体外刺激的T细胞INF-γ、IL-4分泌,^51Cr释放实验观察CTL。(cytotoxic T lymphocytes,CTL)杀伤活性。结果：ox—M—L免疫小鼠的T细胞经丝裂霉素C预处理的LL／2c体外刺激,其INF-γ分泌显著提高：^51Cr释放实验显示出对LL／2c细胞特异性的CTL杀伤活性,且这种杀伤活性具有CD8 T细胞的依赖性：未修饰的LL／2c免疫小鼠的T细胞诱导了较高的IL-4分泌,但未显示出CTL杀伤活性。结论：LL／2c肿瘤细胞作为抗原,经氧化型甘露聚糖修饰有效的诱导了Th1类型的抗瘤免疫反应。     

细胞因子对小鼠结核分枝杆菌感染的免疫保护作用

目的 研究细胞因子IFN-γ和TNF对小鼠结核分枝杆菌感染的免疫保护作用.方法 小鼠结核分枝杆菌感染后第5 d给予IFN-γ和/或TNF治疗,观察治疗后半数死亡时间、一定时间内的死亡率、脾内活菌数,检测脾细胞分泌IFN-γ和IL-4水平(ELLSA法)及MΦ产生NO水平(用Griess试剂).结果 IFN-γ和TNF联用组与单纯攻毒组比较明显延长感染小鼠丰数死亡时间、降低感染小鼠35 d内的死亡率、显著减少感染小鼠脾内活菌数,脾细胞IFN-γ分泌水平明显升高,IL-4分泌水平显著降低,MO产生NO水平明显增加.结论 IFN-γ和TNF联合诱导MΦ产生NO,促进Th1细胞反应,抑制Th2细胞反应,改变了Th1/Th2平衡,对小鼠结核分枝杆菌感染产生保护效应.     

HAC70抗原肽复合物诱导的对艾氏腹水瘤细胞杀伤活性的实验研究

目的观察从艾氏腹水瘤小鼠瘤细胞中提取的热休克蛋白70-抗原肽复合物(HAC70)诱导的抗肿瘤免疫效应,探讨其临床应用。方法提取热休克后的小鼠艾氏腹水瘤细胞经肝素-琼脂糖亲和层析纯化,制成热休克蛋白-肽复合物疫苗,以体内免疫法检测HAC70的抑瘤作用,3H-TdR掺入释放法检测脾细胞对肿瘤细胞的杀伤活性。结果经HAC70免疫的荷瘤小鼠皮下肿瘤结节平均重1.26±0.4 g,较对照组(3.14±0.56 g)明显减轻(P<0.05);当效靶比为200:1时,免疫组脾细胞杀伤活性为49.25±11.25%,显著高于对照组(18.50±9.95%,P<0.01)及荷瘤组(25.75±8.38%,P<0.01)。结论经肝素-琼脂糖亲和层析纯化的HAC70可通过诱导机体产生特异性杀伤艾氏腹水瘤细胞的活性,明显抑制荷瘤小鼠艾氏腹水瘤的生长。     

白细胞介素12:重要的免疫调节因子

IL－12是由巨噬细胞和B细胞产生的一种异二聚体细胞团子,具有多种生物学活性的免疫效应细胞生长刺激因子。能显著增强NK／LAK细胞的杀伤活性,促进特异性CTL细胞的应答能力,诱导γ干扰素,它不仅对细胞免疫具有增强调节作用,且可与其它细胞因子协同作用,有可能成为重要的免疫治疗细胞因子。     

抑制VEGF表达对白血病来源树突状细胞免疫功能的影响

目的：探讨抑制血管内皮生长因子（VEGF）对白血病来源树突状细胞（Dc）免疫功能的影响。方法：利用反义寡核苷酸下调K562白血病细胞VEGF表达，K562细胞经细胞因子作用诱生白血病DC。利用MTT法检测DC刺激正常外周血T细胞增殖的能力及DC激发的细胞毒T淋巴细胞（CTL）杀伤活性；ELISA法检测DC培养上清液中IL-12及DC与外周血单个核细胞（PBMNC）共同培养液中IFN-γ的含量。结果：K562细胞经5μmol／LVEGF反义寡核苷酸处理24h后VEGF蛋白分泌下降59％，其诱生的DC与正常K562来源的DC相比，实验组DC刺激T淋巴细胞增殖能力、CTL杀伤活性以及DC分泌IL-12和协同刺激外周血单个核细胞分泌TNF-γ的能力明显增强。结论：抑制VEGF表达可促进白血病来源DC免疫功能的成熟。     

Multiple antigenic peptides based on H-2K(b)-restricted CTL epitopes from murine heparanase induce a potent antitumor immune response in vivo

