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目的研究表达US3基因重组腺病毒载体对其转染肝细胞介导的免疫逃逸活性。方法首先构建表达US3基因腺病毒载体,扩增纯化后转染HL-7702肝细胞,利用细胞毒性T细胞（CTL）杀伤实验和自然杀伤细胞（NK）杀伤实验,检测表达US3基因重组腺病毒载体的免疫活性。结果表达US3基因的重组腺病毒r-US3成功构建,r-US3能够明显降低CTL和NK细胞对转染肝细胞的杀伤活性。结论表达US3基因腺病毒载体在一定程度上能够介导转染肝细胞的免疫逃逸,降低机体免疫系统对转染肝细胞的排斥反应。

目的构建中国人丙型肝炎病毒（HCV）复合细胞毒性T淋巴细胞（CTL）多表位基因的真核表达载体,并观察其在真核细胞中的表达。方法根据生物信息学方法筛选中国人的HCV多个CTL优势表位,合成复合多表位抗原基因（mcf）;将其克隆入真核表达载体pEGFP-N1,转染COS-7细胞,在荧光显微镜下观察mcf-EGFP融合蛋白在细胞中的分布和定位,RT-PCR及Western blot检测其mRNA及蛋白表达。结果成功构建了真核表达载体pEGFP-mcf。将构建的真核表达载体pEGFP-mcf转染COS-7细胞后,绿色荧光分布于COS-7细胞胞质中;而转染空载体pEGFP-N1后,绿色荧光弥散分布于COS-7细胞胞核及胞质中。RT-PCR检测到转染细胞中mcfmRNA表达;West-ern blot结果证明合成mcf可在COS-7细胞中表达。结论该真核表达载体的成功表达及鉴定,为下一步中国人的HCV复合CTL多表位疫苗的研究奠定了基础。

背景与目的新城疫病毒HN蛋白是新城疫病毒产生溶瘤作用的重要免疫原。在前期体外实验基础上,比较定位表达细胞不同部位的HN蛋白体内抗肿瘤免疫作用。方法通过构建荷瘤小鼠,瘤内注射定位表达于细胞不同部位的新城疫病毒HN蛋白,即胞浆型（Cy-HN）、跨膜型（M-HN）、分泌型（Sc-HN）重组真核表达质粒,比较荷瘤小鼠的肿瘤生长速度,脾淋巴细胞增殖反应和细胞毒T细胞活性。结果瘤内注射跨膜型重组真核表达质粒的荷瘤小鼠肿瘤生长缓慢,与瘤内注射胞浆型和分泌型重组真核表达质粒的荷瘤小鼠相比有统计学差异（第18天：P=0.022;第21天：P〈0.01）,同时,该组荷瘤小鼠的淋巴细胞增殖反应和细胞毒T细胞活性也较高[M-HNvsCy-HN,P=0.019;M-HNvsSc-HN,P=0.043;M-HNvspcDNA3.1（＋）,P〈0.01]。结论定位表达于细胞不同部位的HN蛋白体内抗肿瘤免疫作用存在差异,跨膜型HN蛋白的重组DNA质粒能够提高荷瘤小鼠的特异性细胞免疫应答。

目的 探讨自制的一种白血病疫苗对C57BL／6小鼠细胞毒性T淋巴细胞（CTL）杀伤功能的影响。方法 建立白血病荷瘤小鼠模型，用自制的3种不同的白血病疫苗进行预防或主动免疫治疗，用MTT比色法检测预防或治疗2周、4周后小鼠CTL杀伤活性，并与对照组相比较。结果 ①随着白血病细胞在小鼠体内的生长，小鼠的CTL杀伤功能受到严重抑制。②灭活肿瘤细胞+IFA+细胞因子（rGM-CSF+rIL-2+rIL-6)的肿瘤疫苗在提高小鼠CTL的杀伤功能方面，优于灭活肿瘤细胞+IFA肿瘤疫苗，而仅含灭活肿瘤细胞的疫苗作用不明显。结论 灭活肿瘤细胞+IFA+细胞因子（rGM－CSF+rIL-2+rIL-6)的肿瘤疫苗可以激活以CTL为代表的特异性细胞免疫功能，此种疫苗在血液恶性肿瘤的特异性主动免疫治疗中很有潜力。

