自然杀伤细胞（NK细胞）常通过辐照、工程化的K562喂养细胞来扩展，这些喂养细胞表达膜结合的（mb）IL15和/或mbIL21以及41BBL的核心转基因。以往关于mbIL15和mbIL21的NK扩展的比较缺乏对其产生的NK细胞的关键属性进行比较，包括高维表型、多功能性、细胞毒性的广度和强度、细胞代谢以及异种移植瘤模型的活性。此外，尽管进行了多轮的K562刺激，但关于mbIL15-和mbIL21-基于喂养细胞的连续使用的研究缺乏。我们填补了这些空白，发现使用mbIL15与mbIL21基于喂养细胞驱动出不同的表型和功能特性。仅表达mbIL15的喂养细胞在几乎所有指标上都具有优越的功能，而仅表达mbIL21的喂养细胞则具有优越的产量。在联合使用中，大多数属性类似于mbIL21产生的属性，而在连续使用时，NK产量接近第一个细胞因子带来的产量，并且表型、转录组和功能类似于第二个细胞因子驱动的细胞，突显了NK细胞分化的可塑性。mbIL21接着mbIL15的顺序在2个/ 3个异种移植模型中实现了产量显著的高功能NK细胞，显示出与仅通过mbIL15扩展的细胞相当的体内活性。我们的研究结果确定了mbIL15与mbIL21在NK扩展中的影响，并揭示了NK产量和功能之间以往未被重视的权衡，在许多情况下，连续使用基于mbIL21之后的基于mbIL15的喂养细胞可能是最佳方法。

Natural killer (NK) cells are frequently expanded for the clinic using irradiated, engineered K562 feeder cells expressing a core transgene set of membrane-bound (mb) IL15 and/or mbIL21 together with 41BBL. Prior comparisons of mbIL15 to mbIL21 for NK expansion lack comparisons of key attributes of the resulting NK cells, including their high dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL15- and mbIL21-based feeder cells are absent. We addressed these gaps and found that using mbIL15 versus mbIL21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL15 alone drove superior functionality by nearly all measures, whereas those expressing mbIL21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL21, whereas in sequence, NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine, highlighting the plasticity of NK cell differentiation. The sequence mbIL21 followed by mbIL15 was advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL15 alone in 2 of 3 xenograft models. Our findings define the impact of mbIL15 versus mbIL21 during NK expansion and reveal a previously underappreciated tradeoff between NK yield and function for which sequential use of mbIL21- followed by mbIL15-based feeder cells may be the optimal approach in many settings.