【作者】 肖海燕；

【导师】 富宁； 何玉凯；

【作者基本信息】 南方医科大学 ， 免疫学， 2013， 博士

【摘要】 疫苗在抗微生物免疫中取得的巨大成功使得人们开始考虑是否可以用同样的思路来治疗肿瘤。早在1902年Leyden和Blumentha就尝试将肿瘤细胞疫苗用于治疗病人,但结果并不乐观。1959年Graham使用自身肿瘤疫苗治疗了232例妇科肿瘤患者,结果发现虽然自身疫苗可用于治疗患者,少见不良反应,但并不能改变疾病自然病程。尽管经历了挫折和失败,但人们一直没有放弃对肿瘤疫苗的研究。随着人类肿瘤特异性抗原(Tumor Specific Antigen, TSA)和相关抗原(Tumor Associated Antigen, TAA)被证实及其基因克隆的成功,肿瘤的主动免疫治疗再次受到重视,肿瘤疫苗的研究被推向高潮。肿瘤疫苗通过主动免疫诱导机体特异性细胞免疫和体液免疫反应,发挥特异性杀伤与清除作用,达到治疗肿瘤的作用。与用于预防传染病的经典疫苗不同的是,肿瘤疫苗一般为治疗性疫苗,由于肿瘤免疫抑制性微环境的存在和肿瘤抗原的低免疫原性使得肿瘤疫苗诱导的免疫反应非常有限,提示我们应该开发更好的抗原运载系统以在免疫抑制性的环境内诱导出更强的抗弱免疫原性肿瘤抗原的效应性免疫反应。利用重组病毒载体表达肿瘤抗原诱导机体抗肿瘤免疫反应是非常有效的方法之一。重组牛痘病毒载体(vaccinia virus vector, vv)是第一批用于治疗肿瘤的基因载体,vv具有几个突出的优点：免疫原性强；能够运载较长的基因片段；可感染小鼠和人的所有细胞；具有直接溶瘤效应；安全性良好,不会整合到宿主基因中去；易于制备高浓度的病毒颗粒。因此,vv作为有效实用的病毒载体被广泛用于抗感染和抗肿瘤研究中。例如,有研究者利用vv表达gp160,诱导机体产生抗HIV免疫反应,vv表达HPV则可诱导抗HPV特异性免疫反应；还有很多报道利用vv表达细胞因子如GM-CSF或者肿瘤相关抗原如CEA用于抗肿瘤治疗。但vv载体部分的高免疫原性限制了携带的目的抗原部分的免疫反应。慢病毒载体(lentiviral vector, lv)只表达目标抗原,因而诱导出的免疫反应以针对目标抗原为主,而针对载体部分的免疫却较弱,因而特别适合用于运载肿瘤抗原的病毒载体。而且,1v能够转染非分裂性(non-dividing)的树突状细胞(Dendritic cells, DCs),转染后的DCs将1v携带表达的肿瘤抗原有效递呈给免疫细胞,启动一系列免疫反应；另外,1v能够诱导持续性的抗原表达,进而诱导持续性的免疫反应。将表达黑色素瘤相关抗原如NY-ESO,酪氨酸酶相关蛋白1(Tyrosinase-related protein1,TRP1)的1v直接免疫小鼠,机体可产生很强的抗肿瘤抗原特异性免疫反应,并能抑制肿瘤生长。然而,尽管vv和1v是非常优秀的肿瘤疫苗病毒载体,但单一应用疗效有限。我们考虑,这和它们诱导的免疫反应强度和质量有关。传统的疫苗免疫一般是相同疫苗多次免疫以诱导再次免疫应答并增强机体的免疫反应。但机体产生的抗载体免疫影响了目标免疫反应。近十几年以来,有很多报道证明,利用表达相同抗原的不同载体来加强免疫能够诱导更强的免疫反应,能够更有效地治疗肿瘤。所以我们考虑,将1v和vv这两个不同的载体联合应用也许能够诱导更强更有效的免疫反应,从而提高预防和治疗肿瘤的作用。因此,本研究分别构建了表达人黑色素瘤相关抗原gp100(glycoprotein100, gp100O)的1v和vv,并制备了用于免疫小鼠的病毒颗粒。以小鼠黑色素瘤为肿瘤模型,研究联合应用hgp100-lv和hgp100-vv对黑色素瘤的预防和治疗作用。全部工作分为三个部分简述如下。一、表达人黑色素瘤相关抗原gp100的重组1v和vv构建及诱导免疫应答的初步鉴定基于目前对异源性DNA疫苗诱导保护性免疫反应优于同源性DNA疫苗的共识,本研究采用了人黑色素瘤相关抗原gp100为目的抗原,利用常规高保真PCR技术将人gp100基因的N-末端的340个氨基酸基因片段克隆到慢病毒载体上,成功构建了表达人黑色素瘤相关抗原gp100的重组慢病毒载体hgp100-lv,同时也构建了表达人gp100的pG10载体hgp100-pG10。hgp100-lv DNA经磷酸钙共沉淀法转染人293T细胞,hgp100-lv病毒颗粒释放到上清中,经收集和浓缩上清,成功获得可用于小鼠免疫的病毒颗粒,浓度可达1～5×109Tu/ml。应用hgp100-pG10载体和WR系(Western Reserve strain)的Vaccinia virus经同源重组法制备出hgp100-vv病毒颗粒,浓度也可达1～5×109Tu/ml。经小鼠体内免疫接种证实,hgp100-lv比hgp100-vv能够诱导更多的gp100特异性Pmel-1细胞(Thy1.1+CD8+)增殖,并能诱导更强的内源性CD8免疫反应,即小鼠外周血单个核细胞在hgp100多肽的刺激下分泌IFNy的CD8+细胞比例增高；而且hgp100-lv诱导的特异性CD8+T细胞能够识别小鼠gp100(mgp100)。本部分工作成功获得了能够诱导小鼠机体特异性免疫反应的的hgp100-lv和hgp100-vv的病毒颗粒,hgp100-lv诱导特异性CD8+T细胞反应优于hgp100-vv。二、hgp100-lv初免、hgp100-vv加强免疫对黑色素瘤的预防作用迄今为止,多数肿瘤疫苗主要是用于肿瘤的治疗,亦称为治疗性疫苗。而在本研究中,我们对hgp100-lv和hgp-100-vv疫苗是否能够预防黑色素瘤的发生进行了初步观察。给予小鼠进行hgp100-lv足垫免疫接种的结果显示：在免疫反应高峰期,即1v接种后10到14天时,小鼠外周血中约有5-10%的CD8T细胞可特异性分泌IFNγ。利用hgp100225-23四聚体(tetramer)染色发现40%的CD8+T细胞是四聚体阳性的,并且这部分细胞中有20%表达CD127,提示它们是记忆前体细胞,由此证实了1v有较强的诱导免疫记忆反应的活性。1v初免后30到40天,小鼠外周血中特异性CD8+T细胞数目趋于稳定,约2%的CD8+T细胞能够特异性分泌IFNγ。此时于腹腔注射vv加强免疫(1v-vv,即1v初免-VV加强免疫)能够诱导很强的再次免疫应答,明显增强外周血免疫反应,表现为在vv加强免疫后的5到10天外周血白细胞于体外经hgp100多肽刺激后有15%的CD8+T细胞能够分泌IFNγ,而vv-1v(vv初免-1v加强免疫)组只有5%可分泌IFNγ,和1v单独免疫时高峰期效应一致。lv-lv (lv初免-1v加强免疫)组也和1v单独免疫组效果相似。更重要的现象是,1v-vv诱导的免疫反应持续时间长,甚至在加强免疫后两个月外周血中依然可检出5%的CD8+T细胞能够特异性分泌IFNγ;而1v免疫组和vv-1v免疫组均只有1%左右。1v-vv的免疫方法明显增强了小鼠外周血中gp100特异性CD8+T细胞的总数目,也增加了外周血中记忆性CD8+T细胞数目。令人惊奇的是,1v-vv诱导的CD8+T细胞能够快速识别进入机体的肿瘤细胞,迅速被再次激活和增殖,产生效应,清除肿瘤细胞,达到有效预防黑色素瘤作用。通过应用中和抗体清除不同细胞系的实验,我们发现在这种保护作用中,CD8+细胞起主导作用,但CD4+细胞也参与了这种预防作用。