目的 探讨细胞毒性T淋巴细胞相关抗原4(CTLA-4)基因外显子1的49位点和启动子的318位点多态性与中国人自身免疫性甲状腺病(AITD)的相关性。方法 共收集50例GD，46例HT患者和50例正常对照静脉血标本提取基因组DNA。采用多聚酶链反应—限制性片段长度多态性分析(PCR-RFLP）方法，分别用Bbv I和Mse I检测外显子1和启动子的多态性。计算CTLA-4的基因型和等位基因频率。结果 ①与正常人相比，外显子1GG纯合子基因型发生频率在AITD组显著高于正常对照组；而AA纯合子基因型和AG杂合子基因型发生频率均显著低于正常对照组；G等位基因频率显著高于对照(P＜0．01)。②GD患者启动子的CC纯合子基因型发生频率明显高于正常对照；HT杂合子基因型发生频率明显低于正常对照；C等位基因频率显著高于正常对照(P＜0．01，OR＝2．61)；HT患者虽然有较多的CC基因型及较少的HT基因型，但与正常对照无显著性差异。⑦GD患者启动子为CC纯合子，外显子1为GG基因型显著高于对照(P＜0．01，OR＝2．38)，计算连锁不平衡系数揭示启动子的C等位基因和外显子1的G等位基因有连锁不平衡。结论 CTLA-4基因外显子1G49等位基因与AITD显著相关；CTLA-4基因启动子的多态性(C—T)与GD的相关性是由于其与外显子1连锁不平衡，因此它是GD的一个遗传危险标志，但不具有独立易感作用。

The cytotoxic T lymphocyte antigen-4 （CTLA-4）-blocking antibody ipilimumab induces immune-mediated longterm control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the a and β subunits of the IL-2 receptor （CD25 and CD122, respectively） abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells （Tregs）, which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4^＋ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor u （IL-2Ra, sCD25） inhibited the anticancer effects of CTLA-4 blockade. In 262 meta- static melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.

Regulatory T （Treg） cells may participate in mediating a suppressive microenvironment that blunts successful anti-tumor immunotherapy. Recent studies show that CD8＋ Treg cells might impede effective immune responses to established tumors. However, there is limited research regarding CD8＋ Treg cells in ovarian cancer （OC） patients. Here, we investigated CD8＋ Treg cells in OC patients and their in vitro induction. The immunohistochemistry of tumor-infiltrating lymphocytes revealed a significant correlation between the intratumoral CD8＋ T cells and the forkhead box p3 （Foxp3）＋ cells in the intraepithelial and stromal areas of advanced OC tissues. We examined the expression of Treg markers in CD8＋ T cells from the peripheral blood and fresh tumor tissues of OC patients using flow cytometry. Our results indicated an increase in the CD8＋ Treg cell subsets of OC patients compared with those in patients with benign ovarian tumors and healthy controls, including a n increased expression of CD25, cytotoxic T-lymphocyte-associated protein 4 （CTLA-4）, and Foxp3 and decreased CD28 expression. To demonstrate whether the tumor microenvironment could convert CD8＋ effector T cells into suppressor cells, we used an in vitro transwell culturing system. Compared with the CD8＋ T cells cultured alone, the CD8＋ Treg cells induced in vitro by coculture with SK-OV-3/A2780 showed increased CTLA-4 and Foxp3 expression and decreased CD28 expression. In addition, the in vitro-induced CD8＋ Treg cells inhibited naive CD4＋ T-cell proliferation, which was partially mediated through TGF-β1 and IFN-γ. Our study suggests that CD8＋ Treg cells were increased in OC patients and could be induced in vitro, which may be the way that tumors limit antitumor immunity and evade immune surveillance.

