Long-term expansion using IL-7 and IL-15 increases T cell yield with preserved Tcm/Tscm properties suitable for adoptive T cell therapy. Long-term expansion using IL-7 and IL-15 increases T cell yield with preserved Tcm/Tscm properties suitable for adoptive T cell therapy

Adoptive T cell therapy (ATT) requires activation, expansion and, for certain indications, gene-modification of T cells. Sufficient numbers of T cells and the characteristics of the final T cell product decisively determine the success of treatment. In this study, we addressed the question if prolonged expansion by simply using the cytokine combination IL-7/IL-15 could enhance the T cell yield without sacrifying T cell functionality. Prolonged expansion resulted in a 50-fold increase of CD8+ central memory T cells (Tcm). RNA sequencing and differential analysis of short-term expanded (ST) and long-term expanded (LT) murine CD8+ and CD4+ Tcm revealed that LT T cells retain a gene expression profile related to Tcm/stem central memory T cells (Tscm). Upon anti-CD3/CD28 stimulation, ST and LT T cells proliferated and underwent apoptosis comparably. IL-2 and INF- production was higher for LT T cells. Engraftment, persistence and anti-tumor capacities of LT murine T cells were preserved after in vivo administration. We confirmed our in vitro findings for human T cells. Our study demonstrates the feasibility of manufacturing an increased number of favorable T cells with Tcm/Tscm properties for ATT by a prolonged, minimally manipulative expansion protocol using the cytokines IL-7 and IL-15. Overall design: transcriptome profiling of long-term T-cell cultures

过继性T细胞疗法（Adoptive T cell therapy, ATT）需要对T细胞进行活化、扩增，针对部分适应症还需开展基因修饰。足够数量的T细胞与最终T细胞产品的特性，是决定治疗成功与否的关键因素。本研究旨在探讨：单纯采用细胞因子组合IL-7/IL-15进行长时间扩增，是否可在不牺牲T细胞功能的前提下提升T细胞收获量。

长时间扩增可使CD8+中枢记忆T细胞（central memory T cells, Tcm）的数量提升50倍。对短期扩增（short-term expanded, ST）与长期扩增（long-term expanded, LT）的小鼠CD8+及CD4+ Tcm进行RNA测序与差异表达分析，结果显示LT组T细胞保留了与Tcm/干细胞样中枢记忆T细胞（stem central memory T cells, Tscm）相关的基因表达谱。

经抗CD3/CD28刺激后，ST组与LT组T细胞的增殖与凋亡水平相当。LT组T细胞的IL-2与干扰素γ（IFN-γ）分泌水平更高。小鼠LT组T细胞的体内植入能力、存活持久性与抗肿瘤活性在给药后仍得以保留。我们在人类T细胞中验证了上述体外实验结果。

本研究证实，通过采用IL-7与IL-15细胞因子的低操作量长时间扩增方案，可制备获得数量更多、具备Tcm/Tscm特性的优质T细胞，用于过继性T细胞疗法。总体实验设计：长期T细胞培养物的转录组分析。