此外,无论是1v初免抑或vv加强免疫之前清除CD4+细胞,1v-vv免疫接种诱导的CD8初次免疫反应和再次免疫反应均明显降低。提示此系统中CD8初次免疫反应和再次免疫反应均需要CD4+细胞的帮助。更重要的是,应用小鼠自发肿瘤模型证实hgp100-lv-hgp100-vv初次免疫一加强免疫的方法能够有效预防原发性黑色素瘤,从而证明1v-vv的免疫方法能够有效预防肿瘤。本部分研究证明,与其它免疫方式比较,1v-vv免疫能够诱导更强的(再次)免疫反应,包括特异性CD8+T细胞增殖反应增强及免疫记忆细胞增加,并能够快速感应肿瘤细胞的出现并将其清除,因而能够有效预防黑色素瘤的形成与发展。三、肿瘤局部诱导急性炎症对hgp100-lv免疫的肿瘤浸润效应细胞功能和黑色素瘤治疗效果的影响作用研究近期研究包括我们的前期工作表明肿瘤疫苗对实体瘤的疗效有限,因而应用受限的主要原因之一是肿瘤微环境导致浸润到肿瘤组织的T细胞即肿瘤浸润淋巴细胞(TIL)不能有效发挥其免疫效应。本实验室前期研究也证明了此现象的存在。然而急性感染组织灶中的抗原特异性免疫细胞却能使感染迅速清除。因此,我们提出假设：肿瘤组织引入微生物建立急性炎症环境有可能恢复TIL细胞的抗肿瘤免疫效应。所采用措施是选择对人体安全的溶瘤型牛痘病毒载体,旨在观察牛痘病毒载体能否恢复肿瘤微环境中TIL细胞功能。本研究采用小鼠皮下种植0.3×106小鼠黑色素瘤细胞系B16细胞,生长5天呈进展期的黑色素瘤模型。以hgp100-lv足垫免疫接种,在1v免疫反应效应期(1v免疫后7天。免疫反应始能检测到)于肿瘤局部或腹腔内注射hgp100-vv,然后观察肿瘤的生长情况,并在加强免疫后6到10天分析肿瘤微环境。结果显示,hgp100-lv初免,效应期hgp100-vv (i. p.)加强免疫也能够增强小鼠的系统免疫反应,并且能够增加肿瘤内CD8+和CD4+细胞浸润,但该措施对进展期肿瘤的治疗作用并没有显著提高；而hgp100-lv初免后,效应期肿瘤内注射(i.t.)hgp100-vv并不能增强系统免疫反应,也不能增加肿瘤内CD8+和CD4+细胞浸润,但hgp100-vv (i. t.)的加强免疫能够恢复肿瘤内效应性CD8+T细胞的功能,同时降低Treg的比例,显著增强对进展期肿瘤的治疗效果。为作进一步的机制探讨,我们应用具有相似作用的PolyI:C/CpG (i.t.)以排除vv溶瘤作用的干扰。PolyI:C/CpG为Toll样受体(TLR)激动剂,同样能诱导瘤内急性炎症环境,且已有很多报道证明它们具有促进肿瘤治疗作用。hgp100-lv足垫初免后,效应期PolyI:C/CpG (i.t.)也不能增加肿瘤内CD8+细胞和CD4+细胞的浸润,同时也能够恢复肿瘤内效应性CD8+T细胞的功能,降低瘤内Treg浸润,并增强对进展期肿瘤的治疗作用。机制上,hgp100-vv(i. t.)和PolyⅠ:C/CpG (i.t.)诱导肿瘤内产生的I型IFN起关键作用。PolyⅠ:C/CpG作用于基因敲除I型IFN受体的小鼠,其肿瘤内T细胞功能并不能被恢复；而且PolyⅠ:C/CpG (i.t.)也不能增强hgp100-lv免疫接种的肿瘤治疗作用。此外,PolyⅠ:C/CpG可激活肿瘤内浸润的DC细胞(TIDCs),表现为PolyⅠ:C/CpG(i.t.)处理过的TIDCs表达更高水平的CD40, CD86等DCs成熟分子标志,并具有更强的诱导T细胞增殖能力。激活的TIDCs在肿瘤原位再次激活hgp100-lv激活的CD8+T细胞,恢复CD8+T细胞的功能,进一步促进对肿瘤的治疗作用。本部分研究表明,1v初免后,效应期利用vv或者PolyⅠ:C/CpG于肿瘤局部诱导急性炎症反应能够恢复肿瘤内T细胞的功能,降低Treg细胞的浸润,增强1v初免对肿瘤的治疗作用。综上,本研究证明Iv初免-vv加强的免疫方式可以诱导更强更有效的免疫反应,包括特异性CD8T细胞增殖反应增强及免疫记忆细胞增加,并能够快速感应肿瘤细胞的出现并将其清除,因而能够有效预防黑色素瘤的形成与发展；Iv初免后,效应期vv(i.t.)或者PolyⅠ:C/CpG(i.t.)诱导的肿瘤局部急性炎症能够恢复肿瘤内肿瘤特异性CD8T细胞功能,降低瘤内Treg的浸润,从而增强1V对实体黑色素瘤的治疗作用,为肿瘤的预防和治疗提供了新的方法和理论借鉴。本研究首次报告Iv-vv诱导的记忆性CD8+T细胞能够快速感应和清除肿瘤细胞,有效预防肿瘤,且有效期显著长于用其它方法诱导的预防作用,本研究观察了1v-vv对肿瘤的长期预防作用,观察时间达一年；我们还发现CD4+细胞对hgp100-lv诱导的CD8再次免疫反应也有帮助作用,这和现在的普遍观点不同。本研究还首次提出了诱导肿瘤局部急性炎症可以促进肿瘤特异性CD8+T细胞功能,从而促进对肿瘤的治疗作用。更多还原

【Abstract】 Based on the great success of vaccines in prevention from many infectious diseases, scientists are trying to generate cancer vaccines to treat or prevent tumors. As early as1902, Leyden and Blumentha attempted to treat tumors with tumor cell vaccines. But, their work did not succeed. In1959, Graham treated232women bearing gynecological cancer with autologous cancer vaccines, and found some therapeutic efficiency without any side effects. However this treatment could not change the course of tumor growth. After human tumor specific antigens (TSA) and human tumor associated antigens (TAA) identified and their genes being cloned, active immunotherapy based cancer vaccines has been revitalized and intensively pursued. Cancer vaccines can treat cancers by inducing specific cellular and humoral immune response to kill tumor cells, which differs from the classical vaccine used in prevention of infectious diseases. As therapeutic vaccine, the efficiency of tumor vaccine is restrained by the low magnitude of immune responses