Keever-Taylor C A, Behn B, Konings S, Orentas R, Davies B, Margolis D
Department of Medicine Division of Neoplastic Diseases, Medical College of Wisconsin, WI 53226, USA.
Cytotherapy. 2003;5(4):323-35. doi: 10.1080/14653240310002243.
B lymphoblastoid cell-lines (BLCL), generated by exposure of PBMC to a laboratory strain of EBV, are commonly utilized in the preparation of T cells used for immunotherapy. Although most B cells are latently infected, BLCL contain a subset of cells that harbor infectious virus, which could be released into the infusion product during preparation. To reduce this known risk, laboratories have pretreated BLCL for > or = 14 days with 100 microM acyclovir (ACV), an inhibitor of viral DNA polymerase, prior to use. We tested the effectiveness of ACV in preventing the release of infectious virus from irradiated fresh and previously frozen BLCL, and compared its effects with those of ganciclovir (GCV).
BLCL were grown for 14 days in medium containing various doses of ACV or GCV, washed, irradiated, and tested for the presence of infectious virus in co-culture assays with cord blood mononuclear cells(CBMC) (21 CBMC to BLCL). B-cell transformation was assessed at 3-4 weeks of culture.
Both fresh and previously frozen BLCL released infectious virus, which transformed nearly all (92%) of CBMC co-cultures (n = 52). Transformation was not prevented by treatment with 100 microM ACV (88%, n = 52). Increasing the ACV dose to 200 microM (or 50 microg/mL) still allowed transformation in 4/9 (44%) cultures, while this and higher doses severely reduced the proliferation rate of the BLCL during ACV exposure. Infectious virus release was detectable within 1 day of ACV removal and BLCL irradiation. In contrast, GCV was able to prevent infectious virus release in 12/12 co-cultures at a concentration (15 microM) that only modestly reduced BLCL growth.
These results indicate that GCV is more effective at preventing release of infectious EBV from irradiated BLCL than ACV at concentrations that do not severely inhibit B-cell growth.
通过将外周血单核细胞（PBMC）暴露于实验室株EBV而产生的B淋巴母细胞系（BLCL），常用于制备用于免疫治疗的T细胞。虽然大多数B细胞处于潜伏感染状态，但BLCL含有一部分携带传染性病毒的细胞，这些细胞在制备过程中可能会释放到输注产品中。为降低这一已知风险，实验室在使用前用100微摩尔阿昔洛韦（ACV）（一种病毒DNA聚合酶抑制剂）对BLCL进行了≥14天的预处理。我们测试了ACV在防止传染性病毒从辐照后的新鲜和先前冷冻的BLCL中释放的有效性，并将其效果与更昔洛韦（GCV）进行了比较。
将BLCL在含有不同剂量ACV或GCV的培养基中培养14天，洗涤、辐照，然后在与脐血单核细胞（CBMC）（21个CBMC比1个BLCL）的共培养试验中检测传染性病毒的存在。在培养3 - 4周时评估B细胞转化情况。
新鲜和先前冷冻的BLCL均释放出传染性病毒，这些病毒使几乎所有（92%）的CBMC共培养物发生转化（n = 52）。用100微摩尔ACV处理并不能防止转化（88%，n = 52）。将ACV剂量增加到200微摩尔（或50微克/毫升）仍使4/9（44%）的培养物发生转化，而此剂量及更高剂量在ACV暴露期间严重降低了BLCL的增殖速率。在去除ACV和辐照BLCL后1天内即可检测到传染性病毒的释放。相比之下，GCV在浓度为15微摩尔时能够防止12/12的共培养物中传染性病毒的释放，而该浓度仅适度降低了BLCL的生长。
这些结果表明，在不严重抑制B细胞生长的浓度下，GCV在防止辐照后的BLCL释放传染性EBV方面比ACV更有效。
