慢病毒介导的转化生长因子β不敏感肿瘤特异性细胞毒性T淋巴细胞的构建及鉴定
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摘要
目的肿瘤细胞分泌大量的转化生长因子β(transforming growth factor-β,TGF-β),具有潜在的免疫抑制功能。因此,肿瘤细胞能够逃避宿主细胞的免疫监视而进展和转移。肿瘤分泌的TGF-β抑制宿主的免疫系统,特别是细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)对其的识别杀伤。鉴于TGFβ在肿瘤发展过程中的负性免疫调节作用,使CTL对TGFβ失去敏感性可以恢复其对肿瘤的杀伤活性。因此,TGF-β是抗肿瘤治疗的重要靶点之一。肿瘤免疫治疗大多着眼于增强机体免疫系统的抗肿瘤活性,所以针对肿瘤免疫抑制的免疫疗法是很有前景的。肿瘤特异性CTL是体内抗肿瘤免疫最为有效的效应细胞,但其激活需要抗原呈递细胞(antigen-presenting cells,APC)处理并呈递相应的抗原。树突状细胞(Dendritic cells,DC)是目前已知功能最强的APC,可以激活初始T细胞(naive T cell)和记忆性T细胞。DC可提供T细胞活化所需的共刺激信号,在体内外诱导CTL的生成。肿瘤组织匀浆、细胞裂解物包含全部抗原信息,用其致敏DC细胞,体外可以激发T细胞大量增殖,产生肿瘤特异性CTL。鉴于TGFβ在肿瘤发展过程中的负性免疫调节作用,使CTL对TGFβ失去敏感性可以恢复其对肿瘤的杀伤活性,用含有TβRⅡDNglytk质粒的慢病毒来感染肿瘤特异性CTL使其对TGFβ失去敏感性后发挥抗肿瘤效应。本实验中将HSV-tk引入,当肿瘤细胞被消除或CTL量过大时,给予适量GCV能够使这些CTL自杀,避免其产生毒副作用。
方法我们用TOPO技术将显性负相TGF-βⅡ型受体(dominant negative TGFβtypeⅡreceptor,TDRⅡDN)与单纯疱疹病毒胸苷激酶(herpes simplex virusthymidine kinase,HSV-tk)连接起来后转入慢病毒载体,构建表达TβRⅡDNglytk的慢病毒,并构建TRANSglytk为对照;用DC、肿瘤组织匀浆、T淋巴细胞培养出肿瘤特异性CTL;然后以慢病毒为载体将TβRⅡDNglytk基因转染到肿瘤特异性CTL,从而去除了TGF-β对肿瘤特异性CTL的抑制作用,恢复CTL对肿瘤的识别杀伤;最后通过荧光抗体染色,Western-blot,MTT,流式细胞术等方法对其进行了分析、鉴定。Smad2/3磷酸化是TGFβ活化的直接结果,是TGFβ信号传导通路的关键点所在,TGFβ处理过的CTL若P-Smad2/3表达缺失则认为是TGFβ信号传导通路被阻断,细胞对TGFβ不敏感。Western blot分析不同类型CTL磷酸化的Smad2/3(P-Smad2/3)量可以鉴别其对TGFβ的敏感性。表达TRANSglytk的CTL为对照。
结果1利用重组PCR技术连接TβRⅡDN(TRANS)与HSV-tk连接起来转入慢病毒载体,测序验证正确,成功构建了表达TβRⅡDNglytk及对照TRANSglytk的慢病毒。
2外周血来源PBMC,在IL-4、GM-CSF诱导下,可得到DC。负载肿瘤抗原DC,激活T细胞,可使其大量扩增,得到肿瘤特异性CTL。
3以慢病毒为载体可将TβRⅡDN-tk基因转染到肿瘤特异性CTL,Anti-v5-TIFC荧光抗体染色证实了转染成功,Western-blot检测P-Smad2/3蛋白表达明显减少,证明转染TβRⅡDNglytk基因后TGF-β信号传导通路阻断,对正常表达的TGF-βⅡ型受体产生了竞争性抑制作用。
4转染TβRⅡDN-tk,TRANS基因对淋巴细胞表型,凋亡,周期无明显影响。GCV对携带HSV-tk自杀基因的TGF-β不敏感CTL杀伤活性结果表明GCV能够杀死经慢病毒转染后表达HSV-tk的细胞,而未转染组细胞几乎无死亡。
结论对TGF-β不敏感肿瘤特异性CTL培养成功及鉴定表明:这一方法在肿瘤免疫治疗中具有潜在的应用前景,为肿瘤的过继免疫治疗提供了又一新的思路。
Objective Cancer cells produce large amounts of TGF-β,which is a potent immunosuppressant.As a result,cancer cells are able to escape the host's immune surveillance program,leading to tumor progression and metastasis.Tumor immunotherapy has been focused largely on the enhancement of host immune responses against cancer.Tumor-derived TGF-β,however,is capable of inhibiting the host immune system,especially the cytotoxic T lymphocyte(CTL)at the site of tumor growth.Therefore,TGF-βis an attractive target for anti-cancer therapy.We render immune cells insensitive to TGF-β,these immune cells should be able to reject the tumor.On the other hand,CTLs are the most important effect cell in anti-tumor immune response in vivo.CTLs must be activated through the presentation of appropriate antigen by antigen-present cells(APC).Dendritic cells(DC)are the potentest APC.As we known,they can activate not only na(?)ve T cells but memory T cell.CTL can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells.Our hypothesis states that if we render CTLs insensitive to TGF-β,these immune cells should be able to reject the tumor.We are willing to developed a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGFβtypeⅡreceptor(TβRⅡDNglytk)expression vector.
