Adapting the protocol for studying the functional capacity of T lymphocytes thawed from cryopreservation

Background. Immunological studies are impossible without long-term storage of cryopreserved biomaterial. There are no standard procedures for working with cryopreserved mononuclear leukocytes.
The aim of the study. To optimize the protocol for culturing T lymphocytes thawed after cryopreservation by assessing their viability and proliferative capacity.
Methods. Mononuclear leukocytes were isolated from the peripheral blood of relatively healthy volunteers (n = 18). Cells were subjected to controlled freezing down to –80 °C and were transferred to liquid nitrogen. First step: after thawing, the cells were stained with CFSE (carboxyfluorescein succinimidyl ester), were divided into two parts and cultured in the presence/absence of interleukin 2 (IL-2). Cell proliferation was stimulated with phytohemagglutinin (type P). Cells were incubated for 7 days. Sample analysis was performed using flow cytometry. Second stage: thawed cells were divided into three parts. Two parts were resuspended in a full growth medium with IL-2 and were placed in a thermostat (+37 °C) to “rest” for one hour or overnight. After “resting”, the cells were stained with CFSE. One third of the thawed leukocytes were stained with CFSE immediately after thawing. Cells were stimulated, cultured and analyzed the same way at both stages of the study.
Results. It has been established that adding IL-2 to the culture medium contributes to a better cell survival. In the presence of IL-2, stimulated CD4+ and CD8+ T lymphocytes produced more daughter cell generations. At the end of the 7-day incubation “rested” samples had reduced leukocyte counts compared to the samples that were cultured immediately after thawing. The number of daughter cell generations formed by stimulated CD4+ and CD8+ T cells decreased when the “rest” stage was included into the study protocol.
Conclusion. Adding IL-2 into culture medium can increase the viability and mitotic capacity of thawed T cells, making their state more similar to that of freshly isolated lymphocytes. Cell “rest” after thawing negatively affects the viability and proliferative activity of T lymphocytes during their weekly incubation.

1. Annaratone L, De Palma G, Bonizzi G, Sapino A, Botti G, Berrino E, et al. Basic principles of biobanking: From biological samples to precision medicine for patients. Virchows Arch. 2021; 479(2): 233-246. doi: 10.1007/s00428-021-03151-0

2. Capelle CM, Ciré S, Ammerlaan W, Konstantinou M, Balling R, Betsou F, et al. Standard peripheral blood mononuclear cell cryopreservation selectively decreases detection of nine clinically relevant T cell markers. Immunohorizons. 2021; 5(8): 711-720. doi: 10.4049/immunohorizons.2100049

3. Li B, Yang C, Jia G, Liu Y, Wang N, Yang F, et al. Comprehensive evaluation of the effects of long-term cryopreservation on peripheral blood mononuclear cells using flow cytometry. BMC Immunol. 2022; 23(1): 30. doi: 10.1186/s12865-022-00505-4

4. Freer G, Rindi L. Intracellular cytokine detection by fluorescence- activated flow cytometry: Basic principles and recent advances. Methods. 2013; 61(1): 30-38. doi: 10.1016/j.ymeth.2013.03.035

5. Horton H, Thomas EP, Stucky JA, Frank I, Moodie Z, Huang Y, et al. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods. 2007; 323(1): 39-54. doi: 10.1016/j.jim.2007.03.002

6. Santos R, Buying A, Sabri N, Yu J, Gringeri A, Bender J, et al. Improvement of IFNg ELISPOT performance following overnight resting of frozen PBMC samples confirmed through rigorous statistical analysis. Cells. 2015; 4(1): 1-18. doi: 10.3390/cells4010001

7. Janetzki S, Panageas KS, Ben-Porat L, Boyer J, Britten CM, Clay TM, et al. Results and harmonization guidelines from two large-scale international ELISPOT proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother. 2008; 57(3): 303-315. doi: 10.1007/s00262-007-0380-6

8. Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, et al. The CIMT-monitoring panel: A twostep approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol Immunother. 2008; 57(3): 289-302. doi: 10.1007/s00262- 007-0378-0

9. Kutscher S, Dembek CJ, Deckert S, Russo C, Körber N, Bogner JR, et al. Overnight resting of PBMC changes functional signatures of antigen specific T cell responses: Impact for immune monitoring within clinical trials. PLoS One. 2013; 8(10): 76215. doi: 10.1371/journal.pone.0076215

10. Boaz MJ, Hayes P, Tarragona T, Seamons L, Cooper A, Birungi J, et al. Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents. Clin Vaccine Immunol. 2009; 16(2): 147-155. doi: 10.1128/CVI.00326-08

11. Wang H, Tsao ST, Gu M, Fu C, He F, Li X, et al. A simple and effective method to purify and activate T cells for successful generation of chimeric antigen receptor T (CAR-T) cells from patients with high monocyte count. J Transl Med. 2022; 20(1): 608. doi: 10.1186/s12967-022-03833-6

