Efficient CAR T cell generation starting with leukoreduction system chambers of thrombocyte apheresis sets

Background

During 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. To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells.

Methods

Immunophenotyping of the LRSC derived mononuclear cells (MNCs) was performed via flow cytometry. T lymphocytes were isolated using the Pan T Cell Isolation Kit and stimulated with IL-2 and paramagnetic beads. Upon lentiviral transduction with concentrated self-inactivating viral vector supernatants, CD19 CAR T cells were further enriched through anti-CD19 CAR MACS, co-incubation with irradiated CD19+ Daudi cells, or through a vector containing an EGFP-T2A-puromycin resistance gene and a one-shot 2 day puromycin treatment. CAR T cell cytotoxicity was assessed using Luciferase labeled cell lines. In vitro CAR T cell expansion to clinically relevant numbers was achieved.

Results

Compared to peripheral blood, LRSC yields a 10-fold MNC concentration. Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T cell populations, with low CD19+ B cell counts. T cells showed low expression of exhaustion markers and a normal memory subpopulation distribution. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. Anti-CD19 CAR MACS resulted in 80% CAR+ cell populations, whereas puromycin selection achieved >90% CAR T cell populations. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell lines Nalm6 and Daudi. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 and Daudi cells.

Conclusion

Stable transduction rates of the isolated T cells could be achieved by lentiviral transduction. Puromycin selection or anti-CD19 CAR-MACS can be used for further purification. These CAR T cells can be successfully expanded and show superior cytotoxicity against target cells compared to unmodified T cells. 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.

Offenlegung Interessenkonflikt:

Die Autoren erklären, dass keine Interessenkonflikte bestehen.