Advanced Biomedical Research
Optimization of culture media for ex vivo T-cell expansion for adoptive T-cell therapy
Authors
- Ilnaz Rahimmanesh 1
- Mehrsa Tavangar 2
- Seyedeh Noushin Zahedi 2
- Yadollah Azizi 2
- Hossein Khanahmad Shahreza 2
1 Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences; Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract
Background: Adoptive T-cell therapy is a promising treatment strategy for cancer immunotherapy. The ability of immunotherapy based on the adoptive cell transfer of genetically modified T cells to generate powerful clinical responses has been highlighted by recent clinical success. Techniques which are used to expand large numbers of T cells from different sources are critical in adoptive cell therapy. In this study, we evaluated the expansion, proliferation, activation of T lymphocytes, in the presence of various concentrations of interleukin-2, phytohemagglutinin (PHA), and insulin.
Materials and Methods: The effect of different supplemented culture media on T cell expansion was evaluated using MTT assay. The expression level of the Ki-67 proliferation marker was evaluated by real-time polymerase chain reaction. In addition, flow cytometry analysis was performed to access T cell subpopulations.
Results: Our results showed that supplemented culture media with an optimized concentration of PHA and interleukin-2 increased total fold expansion of T cells up to 500-fold with approximately 90% cell viability over 7 days. The quantitative assessment of Ki-67 in expanded T cells showed a significant elevation of this proliferation marker. Flow cytometry was also used to assess the proportion of CD4+ and CD8+ cells, and the main expanded population was CD3+ CD8+ cells.
Conclusions: Based on these findings, we introduced a low-cost and rapid method to support the efficient expansion of T cells for adoptive cell therapy and other in vivo experiments.
Keywords
| 1. |
Rasmussen AM, Borelli G, Hoel HJ, Lislerud K, Gaudernack G, Kvalheim G, et al. Ex vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy. J Immunol Methods 2010;355:52-60.  |
| 2. |
Lewis MD, de Leenheer E, Fishman S, Siew LK, Gross G, Wong FS. A reproducible method for the expansion of mouse CD8+T lymphocytes. J Immunol Methods 2015;417:134-8. |
| 3. |
Wu TD, Madireddi S, de Almeida PE, Banchereau R, Chen YJ, Chitre AS, et al. Peripheral T cell expansion predicts tumour infiltration and clinical response. Nature 2020;579:274-8. |
| 4. |
Janelle V, Delisle JS. T-cell dysfunction as a limitation of adoptive immunotherapy: Current concepts and mitigation strategies. Cancers (Basel) 2021;13:598. |
| 5. |
Titov A, Zmievskaya E, Ganeeva I, Valiullina A, Petukhov A, Rakhmatullina A, et al. Adoptive immunotherapy beyond CAR T-cells. Cancers (Basel) 2021;13:743. |
| 6. |
Ho PC, Meeth KM, Tsui YC, Srivastava B, Bosenberg MW, Kaech SM. Immune-based antitumor effects of BRAF inhibitors rely on signaling by CD40L and IFNγ. Cancer Res 2014;74:3205-17. |
| 7. |
Bos R, Sherman LA. CD4+T-cell help in the tumor milieu is required for recruitment and cytolytic function of CD8+T lymphocytes. Cancer Res 2010;70:8368-77. |
| 8. |
Zhang DKY, Cheung AS, Mooney DJ. Activation and expansion of human T cells using artificial antigen-presenting cell scaffolds. Nat Protoc 2020;15:773-98. |
| 9. |
Thomas AK, Maus MV, Shalaby WS, June CH, Riley JL. A cell-based artificial antigen-presenting cell coated with anti-CD3 and CD28 antibodies enables rapid expansion and long-term growth of CD4 T lymphocytes. Clin Immunol 2002;105:259-72. |
| 10. |