due to the weak immunogenicity of tumor self-antigen and by the multiple immunosuppressive factors in tumor microenvironment. One approach for resolving above problems is to develop efficient antigen delivery system to elicit potent immune responses against weak antigen in immunosuppressive microenvironments. Here, we would use recombinant viral vectors as TSA/TAA delivery system to elicit the specific anti-tumor immune response.Recombinant vaccinia virus vector (w) is one of the vectors being earliest used for tumor gene therapy. The several unique features of w include1) high immunogenicity;2) high capacity of accommodating large Ag,3) pan-trpoism of infecting different types of human and mouse cells;4) capability of lyzing tumor cells;5) well established safety;6) easy preparation of large amount of virus. Therefore, w has been widely used in developing vaccines for infectious diseases and cancers. For example, w carrying gp160was immunized to induce anti-HIV immune response in human, and w encoded HPV can elicit HPV specific immune response. Moreover, a series of studies proved that w expressing cytokines (e.g.GM-CSF) or TAA (e.g.CEA) can be used to treat tumors. However the dominant immune responses against viral vector will limit the desired immune responses against target tumor Ags. On the other hand, lentiviral vector (lv) is a very promising vector for delivering tumor antigens because lv often only expresses desired antigen and induces dominant specific antigen immune response. Secondly, lv can efficiently transfect the non-dividing dendritic cells (DCs), resulting in the effective processing and presentation of lv encoded Ags to effector T cells to initiate the immune responses. Additionally, lv can induce persistent Ag expression and elicit long lasting immune response. For example, lv expressing melanoma associated Ag like NY-ESO or TRP1can induce effective specific anti-melanoma immune response and prevent tumor growth in vivo.As the application of single virus vector cannot induce ideal therapeutic effect even though both w and lv are good virus vectors for tumor vaccine, our consideration is to use lv and w with a prime-boost strategy which is expected to induce more potent immune response and enhance the tumor prevention and therapeutic effect. In the past decade, heterologous prime-boost immunizations, which was performed by giving different vaccine delivery system carrying the same antigen was found to be more effective than the ’homologous’ prime-boost approach. In current work, we constructed lv and w which expresses human melanoma antigen gp100respectively, and the corresponding virus particles were prepared for immunization in mice. The preventive and therapeutic effects of lv and vv carrying tumor antigen were observed in vaccinated mouse melanoma model. There are three parts in our work, which are briefly described as follows.Part Ⅰ. Construction and immunization of recombinant lv and vv-expressing human melanoma antigen gp100We constructed recombinant lv-expressing melanoma Ag human gp100because it was previously