Blache Ulrich, Weiss Ronald, Boldt Andreas, Kapinsky Michael, Blaudszun André-René, Quaiser Andrea, Pohl Annabelle, Miloud Tewfik, Burgaud Mégane, Vucinic Vladan, Platzbecker Uwe, Sack Ulrich, Fricke Stephan, Koehl Ulrike
Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.
Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany.
Front Immunol. 2021 May 3;12:658314. doi: 10.3389/fimmu.2021.658314. eCollection 2021.
Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient's immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.
使用嵌合抗原受体(CAR)-T细胞的过继性免疫疗法已在难治性B细胞白血病和B细胞淋巴瘤中实现了成功缓解。为了评估CAR-T细胞疗法的疗效和严重副作用,在临床分期的同时,对包括CAR-T细胞在内的患者免疫系统进行纵向监测是很有必要的。为了研究输注的CAR-T细胞的命运和免疫影响,我们建立了标准化的13色/15参数流式细胞术检测方法,该方法适用于在CAR-T细胞治疗期间对外周血中的免疫细胞亚群进行表征。相应的染色技术基于统一格式的预配制干燥抗体板。此外,可以添加其他选择的抗体来解决特定的临床或研究问题。我们设计了用于抗CD19 CAR-T疗法的检测板,作为概念验证,我们评估了一名健康个体和三名接受抗CD19 CAR-T细胞治疗的B细胞淋巴瘤患者。我们分析了抗CD19 CAR-T细胞以及残余CD19+B细胞的存在情况、T细胞区室的激活状态、共刺激信号分子以及细胞毒性剂如穿孔素和颗粒酶B的表达。总之,这项工作介绍了用于CAR-T细胞临床研究的标准化和模块化流式细胞术检测方法,未来在CAR-T细胞制造过程中也可将其用作质量控制方法。