树突状细胞-EBV-LMP2疫苗治疗鼻咽癌的初步研究
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摘要
鼻咽癌(Nasoparyngeal carcinoma, NPC)是我国南方一些省(自治区)的常见恶性肿瘤之一。目前放射治疗是鼻咽癌治疗的基本方法,但中晚期鼻咽癌治疗后常常复发或向远处转移,对于复发和转移的患者在临床上尚无有效治疗方案。树突状细胞(Dendritic cell, DC)为基础的疫苗在治疗肿瘤方面,尤其在某些晚期肿瘤如黑色素瘤、前列腺癌、结肠癌、尿道癌的临床治疗方面已经取得一些的成效,现在已进入Ⅱ期临床试验。但DC在治疗NPC方面,还处于起始阶段。
目前治疗用的DC是其前体细胞在细胞因子作用下体外诱导培养获得的,但对于DC诱导培养条件文献报道不一。本研究首先通过对DC培养条件、前体细胞来源、细胞因子组合、细胞贴壁孵育时间、rAd-LMP2感染DC剂量,细胞冻融方面的进行探索,优化了DC培养条件。
我们课题组以rAd-LMP2以灌胃、肌肉注射和滴鼻的方式感染小鼠,均可诱发小鼠针对LMP2的特异性体液免疫和细胞免疫；EBV-LMP2 DNA疫苗、腺相关病毒(adeno-associated virus,AAV)疫苗、非复制5型腺病毒疫苗(Ad5)联合免疫Balb/C小鼠能够更好地诱导机体产生特异性CTL。rAd-LMP2、rAd5F35-LMP2肌肉免疫恒河猴诱导EBV-LMP2特异性的CTL。pcDNAⅢ-LMP2真核表达质粒免疫小鼠可诱发LMP2特异性细胞和体液免疫。在此基础上,我们以DC-LMP2肌肉免疫方式,研究LMP2诱导的特异性CTL。
通过与广西自治区人民医院合作,在获得伦理委员会批准下,依据自愿原则,选择部分NPC治疗进行初步DC-LMP2皮内免疫治疗,首次以LMP2肽库形式检测DC-LMP2免疫后所激发LMP2特异性CTL,探讨患者特异性CTL与NPC发展进程的关系。
结果显示：普通培养瓶诱导培养DC的效果最差,带滤膜盖的细胞培养瓶次之,六孔细胞培养板诱导培养效果最好。rmGM-CSF+rmIL-4+rmTNF-α组合对骨髓前体细胞诱导培养DC的效果最好,终浓度为rmGM-CSF(200U)+rmIL-4(10U)+rmTNF-α(100U)/mL;对脾脏来源DC进行诱导培养,rmGM-CSF+rmTNF-α组合的阳性率最高,但最终获得DC数量远远少于骨髓来源DC。随着前体细胞孵育时间的增加获得DC细胞数量也随之增多,DC纯度无明显差异。rAd-LMP2感染骨髓细胞诱导培养的DC,以10、100、1000 MOI感染DC,100 MOI感染DC效果最好,40-60%的DC中有LMP2表达。苔盼蓝法检测-80℃、-196℃冻存骨髓和脾脏淋巴细胞复苏后存活率,骨髓细胞存活率(94.8±0.7%和82.85±3.8%)均大于脾脏细胞(80.98±2.2%和68.3±5.2%),DC前体细胞冻存复苏影响其诱导培养。以优化获得的DC培养条件诱导培养骨髓来源的DC,然后DC-LMP2免疫BALB/C鼠可诱导LMP2特异性免疫应答。第5周(308±167/106细胞,对照18±6/106细胞)强于第8周(196±81/106细胞,对照7±2/106细胞)；肌肉注射方式免疫小鼠rAd-LMP2、DC-LMP2所诱导的LMP2特异性CTL, rAd-LMP2组(1178±228/106细胞)大大高于DC-LMP2组((308±167/106细胞)。在小鼠0,2,4周免疫后的第五周,DC-LMP2免疫组CD8+T/CD3+T细胞亚群的比值(22.6±1.7%)高于PBS(20.4±3.5%)和DC组(22.04±1.2%),在第八周DC-LMP2免疫组CD8+T/CD3+T细胞亚群的比值(15.5±1.7%)低于PBS(17.6±2.9%)和DC组(17.5±1.5%)。在小鼠0,2,4周DC-LMP2免疫后,小鼠血清中产生LMP2抗体。
在NPC患者前臂内侧下缘皮内免疫0.1 mL生理盐水,含1-2×105个DC-LMP2,未出现明显过敏反应,表明皮内免疫DC-LMP2的途径、部位、剂量安全可行。参加皮内免疫的部分NPC患者,在在第五周LMP2特异性CTL检测中,15人中9人提高了LMP2特异性CTL(/106),6人在DC-LMP2免疫后LMP2特异性CTL降低。1人在第八周的特异性细胞免疫水平高于治疗前,1人低于治疗前。在NPC免疫前后的IgA/VCA和IgA/EA抗体滴度无明显改变,NPC患者经过初次临床常规治疗后3-21个月内,血清中EBV DNA载量均低于103copies/mL。