Accumulating research suggests that heparanase may be a universal tumor-associated antigen (TAA). Several heparanase T-cell epitopes from humans and mice have already been identified. However, because of low immunogenicity, polypeptide vaccines usually have difficulty inducing effective antitumor immune responses in vivo. In this study, to increase the immunogenicity of polypeptide vaccines, we designed and synthesized two four-branch multiple antigenic peptides (MAP) on the basis of mouse heparanase (mHpa) T-cell epitopes (mHpa398 and mHpa519). The dendritic cells (DC) from mice bone marrow loaded with above MAP vaccines from heparanase were used to evaluate immune response against various tumor cell lines, compared with immune response to their corresponding linear peptides, ex vivo and in vivo. We further assessed IFN-γ release both in CD4(+) T-cell-depleted and nondepleted mice. The results showed that effectors generated from DCs, loaded with MAP-vaccinated mice splenocytes, induced a stronger immune response against target cells expressing both heparanase and H-2K(b) than did effectors generated from mice vaccinated with their corresponding linear peptides. Heparanase-specific CD8(+) T-cell responses induced by MAP and linear peptide vaccination required synergy of CD4(+) T cells. In addition, heparanse-derived MAP vaccines significantly inhibited the growth of B16 murine melanoma in C57BL/6 mice, while also increasing the survival rate of tumor-bearing mice. Our data suggest that MAP vaccines based on T-cell epitopes from heparanase are efficient immunogens for tumor immunotherapy.     

Influenza virus subtype-specific cytotoxic T lymphocytes lyse target cells coated with a protein produced in E. coli

We have tested the ability of the c13 protein, which is a hybrid protein of the first 81 amino acids of the viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutination produced in E. coli, to render target cells susceptible to the lytic activity of influenza virus-specific cytotoxic T lymphocytes (CTL). The results showed that P815 cells coated with c13 protein were lysed by PR8 virus-induced secondary CTL derived from BALB/c mice. Cold-target inhibition tests clearly demonstrated that c13 protein-coated P815 cells were recognized by an H1 subtype-specific CTL population. Furthermore, PR8 virus-induced CTL derived from C3H mice did not lyse c13 protein-coated P815 cells, suggesting that c13 protein was recognized by CTL in conjunction with H-2d products. These findings suggest that this protein interacts with the cellular plasma membrane and makes target cells recognizable by H-2-restricted, influenza virus subtype-specific CTL.     

Role of self and foreign antigenic determinants in allogeneic and self- restricted cytotoxic T cell recognition

Murine spleen cells were sensitized in vitro to H-2 disparate allogeneic spleen cells and assayed on syngeneic target cells conjugated with the trinitrophenyl (TNP)-self or the fluorescein isothiocyanate (FITC)-self haptens, or on syngeneic target cells expressing the male H-Y antigen (H-Y self). The results indicated that allo-induced cytotoxic T lymphocytes (CTL) contained effectors that lysed both hapten-self but not H-Y self targets. Furthermore, it was demonstrated that separate populations of those allogeneic CTL were responsible for the lysis of TNP-self and FITC-self targets. This study also showed that cytotoxic effectors generated against the H-Y antigen with lytic activity equal to or greater than that of an allogeneically induced CTL response were unable to lyse hapten-self targets. These findings provide the first evidence that H-2 alloantigens may be unique in their ability to induce effectors that lyse hapten-conjugated autologous targets. These observations are discussed with respect to the self and foreign antigenic determinants involved in allogeneic and self-restricted CTL models.     

Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.     

Thymus dictates major histocompatibility complex (MHC) specificity and immune response gene phenotype of class II MHC-restricted T cells but not of class I MHC-restricted T cells

Athymic H-2b nude mice received grafts from C57BL/6 (Sendai virus and H-Y antigen cytotoxic T lymphocyte [CTL] responder type), bm1 (H-2Kb mutant, Sendai CTL nonresponder type), or bm12 (H-21-A mutant, H-Y CTL nonresponder type) neonates. In observations of the CTL response to H-Y, both recipients and thymus donors were female. All types of thymus engraftment resulted in mature H-2b splenic T lymphocyte surface phenotype in nude hosts. T cell immunocompetence (as measured by major histocompatibility complex [MHC] CTL responses to allogeneic cells) was restored, and induced nonresponsiveness to the MHC determinants of the engrafted thymus in the nude host. The CTL reaction to Sendai virus in both responder type C57BL/6 and nonresponder type bm1 neonatal thymuses allowed maturation of Sendai-specific, H-2Kb-restricted CTL. For the CTL reaction to H-Y, only responder type C57BL/6 thymuses restored the CTL response, whereas this was not achieved with thymuses from nonresponder type bm12 neonatal females. Results of double thymus (B6 and bm12) engraftment excluded the possibility that this latter effect was caused by suppression. In addition, athymic bm1 mice were engrafted with thymuses from either B6 (Sendai CTL responder type) or syngeneic bm1 neonates (Sendai CTL nonresponder type). Again, both types of neonate thymuses restored T cell competence as measured by MHC/CTL responses to allogeneic cells. However, neither responder B6 nor nonresponder bm1 neonate thymus grafts allowed maturation of Sendai-specific CTL. In conclusion, the thymus dictates MHC specificity and immune response gene phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.     