CD8＋ cytotoxic T lymphocyte （CTL） exhaustion is a chief issue for ineffective virus elimination in chronic infectious diseases. We generated novel ovalbumin （OVA）-specific OVA-Texo and HIV-specific Gag-Texo vaccines inducing therapeutic immunity. To assess their therapeutic effect in chronic infection, we developed a new chronic infection model by i.v. infecting C57BL/6 mice with the OVA-expressing adenovirus AdVova. During chronic AdVova infection, mouse CTLs were found to express the inhibitory molecules programmed cell-death protein-1 （PD-1） and lymphocyte-activation gene-3 （LAG-3） and to be functionally exhausted, showing a significant deficiency in T-cell proliferation, IFN-7 production and cytolytic effects. Naive CD8＋ T cells upregulated inhibitory PD-ligand 1 （PD-L1）, B- and T-lymphocyte attenuator and T-cell anergy-associated molecules （Grail and Itch） while down-regulating the proliferative response upon stimulation in mice with chronic infection. Remarkably, the OVA-Texo vaccine counteracted T-cell anergy and converted CTL exhaustion. The latter was associated with （i） the upregulation of a marker for CTL functionality, diacetylated histone-H3 （diAcH3）, （ii） a fourfold increase in CTLs, occurring independent of host DCs or CD4＋ T cells, and （iii） the restoration of CTL IFN-7 production and cytotoxicity. In vivo OVA-Texo-stimulated CTLs upregulated the activities of the mTORC1 pathway-related molecules Akt, S6, elF4E and T-bet, and treatment of the CTLs with an mTORC1 inhibitor, rapamycin, significantly reduced the OVA-Texo- induced increase in CTLs. Interestingly, OVA-Texo-mediated CD40L signaling played a critical role in the observed immunological effects. Importantly, the Gag-Texo vaccine induced Gag-specific therapeutic immunity in chronic infection. Therefore, this study should have a serious impact on the development of new therapeutic vaccines for human immunodeficiency virus （HIV-1） infection.

Cytotoxic T lymphocyte （CTL） epitope peptide-based vaccines are widely used in cancer and infectious disease therapy. We previously generated an immune-tolerant elastin-like polypeptides （iTEPs）-based carrier to deliver a peptide CTL vaccine and enhance the efficiency of the vaccine. To further optimize the vaccine carrier, we intended to potentiate its function by designing an iTEP-based carrier that was able to deliver adjuvant and a vaccine epitope as one molecule. Thus, we fused a 9-mer H~oo, a peptide derived from the high-mobility group box 1 protein （HMGB1） that could induce activation of dendritic cells （DCs）, with an iTEP polymer to generate a new iTEP polymer named Hloo-iTEP. The Hloo-iTEP still kept the feature of reversible phase transition of iTEPs and should be able to be used as a polymer carrier to deliver peptide vaccines. The expression levels of CD80/CD86 on DCs were assessed using flow cytometry. The iTEP fusion-stimulated IL-6 secretion by DCs was measured with ELISA. Activation of antigen-specific CD8~ T cells induced by iTEP fusions was examined through a B3Z hybridoma cell activation assay. In vivo CTL activation promoted by iTEP fusions was detected by an IFN-v-based ELISPOT assay. The iTEP fused with Hloo could induce maturation of DCs in vitro as evidenced by increased CD80 and CD86 expression. The iTEP fusion also promoted activation of DCs by increasing secretion of a proinflammatory cytokine IL-6. The N-terminus or C-terminus fusion of Hloo to iTEP had a similar effect and a reduced form of cysteine in iTEP fusions was required for DC stimulation, iTEP fusions potentiated a co-administrated CTL vaccine by increasing an antigen-specific CTL response in vitro and in vivo. When the Hzoo-iTEP was fused to a CTL epitope to generate a one-molecule vaccine, this self-adjuvanted vaccine elicited a stronger antigen-specific CTL response than a vaccine adjuvanted by Incomplete Freund＇s Adjuvant. Thus, we have successfully generated a functional, one-molecule iTEP-based self-adjuvanted vaccine.

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 （PGE2） and recruit myeloid-derived suppressor cells （MDSCs）. Activated cytotoxic T lymphocytes （CTLs） induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8^＋ T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.