12. Mora-Buch R, Tomás-Marín M, Enrich E, Antón-Iborra M, Martorell L, Valdivia E, et al. Virus-specific T cells from cryopreserved blood during an emergent virus outbreak for a potential off-theshelf therapy. Transplant Cell Ther. 2023; 29(9): 572.e1-572.e13. doi: 10.1016/j.jtct.2023.06.001

13. Herda S, Heimann A, Obermayer B, Ciraolo E, Althoff S, Ruß J, et al. Long-term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges. Int J Cancer. 2021; 148(12): 3097-3110. doi: 10.1002/ijc.33523

14. Clarkson BD, Johnson RK, Bingel C, Lothaller C, Howe CL. Preservation of antigen-specific responses in cryopreserved CD4(+) and CD8(+) T cells expanded with IL-2 and IL-7. J Transl Autoimmun. 2022; 5: 100173. doi: 10.1016/j.jtauto.2022.100173

15. Sarkar S, Kalia V, Montelaro RC. Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing. Cryobiology. 2003; 47(1): 44-58. doi: 10.1016/s0011-2240(03)00068-3

16. Baust JM, Buskirk RV, Baust JG. Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell Dev Biol Anim. 2000; 36(4): 262. doi: 10.1290/1071-2690(2000)036<0262:cvifio>2.0.co;2

17. Fu Y, Dang W, He X, Xu F, Huang H. Biomolecular pathways of cryoinjuries in low-temperature storage for mammalian specimens. Bioengineering (Basel). 2022; 9(10): 545. doi: 10.3390/bioengineering9100545

18. Benczik M, Gaffen SL. The interleukin (IL)-2 family cytokines: Survival and proliferation signaling pathways in T lymphocytes. Immunol Invest. 2004; 33(2): 109-142. doi: 10.1081/imm-120030732

19. Abbas AK. The surprising story of IL-2: From experimental models to clinical application. Am J Pathol. 2020; 190(9): 1776-1781. doi: 10.1016/j.ajpath.2020.05.007

20. Leonard WJ, O’Shea JJ, Jaks and STATs: Biological implications. Annu Rev Immunol. 1998; 16: 293-322. doi: 10.1146/annurev. immunol.16.1.293

21. Friedmann MC, Migone TS, Russell SM, Leonard WJ. Different interleukin 2 receptor beta-chain tyrosines couple to at least two signaling pathways and synergistically mediate interleukin 2-induced proliferation. Proc Natl Acad Sci U S A. 1996; 93(5): 2077- 2082. doi: 10.1073/pnas.93.5.2077

22. Lali FV, Crawley J, McCulloch DA, Foxwell BM. A late, prolonged activation of the phosphatidylinositol 3-kinase pathway is required for T cell proliferation. J Immunol. 2004; 172(6): 3527- 3534. doi: 10.4049/jimmunol.172.6.3527

23. Mui AL, Wakao H, O’Farrell AM, Harada N, Miyajima A. Interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5 transduce signals through two forms of STAT5. J Leukoc Biol. 1995; 57(5): 799-803. doi: 10.1002/jlb.57.5.799

24. Malek TR, Castro I. Interleukin-2 receptor signaling: At the interface between tolerance and immunity. Immunity. 2010; 33(2): 153-165. doi: 10.1016/j.immuni.2010.08.004

25. Kuerten S, Batoulis H, Recks MS, Karacsony E, Zhang W, Subbramanian RA, et al. Resting of cryopreserved PBMC does not generally benefit the performance of antigen-specific T cell ELISPOT assays. Cells. 2012; 1(3): 409-427. doi: 10.3390/cells1030409

26. Smith JG, Joseph HR, Green T, Field JA, Wooters M, Kaufhold RM, et al. Establishing acceptance criteria for cell-mediatedimmunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol. 2007; 14(5): 527-537. doi: 10.1128/CVI.00435-06

27. Lenders K, Ogunjimi B, Beutels P, Hens N, Van Damme P, Berneman ZN, et al. The effect of apoptotic cells on virusspecific immune responses detected using IFN-gamma ELISPOT. J Immunol Methods. 2010; 357(1-2): 51-54. doi: 10.1016/j.jim.2010.03.00128

28. Zorn E, Nelson EA, Mohseni M, Porcheray F, Kim H, Litsa D, et al. IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood. 2006; 108(5): 1571-1579. doi: 10.1182/blood-2006-02- 004747

29. Oh S, Berzofsky JA, Burke DS, Waldmann TA, Perera LP. Coadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunity. Proc Natl Acad Sci U S A. 2003; 100(6): 3392-3397. doi: 10.1073/pnas.0630592100