demonstrated that xenogenic hgp100DNA could better protect mice from B16tumor cell challenge than mouse gp100(mgp100). The gene fragment containing the N-terminal340aa of hgp100was obtained using high-fidelity PCR and cloned into the lv plasmid. The recombinant lv was designated as hgp100-lv. The hgp100-lv DNA was transfected into human293T cells with calcium phosphate transfection method, and the virus particles could be detected in supernatant. We successfully obtained the highly concentrated virus particles (1-5×109Tu/ml). We also constructed hgp100-pG10vector, which was co-cultured with WR vaccinia virus in CV-1cells. The virus particle of hgp100-w with a high titer of1-5×109Tu/ml was obtained by homologous recombination and several rounds of purification. Compare to hgp100-w, hgp100-lv induced more profound in vivo proliferation of hgp10025-33-specific CD8T cells, pmel-1. In addition, using intracellular staining, we found that the activation of endogenous CD8T cells by lv was substantially higher than of recombinant w. Notably, the lv-expressing xenogenic human gp100could induce potent CD8responses, which showed a cross-react with mouse gp100.In summary, we obtained hgp100-lv and hgp100-vv virus particles which can induce specific immune response. The hgp100-lv is more potent than hgp100-w in inducing specific T cell immune responses.Part Ⅱ The preventive effect of vaccination with hgp100-lv-hgp100-w prime-boost in melanoma model.Thus far, most of cancer vaccines are used to treat tumors, which, not surprisingly, did not generate significant antitumor effect because vaccines are intended to prevent diseases. In this work, we first studied if lv and w can prevent melanoma. The mice were immunized with hgp100-lv by footpad injection, and5to10%of CD8+T cells can secrete IFNy after ex vivo stimulated with hgp100peptide.40%of CD8+T cells are hgp10025-33tetramer positive and～20%of tetramer-positive CD8+T cells with CD127+were observed in the peak of the response,10to14days after immunization, which suggests that lv-activated T cells contain a high level of precursors of effector memory CD8+T cells in the peripheral at early stage of immune responses. The CD8immune response decreases to a plateau, when only2%of CD8+T cells can specifically secrete IFNy in30to40days after lv immunization. Boosting immunization with w followed by primary immunization with lv was performed, and the results showed that vv boosting can induce very strong secondary response and substantially enhance the immune response. About15%of CD8+T cells in PBMC can secrete IFNy after ex vivo specific stimulation at5to10days after vv boost, while vv prime-lv boost only induced the same magnitude of immune response as that of the lv-elicited primary responses. And the lv prime-lv boost group also obtained similar level of response. More importantly, the CD8responses induced by lv prime-vv boost were long lasting. More than2months (70d) after boost, the ratio of IFNγ+CD8+T cells in the PBMC was still at the level of5%, while this number decreased to1%in lv or vv-lv immunized