B7转染人肝癌细胞株的建立及生物学特性的实验研究

研究共刺激信号在肿瘤免疫中的作用。脂质体介导DNA转移法将入B7基因转入人肝癌细胞株Smmc7721,建立新的细胞株B7^ Smmc7721,PCR及逆转录PCR（RT-PCR）法鉴定。四唑盐（MTT）比色试验体外检测白细胞介素-2（IL-2）激活的LAK细胞（LAK-C）对两种肝癌细胞的杀伤活性。PCR,RT-PCR法证实B7^ Smmc7721细胞稳定表达B7基因,LAK细胞对B7^ Smmc7721细胞的杀伤活性明显高于Smmc7721,结果有统计学意义。B7基因可以体外转染人肝癌细胞,并表达有活性的B7分子,并可明显增强IL-2激活的LAK细胞的肿瘤杀伤作用,为进一步开展临床应用奠定基础。     

肝癌细胞裂解物致敏的树突状细胞瘤苗体外诱导特异性抗肝癌免疫

目的 探讨肝癌患者肿瘤细胞裂解物致敏的树突状细胞(DC)瘤苗体外诱导自体T淋巴细胞特异性抗肝癌免疫效应。 方法 从肝癌患者外周血单个核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏。用流式细胞仪检测D C细胞表面分子的表达,酶联免疫吸附法检测T淋巴细胞培养上清液中干扰素(I F N)γ和白细胞介索-12(IL-12)的含量,液体闪烁计数仪测定肝癌细胞裂解物致敏的D C刺激自体T淋巴细胞增殖效应,四甲基偶氮唑盐法检测肝癌细胞裂解物致敏D C诱导的细胞毒T淋巴细胞对自体肝癌细胞的特异性杀伤作用。 结果 肝癌细胞裂解物致敏的DC瘤苗可上调DC表面CD1 a、CD40、CD86和人类白细胞抗原-DR分子表达水平,其与T淋巴细胞共培养产生的IFN γ、IL-12的浓度明显高于未致敏的D C组(t值分别为2.30、2.14,P<0.05),肝癌细胞裂解物组(t值分别为14.01、15.40,P<0.01)和对照组(t值分别为14.85、16.87,P<0.01)。同时肝癌细胞裂解物致敏的瘤苗可明显诱导T淋巴细胞的增殖,其诱导的细胞毒性T淋巴细胞对自体肝癌细胞的杀伤率(81.72％±9.49％)显著高于对HepG2的杀伤率(49.37％±11.21％)和人鼻咽癌肿瘤细胞的杀伤率(17.14％±5.65％),P<0.01。 结论 肝癌细胞     

树突状细胞激活的肿瘤浸润性淋巴细胞抗胃癌活性的研究

目的　树突状细胞 (DC)是目前已知的功能最强的抗原提呈细胞 (APC) ,可以向包括肿瘤浸润性淋巴细胞 (TIL)在内的T淋巴细胞提呈抗原 ,并诱发细胞毒T淋巴细胞 (CTL)反应。该文探讨树突状细胞激活的肿瘤浸润性淋巴细胞体外对胃癌细胞 (SGC 790 1 )的杀伤活性。方法　从胃癌患者外周血获取DC ,应用粒 /巨噬细胞集落刺激因子 (GM CSF)、白介素 4(IL 4)和肿瘤抗原激活DC ,然后用DC激活TIL ,观察TIL在体外对自体胃癌细胞和人胃癌细胞株细胞的杀伤活性。结果　DC激活的TIL具有很高的对自体胃癌细胞杀伤活性 ,杀伤率为 (89.39± 3 .0 5) % ,明显高于未经DC激活的TIL、CD激活的T淋巴细胞和未经DC激活的T淋巴细胞对自体胃癌细胞的杀伤率 [杀伤率分别为 (54 .37±1 .50 ) % ,(53 .92± 1 .46) %和 (3 .55± 0 .2 5) % ]。而它们对SGC 790 1细胞的杀伤活性则相对较低。结论　胃癌患者外周血DC能诱导TIL产生高效而特异的抗胃癌免疫     

葡萄球菌肠毒素A和B7.1双刺激分子共转染人肝癌细胞及其免疫学效应

超抗原(SAg)是来源于细菌和病毒家族的蛋白组份,可未经处理而直接结合于组织相容性复合物Ⅱ分子并激活含特定Vβ成份的T细胞。为开辟超抗原在T细胞介导的肿瘤免疫排斥中的新途径,我们将葡萄球菌肠毒素A(SEA)超抗原分子及共刺激分子B7.1的基因直接转入人肝癌细胞,从而诱发强的免疫排斥反应。     

