Abstract：
BACKGROUND: The growing attention to NK cells for cancer cell therapy is associated with the need to establish highly efficient protocols for their genetic modification, particularly by retroviral transduction.
OBJECTIVE: In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets.
METHODS: Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells.
RESULTS: We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts.
CONCLUSIONS: Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.
OBJECTIVE: In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets.
METHODS: Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells.
RESULTS: We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts.
CONCLUSIONS: Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.
摘要：
背景：对NK细胞用于癌细胞治疗的日益关注与需要建立高效的遗传修饰方案有关，特别是通过逆转录病毒转导。
目的：在这项工作中，我们优化了基于逆转录病毒的修饰过程的几个阶段，并确定了氨基酸转运蛋白ASCT2在NK细胞亚群之间的分布。
方法:使用用磷酸钙方法转染的PhoenixAmpho细胞系产生逆转录病毒颗粒。我们将基于RD114的逆转录病毒转导用于淋巴细胞细胞系和原代NK细胞。
结果:我们已经确定了收集RD114假型病毒上清液的最佳时间,导致有效NK细胞修饰所需的病毒颗粒滴度在48至72小时之间。通过基于retronectin的方法进行的逆转录病毒修饰不会改变NK细胞的功能活性和细胞存活。我们鉴定了部分与ASCT2表面表达相关的细胞系之间感染多重性(MOI)的差异。具有较高ASCT2水平的细胞更易于用RD114假型病毒颗粒转导。与它们的阴性对应物相比,在活化的CD57+和KIR2DL2DL3+NK细胞中显示更高的ASCT2表达水平。
结论：我们的发现为NK细胞转导提供了更细致的理解，为改善涉及NK细胞修饰的治疗应用提供有价值的见解。
目的：在这项工作中，我们优化了基于逆转录病毒的修饰过程的几个阶段，并确定了氨基酸转运蛋白ASCT2在NK细胞亚群之间的分布。
方法:使用用磷酸钙方法转染的PhoenixAmpho细胞系产生逆转录病毒颗粒。我们将基于RD114的逆转录病毒转导用于淋巴细胞细胞系和原代NK细胞。
结果:我们已经确定了收集RD114假型病毒上清液的最佳时间,导致有效NK细胞修饰所需的病毒颗粒滴度在48至72小时之间。通过基于retronectin的方法进行的逆转录病毒修饰不会改变NK细胞的功能活性和细胞存活。我们鉴定了部分与ASCT2表面表达相关的细胞系之间感染多重性(MOI)的差异。具有较高ASCT2水平的细胞更易于用RD114假型病毒颗粒转导。与它们的阴性对应物相比,在活化的CD57+和KIR2DL2DL3+NK细胞中显示更高的ASCT2表达水平。
结论：我们的发现为NK细胞转导提供了更细致的理解，为改善涉及NK细胞修饰的治疗应用提供有价值的见解。