总之,rmGM-CSF+rmIL-4+rmTNF-α组合对骨髓前体细胞诱导培养DC的效果最好,终浓度为rmGM-CSF(200U)+rmIL-4(10U)+rmTNF-α(100U)/mL; DC-LMP2免疫BALB/C鼠可诱导LMP2特异性CTL。CD8+T/CD3+T细胞亚群的比值先升高。DC-LMP2免疫NPC患者的的途径、部位、剂量安全可行,部分NPC患者提高了LMP2特异性CTL。
Nasopharyngeal carcinoma (NPC) is one of the common malignancies in southern regions China. Radiotherapy is the basic method of treatment for NPC cases. For patients with recurrence and metastasis there is still no effective treatment. Recent trials using dendritic cells (DC) for clinical immunotherapy make great progress in such advanced tumors as melanoma, prostate cancer, and urologic cancer. Some of them are in clinical phase II trial. However, immunotherapy based on DC for NPC is still in the initial stage.
DC is derived from progenitor cell cultured with cytokines in vitro, but different procedures were reported about DC's culturing condition. In this study, the optimization including screen of vessel, choice of progenitor cell, combination of cytokines, time of adherence, infection dose of recombinant adenovirus encoding Epstein-Barr virus latent membrane protein 2 (rAd-EBV-LMP2), store and thaw were conducted. Our previous data illustrated that LMP2-specific cytotoxicity lymphocyte (CTL) was induced in BALB/c mice with rAd-LMP2 by the way of lavage, intramuscle, and dropping. And powerful response was achieved in BALB/c mice combining pcDNA-LMP2 and adeno-associated virus encoding LMP2 (rAAV-LMP2) with uncompetent rAd-LMP2. The similar response was also observed in rhesus intrmuscle vaccination with rAd-LMP2 and rAd5F35-LMP2. In this study we examine LMP2 specific CTL in BALB/c mice intramuscle vaccination with rAd-LMP2 infected DC. With consensus of ethnic committee, some volunteers in The People's Hospital Guangxi Zhuang Autonomous Region were enrolled. Intradermal vaccinations DC with rAd-LMP2 infected were conducted on those cases. For the first time full LMP2 specific CTL were detected by enzyme-linked immunospot assay (ELISPOT) with LMP2 library.
