Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery

Abstract

Background. Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. Material and methods. This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. Results and conclusion. Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-β and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.

Acknowledgments

We thank Raja Choudhury and Kajsa Lundberg at Cancer Centre Karolinska, Karolinska Institute, for help with acquisition and analysis of ELISpot data. We would also like to thank Gabriella Paul-Wetterberg for assistance in the qPCR analysis.

Declaration of interest: This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council and an Uppsala University Hospital ALF grant. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. All authors have declared that there is no financial conflict of interest in regards to this work.