Schmueck-Henneresse Michael, Omer Bilal, Shum Thomas, Tashiro Haruko, Mamonkin Maksim, Lapteva Natalia, Sharma Sandhya, Rollins Lisa, Dotti Gianpietro, Reinke Petra, Volk Hans-Dieter, Rooney Cliona M
Institute for Medical Immunology, Charité University Medicine Berlin, D-13353 Berlin, Germany;
Renal and Transplant Research Unit, Department of Nephrology and Internal Intensive Care, Charité University Medicine Berlin, D-13353 Berlin, Germany.
J Immunol. 2017 Jul 1;199(1):348-362. doi: 10.4049/jimmunol.1601494. Epub 2017 May 26.
The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45ROCCR7 effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells.
嵌合抗原受体（CAR）修饰的T细胞治疗结果受到输注T细胞亚群来源的强烈影响。然而，由于多克隆激活的T细胞在扩增后无论亚群来源如何都会获得主要为CD45RO⁺CCR7⁻效应记忆表型，因此无法知道哪些亚群对最终的T细胞产物有贡献。为了确定初始T细胞、记忆性干细胞T细胞、中枢记忆T细胞、效应记忆T细胞和终末分化效应T细胞群体对我们用于治疗的经CD3和CD28激活的CAR修饰T细胞的贡献，我们追踪了经单独分选的CAR修饰T细胞亚群在用CD3和CD28抗体（CD3/28）激活、单独转导和培养后，或重组成相关亚群耗竭群体后的命运和功能。我们发现所有亚群对CAR转导均敏感，并且在培养过程中各自形成了独特的T细胞功能谱。与记忆来源的群体相比，初始来源的T细胞增殖率最高，但效应功能更有限且杀伤能力降低。当与记忆T细胞一起培养时，初始来源的T细胞显示出分化增加、效应细胞因子产生减少以及对CAR刺激的再增殖反应降低。与白细胞介素-2相比，在白细胞介素-7和白细胞介素-15中扩增的CD3/28激活的T细胞能使记忆性干细胞T细胞和中枢记忆T细胞来源的T细胞产生更大程度的扩增。我们的策略提供了一个强大的工具，可阐明CAR修饰T细胞的特征，无论用于扩增的方案如何，揭示每个扩增的T细胞亚群的功能特性，并为更详细评估生产变化对体外扩增T细胞亚群贡献的影响铺平道路。
