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.
Chen HW, Liao CH, Ying C, Chang CJ, Lin CM. Chen HW, et al. Clin Immunol. 2006 Apr;119(1):21-31. doi: 10.1016/j.clim.2005.11.003. Epub 2006 Jan 9. Clin Immunol. 2006. PMID: 16406844
Li Y, Liu S, Hernandez J, Vence L, Hwu P, Radvanyi L. Li Y, et al. J Immunol. 2010 Jan 1;184(1):452-65. doi: 10.4049/jimmunol.0901101. Epub 2009 Nov 30. J Immunol. 2010. PMID: 19949105
Foster AE, Forrester K, Gottlieb DJ, Barton GW, Romagnoli JA, Bradstock KF. Foster AE, et al. Biotechnol Bioeng. 2004 Jan 20;85(2):138-46. doi: 10.1002/bit.10801. Biotechnol Bioeng. 2004. PMID: 14704996
Schulz JC, Germann A, Kemp-Kamke B, Mazzotta A, von Briesen H, Zimmermann H. Schulz JC, et al. J Immunol Methods. 2012 Aug 31;382(1-2):24-31. doi: 10.1016/j.jim.2012.05.001. Epub 2012 May 8. J Immunol Methods. 2012. PMID: 22580762
Baust JM, Buehring GC, Campbell L, Elmore E, Harbell JW, Nims RW, Price P, Reid YA, Simione F. Baust JM, et al. In Vitro Cell Dev Biol Anim. 2017 Sep;53(8):669-672. doi: 10.1007/s11626-017-0177-7. Epub 2017 Aug 14. In Vitro Cell Dev Biol Anim. 2017. PMID: 28808859 Review.
Sharma G, Round J, Teng F, Ali Z, May C, Yung E, Holt RA. Sharma G, et al. NPJ Precis Oncol. 2024 Aug 19;8(1):182. doi: 10.1038/s41698-024-00669-9. NPJ Precis Oncol. 2024. PMID: 39160299 Free PMC article.
Sharma G, Round J, Teng F, Ali Z, May C, Yung E, Holt RA. Sharma G, et al. bioRxiv [Preprint]. 2023 Nov 21:2023.11.20.567960. doi: 10.1101/2023.11.20.567960. bioRxiv. 2023. Update in: NPJ Precis Oncol. 2024 Aug 19;8(1):182. doi: 10.1038/s41698-024-00669-9. PMID: 38045272 Free PMC article. Updated. Preprint.
Amini L, Kaeda J, Fritsche E, Roemhild A, Kaiser D, Reinke P. Amini L, et al. Front Cell Dev Biol. 2023 Jan 30;10:1081644. doi: 10.3389/fcell.2022.1081644. eCollection 2022. Front Cell Dev Biol. 2023. PMID: 36794233 Free PMC article. Review.
Sparger EE, Chang H, Chin N, Rebhun RB, Withers SS, Kieu H, Canter RJ, Monjazeb AM, Kent MS. Sparger EE, et al. Front Vet Sci. 2021 Dec 2;8:772932. doi: 10.3389/fvets.2021.772932. eCollection 2021. Front Vet Sci. 2021. PMID: 34926643 Free PMC article.
Chandrasekaran S, Funk CR, Kleber T, Paulos CM, Shanmugam M, Waller EK. Chandrasekaran S, et al. Front Immunol. 2021 Aug 26;12:718621. doi: 10.3389/fimmu.2021.718621. eCollection 2021. Front Immunol. 2021. PMID: 34512641 Free PMC article. Review.