趋化因子Fractalkine对小鼠实验性肝癌的基因治疗

目的 通过将趋化因fFractalkine（FK）基因转染小鼠肝癌细胞株MM45T．Li，探讨FK用于诱导抗肝癌主动免疫的可行性。方法 用脂质体将小鼠FK基因导入小鼠肝癌细胞株MM45T．Li，G418筛选抗性克隆。逆转录聚合酶链反应检测FK的表达，体内实验观察野生型及FK基因修饰肿瘤细胞的致瘤性，组织病理学检测肿瘤中免疫细胞的浸润，流式细胞仪检测外周血中CD4^＋、CD8^＋T淋巴细胞水平。结果 逆转录聚合酶链反应检测发现G418筛选得到的转染FK的抗性克隆MM45T．Li-FK表达FK，而野生型MM45T．Li不表达FK。体内成瘤实验显示，与野生型瘤细胞相比，MM45T．Li-FK的成瘤率显著下降。MM45T．Li-FK所形成肿瘤中有明显的免疫细胞浸润，对照组肿瘤中免疫细胞的浸润较少。接种MM45T．Li-FK瘤细胞的小鼠，外周血中CD4、CD8^＋T淋巴细胞水平明显增高，与对照组相比差异均有统计学意义（P〈0．01）。结论 转染FK皋因能促进机体的抗肝痛主动免疫反应。     

经kinectin融合蛋白刺激的人B细胞诱导效应T细胞对肝癌细胞的杀伤

目的:探讨经人动力素(kinectin)融合蛋白刺激的人外周血B细胞诱导效应T细胞杀伤肝癌细胞的效果.方法:取健康人外周血,分离出单个核细胞,加B细胞刺激因子及kinectin麦芽糖融合蛋白进行体外培养,尔后用尼龙毛柱分离T细胞,将其与肝癌细胞株7404混合培养.用乳酸脱氢酶法检测杀伤肝癌细胞的活性.结果:T细胞对肝癌细胞的杀伤率:空白对照组:3.40%±0.27%;实验组:38.48%±2.64%;阴性对照组:13.03%±2.38%.经统计软件分析,实验组杀伤率与其他组有明显差异.结论:经kinectin融合蛋白刺激的人外周血B细胞诱导的效应T细胞在体外对肝癌细胞株7404有杀伤的作用.     

人Midkine启动子驱动的HSV-TK自杀基因体外抗肝癌作用

目的 观察以人Midkine(MK)启动子调控单纯疱疹病毒胸苷激酶基因(HSV-TK)/丙氧鸟苷(GCV)自杀基因系统在体外对人肝癌细胞的杀伤效应.方法 以含有人MK启动子调控的HSV-TK基因的重组腺病毒分别感染体外培养的甲胎蛋白(AFP)阳性人肝癌细胞BEL-7402和AFP阴性人肝癌细胞SMMC-7721,RT-PCR法检测HSV-TK基因在上述两种细胞中的转录表达,观察GCV对人肝癌细胞的杀伤作用.结果 GCV在体外对重组腺病毒感染的BEL-7402及SMMC-7721均有明显的杀伤作用,又以前者为著.相同的病毒滴度,其杀伤作用随着GCV浓度的增高而增强.均具有旁观者效应.结论 在体外表现HSV-TK基因的AFP阳性及阴性人肝癌细胞均可被人MK启动子调控的自杀基因HSV-TK杀伤.     

CD95基因抑制胃癌细胞生长的体外抑瘤效应

目的　将CD95基因导入胃癌细胞 ,建立CD95基因表达株 ,并比较转导前后mRNA与蛋白的表达水平。观察CD95蛋白对体外培养胃癌细胞的抑制作用。方法　采用分子克隆技术将CD95基因插入真核表达载体pBK CMV的多克隆克隆位点之间 ,以脂质体介导法将目的基因导入受体细胞SGC790 1,用G418筛选克隆细胞 ;以Northernblot,Westernblot检测CD95基因的表达。MTT法检测转导株对化疗药物的敏感性 ;直接记数法描述转导株的细胞生长曲线 ;软琼脂集落形成实验观察基因转导前后细胞的克隆形成力。结果　成功地构建了真核表达载体pBK CD95cDNA。转导细胞后 ,从 1× 10 5细胞中筛选出 10 0个抗性克隆以上 ,转导率大于 0 1% ,随机挑选 2个克隆扩增培养 ,获得了 1株稳定的抗性细胞 ,从而有效地建立了CD95基因表达株 (SGC790 1CD95cells)。杂交结果表明 ,转导株在mRNA及蛋白水平的表达均明显高于非转导株。转导细胞的细胞倍增时间、对数生长期等均体现了比非转导株更为缓慢和处于抑制状态 ,集落形成能力低下 ,而对VCR、5 FU等化疗药物的敏感性明显增强。结论　CD95基因在胃癌细胞中处于低表达状态 ;通过真核表达载体的介导 ,CD95基因导入胃癌细胞后 ,能有效地表达CD95mRNA及其蛋白。CD95基因转导株的表达蛋白能有效地抑制体外培     

