Division of Stem Cell Transplantation and Immunology, Department of Children and Adolescents, University Hospital Frankfurt, Frankfurt, Germany.
Department of Children and Adolescents, University Hospital Frankfurt, Frankfurt, Germany.
Front Immunol. 2022 Mar 2;13:830773. doi: 10.3389/fimmu.2022.830773. eCollection 2022.
Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given.
嵌合抗原受体 (CAR) T 细胞疗法是治疗复发或难治性血液系统恶性肿瘤的一种有效新方法。由于监测 CAR T 细胞动力学可以深入了解治疗的活性，因此适当的 CAR T 细胞检测方法至关重要。在这里，我们报告了一种用于外周血 CD19 CAR T 细胞检测的流式细胞术检测的全面验证。此外，还对 CAR T 细胞和 B 细胞水平随时间的变化进行了回顾性分析（n = 30），并对 CAR T 细胞表型进行了特征分析。系列稀释实验表明，精确和线性定量可低至 0.05%的 T 细胞或 22 个 CAR T 细胞事件。在使用 CAR T 细胞阴性对照样本确认了计算得出的 13 个事件的检测限。与实时 PCR 的方法比较显示出相当大的相关性。稳定性测试表明，在样本采集后一天，CAR T 细胞值就已经下降。虽然我们发现大多数患者（12/17）具有长期的 CAR T 细胞可检测性和 B 细胞无功能，但有些患者（5/17）经历了 B 细胞恢复。在其中三名患者中观察到 CAR T 细胞和再生 B 细胞共存。重复 CAR T 细胞输注可导致可检测但有限的再扩张。将 CAR T 细胞亚群与其在所有 T 细胞中的对应物进行比较，结果表明 CAR T 细胞中效应记忆 T 细胞的百分比明显更高，而幼稚 T 细胞和 T EMRA 细胞的百分比明显更低。总之，如果测量能够及时开始并且有足够的 T 细胞计数，则流式细胞术 CAR T 细胞检测是一种可靠的监测 CAR T 细胞的方法。