Result:Comparing to DC in the flask vessel, DC induced in the six-well plastic plate is optimal. The quality of mouse bone marrow-derived DC induced in the culture medium including the combination recombinant murine granulocyte macrophage colony-timulating factor (rmGM-CSF) and recombinant murine inerleukin 4 (rmIL-4) with recombinant murine tumor necrosis factor alpha (rmTNF-α) is optimal. Final concentrations of combining cytokines are rmGM-CSF (200U), rmIL-4(10U), and rmTNF-α(100) per milliliter. The combination rmGM-CSF with rmTNF-αis suit for spleen-derived DC. The number of DC from bone marrow is significantly increased by prolongable hours of conglutination, but the percentage of DC is not obviously discrepant. DC from mouse bone marrow was infected by rAd-LMP2 at doses of 10,100 and 1000 multiplicity of infection (MOI) respectively, and the LMP2 expression was detected by immunoenzyme up to 60 percent DC at 100 MOI. The survival of mouse bone marrow and spleen cells through freezing stock(-80℃and -196℃) and thaw was identified by staining of typan blue, and surviving cell from bone marrow (82.85±3.8% and 94.8±0.7%) is higher than that from spleen(68.3±5.2% and 80.98±2.2%). The procedure of DC deriving from progenitor cells is significantly interrupted through freezing stock and thaw. LMP2 specific CTL in BALB/c mice is detected at 5th week and 8th week and LMP2 specific CTL in BALB/c mice is more powerful at 5th week(308±167 per 106cells, negative control 18±6 per 106cells) than at 8th(196±81 per 106cells, negative control 7±2 per 106cells). Vaccination intramuscle with rAd-LMP2 stimulated more quantity of LMP2 specific CTL (1178±228 per 106cells) than that with DC-LMP2 (308±167 per 106cells) in mice. The ratio of CD8+T to CD3+ T subset is higher at DC-LMP2 group (22.6±1.7%) than injected PBS (20.4±3.5%) and DC (22.04±1.2%) groups at 5th week, but lower (15.5±1.7%) than in PBS(17.6±2.9%) and DC groups(17.5±1.5%) at 8th week. The antibody to LMP2 is detected in mice that DC-LMP2 or rAd-LMP2 was vaccinated.
It is enough safe for NPC cases to vaccinate DC-LMP2 intradermal, and the treatment had minimal side-effects and was well tolerated by all patients. LMP2 CTL significantly increased in 9 of 15 and decreased in 6 of 15 at 5th week, and increased in 1 of 2 and decreased another at 8th week. IgA antibody titers to both virus capsid antigen (VCA) and early antigen (EA) is not significantly increased or decreased, and quantity of EBV DNA is less than 103copies per milliliter in serum comparison pre-and post-vaccination.