Methods We developed a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGF-βtypeⅡreceptor(TβRⅡDN)expression vector.First we construct the Lentiviral plenti6/v5-D-TOPO vector containing TβRⅡDnglytk.TβRⅡDN were obtained from the plasmid pAdTrack-CMV by PCR,then connected with HSV-tk by recombinant PCR.After the agarose gel electrophoresis was performed,the TβRⅡDNglytk was retrieved,and then were cloned into Lentiviral vector pLenti6/V5-TOPO by TOPO Cloning technique.The present study also involved isolation and generation of dendritic cells,T cells from patients with bladder cancer.We primed the CTL with tumor cell lysates and dendritic cells.Tumor specific CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of tumor cells and IL-2,IL-4,GM-CSF.These CTLs were rendered insensitive to TGF-βby introducing the dominant negative TGF-βtypeⅡreceptor with the herpes simplex virus thymidine kinase(HSV-tk)connected in the intra-cellular domains (TβRⅡDNglytk)in the lentiviral construct.The following tests will be carried out Western blot analysis for SMAD-2 phosphorylation was peformed with different types of CTLs to verify that the TβRⅡDNglytk(and TRANSglytk)transfected CTLs are insensitive to TGF-β.CTLs transfected with TRANSglytk will be used as the control.
Results 1.The present experiment demonstrate a successful cloning of Tβ3RⅡDNglytk(TRANSglytk)into the Lentiviral vector pLenti6/V5-TOPO.The Sequencing map of plenti6/v5-D-TOPO-TβRⅡDNglytk vector accorded with theoretic analyses,and a successful construction Lentivirals contain TβRⅡDNglytk (TRANSglytk)by 293FT cells.
2.Tumor specific CTLs were generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of tumor cells and IL-2,IL-4,GM-CSF.
3.Since Smad-2 phosphorylation is a direct result of activation of TGF-βactivation,an absence of Smad-2 phosphorylation following TGF-βtreatment of CTL will be an indication that the cells are insensitive to TGF-β.The effectiveness of tumor-specific TGF-βinsensitive CTL were determined using FACS, immunofluorescence,western-blot,MTT and so on.
4.The transfection of Lentiviral containd TβRⅡDNglytk(TRANSglytk) were innocuity to the CTLs.CTLs which express TβRⅡDNglytk could be killed by GCV,and the control were still alive.
Conclusion Our experiment made a foundation for further application of tumor-specific TGF-βinsensitive CTL in adoptive immunotherapy of tumors.These CTL will be used for adoptive transfer in the subsequent studies.
方法我们用TOPO技术将显性负相TGF-βⅡ型受体(dominant negative TGFβtypeⅡreceptor,TDRⅡDN)与单纯疱疹病毒胸苷激酶(herpes simplex virusthymidine kinase,HSV-tk)连接起来后转入慢病毒载体,构建表达TβRⅡDNglytk的慢病毒,并构建TRANSglytk为对照;用DC、肿瘤组织匀浆、T淋巴细胞培养出肿瘤特异性CTL;然后以慢病毒为载体将TβRⅡDNglytk基因转染到肿瘤特异性CTL,从而去除了TGF-β对肿瘤特异性CTL的抑制作用,恢复CTL对肿瘤的识别杀伤;最后通过荧光抗体染色,Western-blot,MTT,流式细胞术等方法对其进行了分析、鉴定。Smad2/3磷酸化是TGFβ活化的直接结果,是TGFβ信号传导通路的关键点所在,TGFβ处理过的CTL若P-Smad2/3表达缺失则认为是TGFβ信号传导通路被阻断,细胞对TGFβ不敏感。Western blot分析不同类型CTL磷酸化的Smad2/3(P-Smad2/3)量可以鉴别其对TGFβ的敏感性。表达TRANSglytk的CTL为对照。
结果1利用重组PCR技术连接TβRⅡDN(TRANS)与HSV-tk连接起来转入慢病毒载体,测序验证正确,成功构建了表达TβRⅡDNglytk及对照TRANSglytk的慢病毒。
2外周血来源PBMC,在IL-4、GM-CSF诱导下,可得到DC。负载肿瘤抗原DC,激活T细胞,可使其大量扩增,得到肿瘤特异性CTL。
3以慢病毒为载体可将TβRⅡDN-tk基因转染到肿瘤特异性CTL,Anti-v5-TIFC荧光抗体染色证实了转染成功,Western-blot检测P-Smad2/3蛋白表达明显减少,证明转染TβRⅡDNglytk基因后TGF-β信号传导通路阻断,对正常表达的TGF-βⅡ型受体产生了竞争性抑制作用。
4转染TβRⅡDN-tk,TRANS基因对淋巴细胞表型,凋亡,周期无明显影响。GCV对携带HSV-tk自杀基因的TGF-β不敏感CTL杀伤活性结果表明GCV能够杀死经慢病毒转染后表达HSV-tk的细胞,而未转染组细胞几乎无死亡。
结论对TGF-β不敏感肿瘤特异性CTL培养成功及鉴定表明:这一方法在肿瘤免疫治疗中具有潜在的应用前景,为肿瘤的过继免疫治疗提供了又一新的思路。