Friend virus-specific cytotoxic T lymphocytes recognize both gag and env gene-encoded specificities

We have constructed a series of "synthetic" target cell lines for an analysis of the specificity of anti-Friend virus (FV) CTL. Our results show that murine H-2 genes and individual retroviral genes can be stable expressed in Fisher rat embryo (FRE) cells, and that their products have the potential to form target structures recognized by mouse CTL. Cells expressing H-2Db and either the env or gag genes of one component of FV, helper Friend murine leukemia virus (FMuLV), were lysed by anti-FV CTL and by CTL generated against FMuLV alone. Experiments with Db-transfected FRE clones infected only with the replication-defective spleen focus-forming virus (SFFV) component of FV indicate that the SFFV genome also provides specificities recognized by both anti-FV and anti-FMuLV CTL, thus demonstrating the existence of a crossreactive CTL population. An unexpected finding was that anti-FMuLV CTL, but not anti-FV CTL were also able to lyse FRE clones that expressed H-2Kb in either the presence or absence of FV. The use of heterologous cell lines for the construction of synthetic target cells thus offers a useful approach for the analysis of T cell specificity.     

Triggering T cells by otherwise inert hybrid anti-CD3/antitumor antibodies requires encounter with the specific target cell

We have previously demonstrated that H-2Kd-restricted CTL specific for HLA-CW3 or HLA-A24 can recognize synthetic peptides corresponding to residues 170-182 of the HLA molecules. Synthetic oligonucleotides encoding region 170-182 of CW3 or A24 were inserted into the influenza nucleoprotein (NP) gene. We demonstrate herein that P815 (H-2d) cells transfected with the NP-oligo recombinant genes are specifically lysed by HLA-specific Kd-restricted CTL clones. Our results imply that there must be a high degree of flexibility for the expression of T cell epitopes in different molecular contexts.     

Use of DNA-mediated gene transfer to analyze the role of H-2Ld in controlling the specificity of anti-vesicular stomatitis virus cytotoxic T cells

Mouse thymidine kinase (tk-) C3H L (H-2k) cells transformed by the technique of DNA-mediated gene transfer with the herpes simplex virus tk gene together with the BALB/c H-2Ld gene express H-2Ld molecules indistinguishable from their counterparts on spleen cells. An established cloned cell line (8-5) was used to assess the function of the H-2Ld antigen in determining the specificity of alloreactive as well as anti-vesicular stomatitis virus (VSV) cytotoxic T cells (CTL). Both anti-H-2d and anti-H-2Ld CTL displayed a cytotoxic effect against 8-5 cells but not a control cell line transformed with the tk gene only (tk+ cells). Further evidence that 8-5 cells express H-2Ld was provided by the finding that monoclonal anti-H-2Ld but not H-2Dd antibodies blocked target cell lysis by the effector cells. Both BALB/c (H-2d) and DBA/2 (H-2d) animals generated anti-VSV CTL that lysed infected 8-5 but not tk+ cells. To further establish that H-2Ld controlled the specificity of the effector cells, a monoclonal antibody directed against H-2Ld was shown to inhibit lysis of infected 8-5 target cells. To determine whether other H-2d-encoded gene products could serve as restricting antigens for anti-VSV CTL in BALB/c animals, unlabeled VSV infected 8-5 cells were tested for their ability to block lysis of 51chromium-labeled P815 (H-2d)-infected target cells. The 8-5-VSV inhibitor cells inhibited lysis to a slightly lesser extent than unlabeled P815-VSV cells, indicating that H-2Ld plays a major if not exclusive role in restricting anti-VSV CTL in H-2d animals.     

Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity

To increase the antitumor effects of cytosine deaminase (AdCD) gene therapy and induce more potent antitumor immunity, Th1 cytokine interleukin-18 encoded adenovirus (AdIL18) was combined with adenovirus encoding CD (AdCD) for the therapy of established murine B16 melanoma. Combination therapy of the tumor-bearing mice with AdIL 18 and AdCD/5FC inhibited the growth of the subcutaneous B16 tumors more significantly, compared with AdIL 18 or AdCD/5FC alone. In vivo depletion analysis with anti-CD4, anti-CD8 or anti-NK 1.1 McAb illustrated that both CD8+ T cells and CD4+ T cells played key roles in the augmented antitumor response of the combined therapy. Peptide/MHC tetramer represents a powerful and general tool for rapid, highly sensitive, and direct analysis of antigen-specific T cells. In this study, we prepared H-2Kb/TRP-2180-188 tetramer, which was demonstrated to bind H-2Kb-restricted, B16 melanoma-specific CD8+ T cells. B16 specific H-2Kb/TRP2180-188 tetramer was used to stain the tumor-specific CD8+ T cells and the results showed that CD8+ tetramer+ T cells were about 3-5% of the splenic CD8+ T cells derived from tumor-bearing mice after combined therapy. The CTL cytotoxicity was markedly induced in mice after combined therapy, suggesting efficient induction of tumor-specific CD8+ T cells after combined gene therapy with AdCD/5FC/AdIL18. IL-18 gene transfer could significantly augment the cytotoxicity of NK cells and macrophages, and increase the production of interleukin-2 and interferon-gamma, as compared with treatments with AdCD/5FC, AdlacZ/5FC or PBS. These data suggested that in vivo IL-18 gene transfer could augment the antitumor effects of CD suicide gene therapy through efficient induction of antitumor immunity.