Efficient Chimeric Antigen Receptor T-Cell Generation Starting with Leukoreduction System Chambers of Thrombocyte Apheresis Sets.

Introduction: Primary human blood cells represent an essential model system to study physiology and disease. However, human blood is a limited resource. During healthy donor plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. Compared to peripheral blood, LRSC yields 10-fold mononuclear cell concentration.
Methods: To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the usually discarded LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells.
Results: Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T-cell populations within LRSC, with low CD19+ B cell counts. Magnetic-activated cell sorting (MACS) purified CD3+ T cells were transduced with CD19 CAR-encoding lentiviral self-inactivating vectors using concentrated viral supernatants. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. CD19 CAR T cells were further enriched through anti-CAR MACS, yielding 80% CAR+ T-cell populations. In vitro CAR T cell expansion to clinically relevant numbers was achieved. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell line Nalm6. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 cells.
Conclusion: Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes.
Competing Interests: The authors confirm that none of the material has been published or is under consideration elsewhere. S.K. has received honoraria from TCR2 Inc., Novartis, BMS, and GSK SK and is inventor of several patents in the field of immuno-oncology. S.K. received license fees from TCR2 Inc. and Carina Biotech. S.K. received research support from TCR2 Inc., Tabby Therapeutics, Plectonic GmbH, and Arcus Bioscience for work unrelated to the manuscript. A.G. received research support from Tabby Therapeutics. The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
(© 2023 The Author(s). Published by S. Karger AG, Basel.)