groups. The immunization with lv-vv markedly increased the number of hgp100specific CD8+T cells and memory CD8+T cells. Surprisingly, memory CD8+T cells in the lv-vv prime-boosted mice could immediately sense the B16tumor cell challenge, be expanded significantly, and exert the cytotoxic function to prevent tumor occurrence.In order to study the role of CD8+and CD4+effector T cells in cancer immune prevention, CD8, CD4, or both were depleted at2weeks after lv immunization. We found that both CD4+and CD8+effector cells were required for immune prevention while CD8+T cells played dominant role. On the other hand, both primary and secondary immune response were greatly impaired by CD4+T cells depletion, either prior to lv prime or prior to vv boost immunization, indicating that CD4help is required for both primary and secondary CD8immune response in this system. More importantly, the lv-vv prime-boost immunization strategy can prevent tumor growth in autochthonous tumor model, the Grm1Tg mice, suggesting the applicability of this strategy in clinical cancer therapy.In this part, we demonstrated that lv-vv prime-boost immunization can induce stronger (secondary) immune responses. This strategy can potently promote CD8+T cell proliferation and generates a large number of memory CD8+T cells, which can rapidly sense and eliminate the tumor cells and results in effective tumor prevention.Part Ⅲ. The affect of local acute inflammation in TIL function and therapeutic effect of hgp100-lv immunization in melanoma modelThe recent research including our previous work reveals that tumor vaccines could not provoke the efficient antitumor activity on solid tumor, which has been attributed to the tumor microenvironment inducing failure of tumor infiltration lymphocytes (TILs) in tumor tissue. Our previous work also observed the same phenomena. Based on the antigen specific lymphocytes in acute infection tissue can quickly eliminate pathogenic microbial, we hypothesized that induction of acute inflammation in tumor lesions may be capable of rescuing and restoring the effector function of TILs to achieve enhanced antitumor effect. The oncloytic vaccinia vector as an excellent candidate can be used for such purpose. In the current study, the effect of vaccinia vector on rescuing the effector function of TILs in tumor milieu were observed.In this study, a mouse advanced melanoma model was established by which0.3×106B16cells were transplanted to grow in the mice for5days. The mice bearing tumor were immunized with hgp100-lv by footpad injection and boosted in effector phase (7days after lv prime when the immune response can be detected) with hgp100-vv intratumorally (i.t.) or i.p.. The tumor growth curve was recorded and the immunological changes of tumor lesions were analyzed in6to10days after boost. The results showed that hgp100-lv prime and hgp100-vv boost by i.p. at effector phase can enhance the systemic immune response, and also increase the number of tumor infiltrating CD8+and CD4+cells. However, this strategy did not improve the anti-tumor effect