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.

Author Info: (1) Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. (2) Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany. (3) Inst itute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany. (4) Beckman Coulter Life Sciences GmbH, Flow Cytometry Business Unit, Krefeld, Germany. (5) Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. (6) Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. (7) Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany. (8) Beckman Coulter Life Sciences, Flow Cytometry R&D, Marseille, France. (9) Beckman Coulter Life Sciences, Flow Cytometry R&D, Marseille, France. (10) Medical Faculty, Department of Hematology and Cell Therapy, University of Leipzig, Leipzig, Germany. (11) Medical Faculty, Department of Hematology and Cell Therapy, University of Leipzig, Leipzig, Germany. (12) Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany. (13) Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. (14) Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany. Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany. Institute for Cellular Therapeutics, Hannover Medical School, Hannover, Germany.