Conclusion, combination rmGM-CSF, rmIL-4 with rmTNF-αis optimal choice for mouse bope marrow-derived DC. Final concentrations of combining cytokines are rmGM-CSF (200V), rmIL-4(10U) and rmTNF-α(100) per milliliter. LMP2 specific CTL in BALB/c mice is detected at 5th week and 8th week. The ratio of CD8+T to CD3+ T subset is higher at DC-LMP2 group than PBS and DC groups at 5th week, but lower than in PBS and DC groups at 8th week. It is enough safe for NPC cases to vaccinate DC-LMP2 intradermal, and the treatment had minimal side-effects and was well tolerated by all patients. LMP2 CTL significantly increased in NPC cases.
目前治疗用的DC是其前体细胞在细胞因子作用下体外诱导培养获得的,但对于DC诱导培养条件文献报道不一。本研究首先通过对DC培养条件、前体细胞来源、细胞因子组合、细胞贴壁孵育时间、rAd-LMP2感染DC剂量,细胞冻融方面的进行探索,优化了DC培养条件。
我们课题组以rAd-LMP2以灌胃、肌肉注射和滴鼻的方式感染小鼠,均可诱发小鼠针对LMP2的特异性体液免疫和细胞免疫；EBV-LMP2 DNA疫苗、腺相关病毒(adeno-associated virus,AAV)疫苗、非复制5型腺病毒疫苗(Ad5)联合免疫Balb/C小鼠能够更好地诱导机体产生特异性CTL。rAd-LMP2、rAd5F35-LMP2肌肉免疫恒河猴诱导EBV-LMP2特异性的CTL。pcDNAⅢ-LMP2真核表达质粒免疫小鼠可诱发LMP2特异性细胞和体液免疫。在此基础上,我们以DC-LMP2肌肉免疫方式,研究LMP2诱导的特异性CTL。
通过与广西自治区人民医院合作,在获得伦理委员会批准下,依据自愿原则,选择部分NPC治疗进行初步DC-LMP2皮内免疫治疗,首次以LMP2肽库形式检测DC-LMP2免疫后所激发LMP2特异性CTL,探讨患者特异性CTL与NPC发展进程的关系。
结果显示：普通培养瓶诱导培养DC的效果最差,带滤膜盖的细胞培养瓶次之,六孔细胞培养板诱导培养效果最好。rmGM-CSF+rmIL-4+rmTNF-α组合对骨髓前体细胞诱导培养DC的效果最好,终浓度为rmGM-CSF(200U)+rmIL-4(10U)+rmTNF-α(100U)/mL;对脾脏来源DC进行诱导培养,rmGM-CSF+rmTNF-α组合的阳性率最高,但最终获得DC数量远远少于骨髓来源DC。随着前体细胞孵育时间的增加获得DC细胞数量也随之增多,DC纯度无明显差异。rAd-LMP2感染骨髓细胞诱导培养的DC,以10、100、1000 MOI感染DC,100 MOI感染DC效果最好,40-60%的DC中有LMP2表达。苔盼蓝法检测-80℃、-196℃冻存骨髓和脾脏淋巴细胞复苏后存活率,骨髓细胞存活率(94.8±0.7%和82.85±3.8%)均大于脾脏细胞(80.98±2.2%和68.3±5.2%),DC前体细胞冻存复苏影响其诱导培养。以优化获得的DC培养条件诱导培养骨髓来源的DC,然后DC-LMP2免疫BALB/C鼠可诱导LMP2特异性免疫应答。第5周(308±167/106细胞,对照18±6/106细胞)强于第8周(196±81/106细胞,对照7±2/106细胞)；肌肉注射方式免疫小鼠rAd-LMP2、DC-LMP2所诱导的LMP2特异性CTL, rAd-LMP2组(1178±228/106细胞)大大高于DC-LMP2组((308±167/106细胞)。在小鼠0,2,4周免疫后的第五周,DC-LMP2免疫组CD8+T/CD3+T细胞亚群的比值(22.6±1.7%)高于PBS(20.4±3.5%)和DC组(22.04±1.2%),在第八周DC-LMP2免疫组CD8+T/CD3+T细胞亚群的比值(15.5±1.7%)低于PBS(17.6±2.9%)和DC组(17.5±1.5%)。在小鼠0,2,4周DC-LMP2免疫后,小鼠血清中产生LMP2抗体。
在NPC患者前臂内侧下缘皮内免疫0.1 mL生理盐水,含1-2×105个DC-LMP2,未出现明显过敏反应,表明皮内免疫DC-LMP2的途径、部位、剂量安全可行。参加皮内免疫的部分NPC患者,在在第五周LMP2特异性CTL检测中,15人中9人提高了LMP2特异性CTL(/106),6人在DC-LMP2免疫后LMP2特异性CTL降低。1人在第八周的特异性细胞免疫水平高于治疗前,1人低于治疗前。在NPC免疫前后的IgA/VCA和IgA/EA抗体滴度无明显改变,NPC患者经过初次临床常规治疗后3-21个月内,血清中EBV DNA载量均低于103copies/mL。
总之,rmGM-CSF+rmIL-4+rmTNF-α组合对骨髓前体细胞诱导培养DC的效果最好,终浓度为rmGM-CSF(200U)+rmIL-4(10U)+rmTNF-α(100U)/mL; DC-LMP2免疫BALB/C鼠可诱导LMP2特异性CTL。CD8+T/CD3+T细胞亚群的比值先升高。DC-LMP2免疫NPC患者的的途径、部位、剂量安全可行,部分NPC患者提高了LMP2特异性CTL。
Nasopharyngeal carcinoma (NPC) is one of the common malignancies in southern regions China. Radiotherapy is the basic method of treatment for NPC cases. For patients with recurrence and metastasis there is still no effective treatment. Recent trials using dendritic cells (DC) for clinical immunotherapy make great progress in such advanced tumors as melanoma, prostate cancer, and urologic cancer. Some of them are in clinical phase II trial. However, immunotherapy based on DC for NPC is still in the initial stage.