significantly by comparing the tumor weight. Interestingly, the boosting with hgp100-w (i.t.) can rescue the effector function of tumor infiltrating CD8T cells which were primed by hgp100-lv, and decrease the Treg percentage in CD4cells and greatly improve the anti-tumor effect, although this i.t. boosting cannot enhance systemic immune response and neither changes the number of TILs cells.Furthermore, we replaced w with TLR3and9ligands (PolyI:C and CpG) that can induce acute inflammation and antitumor effect. The results showed that intratumoral (i.t) injection of TLR3/9ligands (PolyI:C/CpG) during the effector phase effectively rescued the function of lentivector (lv)-activated CD8TILs, decreased the Treg ratio in tumor lesions, and markedly improved the antitumor efficacy of lv immunization. It was proved that the rescue of TILs’ effector function was dependent on type-1IFN that was induced in situ in the tumor lesion by hgp100-w(i.t.) or PolyI:C/CpG (i.t.). But i.t injection of PolyI:C/CpG failed to restore the effector function of lv primed tumor-infiltrating Pmel-1cells in the IFNαPR-/-mice and resulted in similar antitumor effect as lv immunized group. In addition, PolyI:C/CpG can mature and activate tumor infiltrating dendritic cells (TIDCs). For example, the TIDCs expressed higher level of mature markers such as CD40, and CD86, and they were more potent to induce T cell proliferation. Moreover, the activated TIDCs restored the effector function by re-activating hgp100-lv primed CD8T cells in situ and increased the antitumor efficacy.In this part, we proved that i.t injection of vv or PolyI:C/CpG during the effector phase could rescue the function of lv-activated CD8TILs, decreased the Treg ratio in the tumor lesion, and markedly improved the antitumor efficacy of lv immunization.Taken together, lv-vv prime-boost immunization strategy can provoke stronger immune responses, by inducing more potent CD8+T cell proliferation and larger numbers of memory CD8T cells, which can rapidly sense and eliminate the tumor cells to result in effective tumor prevention. In the therapeutic setting, i.t injection of w or PolyI:C/CpG during the effector phase can effectively rescue the function of lv-activated CD8TILs, decrease the Treg ratio in the tumor lesion, and markedly improve the anti-solid tumor efficacy resulted from lv immunization. The whole study has provided new strategy and insight for cancer immunotherapy based tumor vaccine. It is the first time to prove that the memory CD8T cells induced by lv-vv prime-boost can rapidly sense and eliminate the tumor cells as well as effectively prevent tumors growth. The preventive protection can protect the mice for at least one year, which is much more persistent than that induced by other methods. We also found that CD4T cell help is required for CD8secondary response during hgp100-lv immunization, which is different from the prevailing view. It is the first report to prove that the acute inflammation in tumor lesions can rescue the CD8T cell effector function and improve the antitumor efficacy.更多还原