| 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.
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