Objective Cancer cells produce large amounts of TGF-β,which is a potent immunosuppressant.As a result,cancer cells are able to escape the host's immune surveillance program,leading to tumor progression and metastasis.Tumor immunotherapy has been focused largely on the enhancement of host immune responses against cancer.Tumor-derived TGF-β,however,is capable of inhibiting the host immune system,especially the cytotoxic T lymphocyte(CTL)at the site of tumor growth.Therefore,TGF-βis an attractive target for anti-cancer therapy.We render immune cells insensitive to TGF-β,these immune cells should be able to reject the tumor.On the other hand,CTLs are the most important effect cell in anti-tumor immune response in vivo.CTLs must be activated through the presentation of appropriate antigen by antigen-present cells(APC).Dendritic cells(DC)are the potentest APC.As we known,they can activate not only na(?)ve T cells but memory T cell.CTL can be induced by DCs which can provide co-stimulating signal that is indispensable to activation of T cells.Our hypothesis states that if we render CTLs insensitive to TGF-β,these immune cells should be able to reject the tumor.We are willing to developed a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGFβtypeⅡreceptor(TβRⅡDNglytk)expression vector.
Methods We developed a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGF-βtypeⅡreceptor(TβRⅡDN)expression vector.First we construct the Lentiviral plenti6/v5-D-TOPO vector containing TβRⅡDnglytk.TβRⅡDN were obtained from the plasmid pAdTrack-CMV by PCR,then connected with HSV-tk by recombinant PCR.After the agarose gel electrophoresis was performed,the TβRⅡDNglytk was retrieved,and then were cloned into Lentiviral vector pLenti6/V5-TOPO by TOPO Cloning technique.The present study also involved isolation and generation of dendritic cells,T cells from patients with bladder cancer.We primed the CTL with tumor cell lysates and dendritic cells.Tumor specific CTL will be generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of tumor cells and IL-2,IL-4,GM-CSF.These CTLs were rendered insensitive to TGF-βby introducing the dominant negative TGF-βtypeⅡreceptor with the herpes simplex virus thymidine kinase(HSV-tk)connected in the intra-cellular domains (TβRⅡDNglytk)in the lentiviral construct.The following tests will be carried out Western blot analysis for SMAD-2 phosphorylation was peformed with different types of CTLs to verify that the TβRⅡDNglytk(and TRANSglytk)transfected CTLs are insensitive to TGF-β.CTLs transfected with TRANSglytk will be used as the control.
Results 1.The present experiment demonstrate a successful cloning of Tβ3RⅡDNglytk(TRANSglytk)into the Lentiviral vector pLenti6/V5-TOPO.The Sequencing map of plenti6/v5-D-TOPO-TβRⅡDNglytk vector accorded with theoretic analyses,and a successful construction Lentivirals contain TβRⅡDNglytk (TRANSglytk)by 293FT cells.
2.Tumor specific CTLs were generated by isolate lymphocytes cocultured with dendritic cells in the presence of lysates of tumor cells and IL-2,IL-4,GM-CSF.
3.Since Smad-2 phosphorylation is a direct result of activation of TGF-βactivation,an absence of Smad-2 phosphorylation following TGF-βtreatment of CTL will be an indication that the cells are insensitive to TGF-β.The effectiveness of tumor-specific TGF-βinsensitive CTL were determined using FACS, immunofluorescence,western-blot,MTT and so on.
4.The transfection of Lentiviral containd TβRⅡDNglytk(TRANSglytk) were innocuity to the CTLs.CTLs which express TβRⅡDNglytk could be killed by GCV,and the control were still alive.
Conclusion Our experiment made a foundation for further application of tumor-specific TGF-βinsensitive CTL in adoptive immunotherapy of tumors.These CTL will be used for adoptive transfer in the subsequent studies.
引文
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