支气管哮喘患者记忆CD+4T淋巴细胞活化相关基因的研究

目的 筛选及鉴定支气管哮喘(简称哮喘)患者记忆CD+4 T淋巴细胞活化相关基因.方法 使用差异显示聚合酶链反应(DDPCR)方法筛选哮喘患者和健康人记忆CD+4 T淋巴细胞差异表达基因,采用逆转录-聚合酶链反应(RT-PCR)法检测差异表达基因的mRNA表达水平.统计学处理采用SPSS 10.0软件.计量资料采用-x±s表示,组间比较采用双因素方差分析,P＜0.05为差异有统计学意义.结果 经DDPCR克隆获得了19条差异片段,经同源性分析显示其中2条片段分别与白细胞介素-7(IL-7)和MM-1基因高度同源,采用RT-PCR检测进一步证实了这2个基因在哮喘患者激活的记忆CD+4 T淋巴细胞中表达白细胞介素-7和MMI基因激活后分别为0.390±0.029、0.629±0.047(F值分别为983、1264,P均＜0.05).结论 哮喘患者激活的记忆CD+4 T淋巴细胞中IL-7和MM-1基因的上调表达可能是哮喘患者与健康人对于变应原出现不同反应的分子机制之一.     

CD95抑制大肠癌细胞生长的实验研究

目的建立表达外源性CD95基因的大肠癌细胞株,观察CD95表达细胞株在CD95抗体作用下对体外培养的大肠癌细胞的抑制效应。方法采用分子克隆技术将CD95基因插入真核表达载体pBK-CMV的多克隆位点之间。以脂质体介导法将目的基因导入受体细胞的HT-29,用G418筛选克隆细胞。以Northern blot,Western blot检测转导细胞CD95基因的表达。MTT法和直接记数法以及软琼脂集落形成实验检测转导株在CD95抗体作用下的细胞增殖水平、生长曲线及细胞克隆形成率。结果成功地构建了真核表达载体pBK-CMV/CD95 cDNA。转导细胞并经筛选后,获得了2株稳定的抗性细胞,从而建立了CD95基因表达株(HT-29 CD95 cells)。杂交结果表明、转导株CD95mRNA及其蛋白水平的表达均明显高于非转导株,转导细胞增殖速度、倍增时间、对数生长期等均比非转导株更为缓慢和处于抑制状态,集落形成能力低下,但差异无显著性意义,而在CD95抗体作用下效果更为显著,差异有非常显著性意义。结论 CD95基因在大肠癌细胞中处于低表达状态;通过真核表达载体的介导,CD95基因导入大肠癌细胞后,能有效地表达CD95mRNA及其蛋白。CD95表达细胞株在CD95抗体的作用下可明显抑制体外培养的大肠癌细胞的生长增殖,其作用机制与CD95诱导细胞凋亡有关。     

Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and enhanced green fluorescence protein genes in primary T lymphocytes

The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41.8 +/- 9.3% SD (range 31.1-48.4%) EGFP-positive cells and a mean of 92 +/- 2% SD (range 90-94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 microg/ml x 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity.     

Superantigen-SEA gene modified tumor vaccine for hepatocellular carcinoma： An in vitro study

AIM: To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to express staphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC. METHODS: SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80. Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine, positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFgamma-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells. Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by (51)Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control. RESULTS: Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05). CONCLUSION: Tumor cell membrane expressed superantigen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.     

Lymphocytes as cellular vehicles for gene therapy in mouse and man.

The application of bone marrow gene therapy has been stalled by the inability to achieve stable high-level gene transfer and expression in the totipotent stem cells. We show that retroviral vectors can stably introduce genes into antigen-specific murine and human T lymphocytes in culture. Murine helper T cells were transduced with the retroviral vector SAX to express both neomycin-resistance and human adenosine deaminase genes. These cells were expanded in culture and selected for expression of neomycin resistance with G418. The gene insertion, selection, and culture expansion did not alter antigen specificity or growth characteristics of the T cells in vitro. To determine if cultured T cells might be used for gene therapy, their persistence and continued expression of the introduced genes was evaluated in nude mice transplanted with the SAX-transduced T cells. G418-resistant cells could be readily recovered from the spleens of recipients of transduced T cells for several months. In addition, recovered cells continued to produce human adenosine deaminase. Based on these observations, we studied cultured human tumor-infiltrating lymphocytes as a candidate cell for a trial of gene transfer in man. Exponential cultures of interleukin-2-stimulated tumor-infiltrating lymphocytes were efficiently transduced with the neomycin-resistance gene using the retroviral vector N2. Gene insertion and subsequent G418 selection did not substantially alter the growth characteristics, interleukin 2 dependence, membrane phenotype, or cytotoxicity profile of the transduced T cells. These studies provided a portion of the experimental evidence supporting the feasibility of the presently ongoing clinical trials of lymphocyte gene therapy in cancer as well as in patients with adenosine deaminase deficiency.     

Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT-29 cells. METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo. RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditions in vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed. CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo. These cells with EGFP are a valuable tool for in vivo research of tumor metastatic spread.     

Generation of cytotoxic T cell against HBcAg using retrovirally transduced dendritic cells

AIM: Cytotoxic T lymphocytes (CTLs) play an important role in resolving HBV infection. In the present study, we attempted to evaluate the efficiency of bone marrow-derived dendritic cells (DCs) transduced with recombinant retroviral vector bearing hepatitis B virus (HBV) core gene and the capability of generating CTLs against HBcAg by genetically modified DCs in vivo. METHODS: A retroviral vector containing HBV core gene was constructed. Replicating DC progenitor of C57BL/6 mice was transduced by retroviral vector and continually cultured in the presence of recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin-d(IL-4) for 6 days. LPS was added and cultured for additional two days. The efficiency of gene transfer was determined by PCR, Western blot and FACS. Transduced DCs immunized C57BL/6 mice subcutaneously 2 times at an one-week interval. Intracellular IFN-γ, and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphocytes stimulated with mitomycin C-treated EL4-C cell which stably expresses HBcAa was determined bv LDH release assays. RESULTS: Recombinant retroviral expression vector (pLCSN) was positively detected by PCR as well as enzyme digestion with EcoRI and BamH I. Retroviruses were generated by pLCSN transfection packing cell and the virus titer was 3&#215;10^5 CFU/ml. Indirect immunofluorescence and FACS showed that HBV core gene was expressed in murine fibroblasts. Transduced bone marrow cells had capability of differentiating into DCs in vitro in the presence of rmGM-CSF and rmIL-4. The result of PCR showed that HBV core gene was integrated into the genome of transduced DCs. Western blot analysis showed that HBV core gene was expressed in DCs. The transduction rate was 28 % determined by FACS. Retroviral transduction had no influence on DCs expressions of CD80 and MHC class II. HBcAg specific CTLs and Thl type immune responses could be generated in the mice by using transduced DCs as antigen presenting cells (APCs). CONCLUSION: Retroviral transduction of myeloid DCs progenitors expresses efficiently HBcAg, and genetically modified DCs evoke a higher CTLs response than HBcAg in vivo.     

Peripheral blood lymphocytes as target cells of retroviral vector- mediated gene transfer

Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T- cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.     

Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions.

Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.     

An in vivo assay for retrovirally transduced human peripheral T lymphocytes using nonobese diabetic/severe combined immunodeficiency mice

OBJECTIVE: Availability of a mouse model to analyze human peripheral lymphocytes genetically modified with retroviral vectors would be useful in T-cell-directed gene transfer studies. To address this issue, we assessed the ability of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to maintain such cells in their peripheral blood. MATERIALS AND METHODS: Human peripheral lymphocytes stimulated with recombinant human interleukin-2 (rhIL-2) and anti-CD3 and CD28 antibodies were transduced with the enhanced green fluorescent protein (EGFP) gene using the retroviral vector GCsap(MSCV) and then transplanted into NOD/SCID mice at 1 x 10(8) cells per mouse. RESULTS: Transplanted human peripheral lymphocytes survived and expressed EGFP in the mice over the 6- to 8-week posttransplant period without any signs of graft-vs-host disease. Of importance was that these cells remained at the G(0)/G(1) stage and again proliferated in response to cytokines when cultured in vitro. Interestingly, the mice in which the transduced T lymphocytes remained at the resting stage clearly elucidated the superiority of the murine stem cell virus (MSCV) LTR to maintain the transgene expression by nonproliferating T lymphocytes over the Moloney murine leukemia virus (MoMLV)- and myeloproliferative sarcoma virus (MPSV)-derived LTRs, which was obscure in in vitro culture where the transduced lymphocytes was being stimulated with rhIL-2. CONCLUSIONS: The mouse model and GCsap(MSCV) vector described herein comprise a simple and reliable in vivo assay system for studies of gene and cell therapies employing human peripheral lymphocytes.     

In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.     

Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells

Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.