DC is derived from progenitor cell cultured with cytokines in vitro, but different procedures were reported about DC's culturing condition. In this study, the optimization including screen of vessel, choice of progenitor cell, combination of cytokines, time of adherence, infection dose of recombinant adenovirus encoding Epstein-Barr virus latent membrane protein 2 (rAd-EBV-LMP2), store and thaw were conducted. Our previous data illustrated that LMP2-specific cytotoxicity lymphocyte (CTL) was induced in BALB/c mice with rAd-LMP2 by the way of lavage, intramuscle, and dropping. And powerful response was achieved in BALB/c mice combining pcDNA-LMP2 and adeno-associated virus encoding LMP2 (rAAV-LMP2) with uncompetent rAd-LMP2. The similar response was also observed in rhesus intrmuscle vaccination with rAd-LMP2 and rAd5F35-LMP2. In this study we examine LMP2 specific CTL in BALB/c mice intramuscle vaccination with rAd-LMP2 infected DC. With consensus of ethnic committee, some volunteers in The People's Hospital Guangxi Zhuang Autonomous Region were enrolled. Intradermal vaccinations DC with rAd-LMP2 infected were conducted on those cases. For the first time full LMP2 specific CTL were detected by enzyme-linked immunospot assay (ELISPOT) with LMP2 library.
Result:Comparing to DC in the flask vessel, DC induced in the six-well plastic plate is optimal. The quality of mouse bone marrow-derived DC induced in the culture medium including the combination recombinant murine granulocyte macrophage colony-timulating factor (rmGM-CSF) and recombinant murine inerleukin 4 (rmIL-4) with recombinant murine tumor necrosis factor alpha (rmTNF-α) is optimal. Final concentrations of combining cytokines are rmGM-CSF (200U), rmIL-4(10U), and rmTNF-α(100) per milliliter. The combination rmGM-CSF with rmTNF-αis suit for spleen-derived DC. The number of DC from bone marrow is significantly increased by prolongable hours of conglutination, but the percentage of DC is not obviously discrepant. DC from mouse bone marrow was infected by rAd-LMP2 at doses of 10,100 and 1000 multiplicity of infection (MOI) respectively, and the LMP2 expression was detected by immunoenzyme up to 60 percent DC at 100 MOI. The survival of mouse bone marrow and spleen cells through freezing stock(-80℃and -196℃) and thaw was identified by staining of typan blue, and surviving cell from bone marrow (82.85±3.8% and 94.8±0.7%) is higher than that from spleen(68.3±5.2% and 80.98±2.2%). The procedure of DC deriving from progenitor cells is significantly interrupted through freezing stock and thaw. LMP2 specific CTL in BALB/c mice is detected at 5th week and 8th week and LMP2 specific CTL in BALB/c mice is more powerful at 5th week(308±167 per 106cells, negative control 18±6 per 106cells) than at 8th(196±81 per 106cells, negative control 7±2 per 106cells). Vaccination intramuscle with rAd-LMP2 stimulated more quantity of LMP2 specific CTL (1178±228 per 106cells) than that with DC-LMP2 (308±167 per 106cells) in mice. The ratio of CD8+T to CD3+ T subset is higher at DC-LMP2 group (22.6±1.7%) than injected PBS (20.4±3.5%) and DC (22.04±1.2%) groups at 5th week, but lower (15.5±1.7%) than in PBS(17.6±2.9%) and DC groups(17.5±1.5%) at 8th week. The antibody to LMP2 is detected in mice that DC-LMP2 or rAd-LMP2 was vaccinated.
It is enough safe for NPC cases to vaccinate DC-LMP2 intradermal, and the treatment had minimal side-effects and was well tolerated by all patients. LMP2 CTL significantly increased in 9 of 15 and decreased in 6 of 15 at 5th week, and increased in 1 of 2 and decreased another at 8th week. IgA antibody titers to both virus capsid antigen (VCA) and early antigen (EA) is not significantly increased or decreased, and quantity of EBV DNA is less than 103copies per milliliter in serum comparison pre-and post-vaccination.
Conclusion, combination rmGM-CSF, rmIL-4 with rmTNF-αis optimal choice for mouse bope marrow-derived DC. Final concentrations of combining cytokines are rmGM-CSF (200V), rmIL-4(10U) and rmTNF-α(100) per milliliter. LMP2 specific CTL in BALB/c mice is detected at 5th week and 8th week. The ratio of CD8+T to CD3+ T subset is higher at DC-LMP2 group than PBS and DC groups at 5th week, but lower than in PBS and DC groups at 8th week. It is enough safe for NPC cases to vaccinate DC-LMP2 intradermal, and the treatment had minimal side-effects and was well tolerated by all patients. LMP2 CTL significantly increased in NPC cases.
引文
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