Sato K, Kondo M, Sakuta K, Hosoi A, Noji S, Sugiura M, et al. Impact of culture medium on the expansion of T cells for immunotherapy. Cytotherapy 2009;11:936-46. |
| 11. |
Tay RE, Richardson EK, Toh HC. Revisiting the role of CD4+T cells in cancer immunotherapy-new insights into old paradigms. Cancer Gene Ther 2021;28:5-17. |
| 12. |
Naota H, Miyahara Y, Okumura S, Kuzushima K, Akatsuka Y, Hiasa A, et al. Generation of peptide-specific CD8+T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+T cells. J Immunol Methods 2006;314:54-66. |
| 13. |
Zamani A, Vahidinia A, Ghannad MS. The effect of garlic consumption on Th1/Th2 cytokines in phytohemagglutinin (PHA) activated rat spleen lymphocytes. Phytother Res 2009;23:579-81. |
| 14. |
Oppenheim JJ. IL-2: More than a T cell growth factor. J Immunol 2007;179:1413-4. |
| 15. |
Fischer HJ, Sie C, Schumann E, Witte AK, Dressel R, van den Brandt J, et al. The insulin receptor plays a critical role in T cell function and adaptive immunity. J Immunol 2017;198:1910-20. |
| 16. |
Choi E, Yu H. Spindle checkpoint regulators in insulin signaling. Front Cell Dev Biol 2018;6:161. |
| 17. |
Ti D, Bai M, Li X, Wei J, Chen D, Wu Z, et al. Adaptive T cell immunotherapy in cancer. Sci China Life Sci 2021;64:363-71. |
| 18. |
Torabi-Rahvar M, Aghayan HR, Ahmadbeigi N. Antigen-independent killer cells prepared for adoptive immunotherapy: One source, divergent protocols, diverse nomenclature. J Immunol Methods 2020;477:112690. |
| 19. |
Disis ML, Bernhard H, Jaffee EM. Use of tumour-responsive T cells as cancer treatment. Lancet 2009;373:673-83. |
| 20. |
Ye J, Cao W, Tao Z, Zhao S, Wang C, Xu X, et al. Novel method for effectively amplifying human peripheral blood T cells in vitro. Exp Cell Res 2021;399:112451. |
| 21. |
Kondo M, Sakuta K, Noguchi A, Ariyoshi N, Sato K, Sato S, et al. Zoledronate facilitates large-scale ex vivo expansion of functional γδ T cells from cancer patients for use in adoptive immunotherapy. Cytotherapy 2008;10:842-56. |
| 22. |
Yannelli JR. The preparation of effector cells for use in the adoptive cellular immunotherapy of human cancer. J Immunol Methods 1991;139:1-16. |
| 23. |
Vormittag P, Gunn R, Ghorashian S, Veraitch FS. A guide to manufacturing CAR T cell therapies. Curr Opin Biotechnol 2018;53:164-81. |
| 24. |
Nankervis B, Jones M, Vang B, Brent Rice R Jr, Coeshott C, Beltzer J. Optimizing T cell expansion in a hollow-fiber bioreactor. Curr Stem Cell Rep 2018;4:46-51. |
| 25. |
Coeshott C, Vang B, Jones M, Nankervis B. Large-scale expansion and characterization of CD3+T-cells in the Quantum® Cell Expansion System. J Transl Med 2019;17:258. |
| 26. |
Brizova H, Kalinova M, Krskova L, Mrhalova M, Kodet R. A novel quantitative PCR of proliferation markers (Ki-67, topoisomerase IIalpha, and TPX2): An immunohistochemical correlation, testing, and optimizing for mantle cell lymphoma. Virchows Arch 2010;456:671-9. |
| 27. |
Sinn HP, Schneeweiss A, Keller M, Schlombs K, Laible M, Seitz J, et al. Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer. BMC Cancer 2017;17:124. |
| 28. |
Dudley ME, Rosenberg SA. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer 2003;3:666-75. |
| 29. |
Kaartinen T, Luostarinen A, Maliniemi P, Keto J, Arvas M, Belt H, et al. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion. Cytotherapy 2017;19:689-702. |
| 30. |
Cho JH, Kim HO, Kim KS, Yang DH, Surh CD, Sprent J. Unique features of naive CD8+ T cell activation by IL-2. J Immunol 2013;191:5559-73. |
| 31. |
Voisinne G, Nixon BG, Melbinger A, Gasteiger G, Vergassola M, Altan-Bonnet G. T cells integrate local and global cues to discriminate between structurally similar antigens. Cell Rep 2015;11:1208-19.
|
)