Generation and characterization of stable antigen-specific T-regulatory cells using blockers of cyclin-dependent protein kinases
AbstractIntroduction. In the immune system, regulatory CD4+CD25highFoxP3+T cells (Treg) are important players in tolerance induction. Treg suppress inflammatory reactions of various etiologies, including autoimmune and transplant reactions. Using the inherent regulatory potential of Treg induced in vitro, it is possible to achieve suppression of inflammatory responses to a specific antigen without affecting the immune response as a whole.
Aim of the study – obtaining antigen-specific FoxP3+Treg with characteristic immunosuppressive properties in mixed lymphocyte culture.
Material and methods. Object of this study were male mice of C57BL/6 and Balb/c lines. The resulting dendritic cells from C57BL/6 mice and CD4+ splenocytes from Balb/c mice were co-cultured to obtain antigen-specific CD4+ T cells. After that, CD4+ T cells were cultured with AS2863619, TGFβ and IL-2 to transdifferentiate antigen-specific effector T lymphocytes to antigen-specific CD4+CD25highFoxP3+Treg. The phenotype of the resulting Treg was assessed using flow cytometry. The tolerogenic potential of Treg was assessed using a mixed lymphocyte culture.
Results. It was shown that the combination of anti-CD3 and AS2863619 leads to a 10-fold increase in the relative number of CD25highFoxP3+Tregs in a culture of CD4+ T lymphocytes compared to unstimulated lymphocytes. Flow cytometrшс analysis showed that the population of effector memory T cells predominate in the antigen-specific Treg culture. At the same time, in the culture of non-antigen-specific Treg, only about half of the subpopulations consisted of resident memory T cells. In a mixed culture of lymphocytes, it was found that in the presence of non-Ag-Treg the proliferation of Balb/c CD4+ T lymphocytes increased by 2.5 times in response to the addition of the C57Bl/6 antigen. However, in the presence of Ag-Treg, even with the addition of C57Bl/6 antigens, the level of proliferation of Balb/c CD4+ T lymphocytes does not differ from the control group.
Conclusion. The use of AS2863619 in the cultivation of antigen-specific CD4+ lymphocytes enhances the generation of Ag-Treg. At the same time, the resulting Ag-Tregs have the phenotypic properties of effector memory Treg and functionally suppress the proliferation of CD4+ lymphocytes against antigens, which makes it reasonable to study their ability to induce immunological tolerance and suppress the immune response.
Keywords:CD4+T cells; regulatory T cells; antigens; FoxP3; CDK8/19; AS2863619
For citation: Bulygin A.S., Khantakova J.N., Fisher M.S., Tereshchenko V.P., Kurilin V.V., Shkaruba N.S., Sennikov S.V. Generation of stable antigen-specific T-regulatory cells using blockers of cyclin-dependent protein kinases. Immunology. 2023; 44 (2): 147–56. DOI: https://doi.org/10.33029/0206-4952-2023-44-2-147-156 (in Russian)
Funding. The research was supported by the grant of the Russian Science Foundation (project No. 21-75-10089), https://rscf.ru/project/21-75-10089
Conflict of interests. The authors declare no conflict of interests.
Authors’ contributions. The concept and design of the study – Bulygin A.S., Khantakova J.N., Sennikov S.V.; collection and processing of material – Fisher M.S., Kurilin V.V., Bulygin A.S.; statistical processing – Bulygin A.S., Shkaruba N.S.; writing the text – Bulygin A.S., Khantakova J.N.; editing – Sennikov S.V., Khantakova J.N.
References
1. Esensten J.H., Helou Y.A., Chopra G., Weiss A., Bluestone J.A. CD28 costimulation: from mechanism to therapy. Immunity. 2016; 44 (5): 973–88. DOI: https://www.doi.org/10.1016/j.immuni.2016.04.020
2. Khantakova J.N., Bulygin A.S., Sennikov S.V. The regulatory-T-cell memory phenotype: what we know. Cells. 2022; 11 (10): 1687. DOI: https://www.doi.org/10.3390/cells11101687
3. Gratz I.K., Campbell D.J. Organ-specific and memory treg cells: specificity, development, function, and maintenance. Front Immunol. 2014; 5: 333. DOI: https://www.doi.org/10.3389/fimmu.2014.00333
4. Schmidt A., Oberle N., Krammer P.H. Molecular mechanisms of Treg-mediated T cell suppression. Front. Immun. 2012; 3: 51. DOI: https://www.doi.org/10.3389/fimmu.2012.00051
5. Sonar S.A., Lal G. Differentiation and transmigration of CD4 T cells in neuroinflammation and autoimmunity. Front. Immunol. 2017; 8: 1695. DOI: https://www.doi.org/10.3389/fimmu.2017.01695
6. Ohkura N., Sakaguchi S. Transcriptional and epigenetic basis of Treg cell development and function: its genetic anomalies or variations in autoimmune diseases. Cell Res. 2020; 30 (6): 465–74. DOI: https://www.doi.org/10.1038/s41422-020-0324-7
7. Sennikov S.V., Khantakova J.N. Role of T-cell subpopulations in the immunologic tolerance induction. Immunologiya. 2017; 38 (4): 239–44. DOI: http://dx.doi.org/10.18821/0206-4952-2017-38-4-239-244 (in Russian)
8. d’Hennezel E., Yurchenko E., Sgouroudis E., Hay V., Piccirillo C.A. Single-cell analysis of the human T regulatory population uncovers functional heterogeneity and instability within FOXP3+ cells. J Immunol. 2011; 186 (12): 6788–97. DOI: https://www.doi.org/10.4049/jimmunol.1100269
9. Selck C., Dominguez-Villar M. Antigen-specific regulatory T cell therapy in autoimmune diseases and transplantation. Front Immunol. 2021; 12: 661875. DOI: https://www.doi.org/10.3389/fimmu.2021.661875
10. Guo Z., Wang G., Lv Y., Wan Y.Y., Zheng J. Inhibition of Cdk8/Cdk19 activity promotes Treg cell differentiation and suppresses autoimmune diseases. Front. Immunol. 2019; 10: 1988. DOI: https://www.doi.org/10.3389/fimmu.2019.01988
11. Akamatsu M., Mikami N., Ohkura N., Kawakami R., Kitagawa Y., Sugimoto A., Hirota K., Nakamura N., Ujihara S., Kurosaki T., Hamaguchi H., Harada H., Xia G., Morita Y., Aramori I., Narumiya S., Sakaguchi S. Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19. Sci Immunol. 2019; 4 (40): eaaw2707. DOI: https://www.doi.org/10.1126/sciimmunol.aaw2707
12. European convention for the protection of vertebrate animals used for experimental and other scientific purposes. ETS 123 – Protection of Vertebrate Animals, 18.III.1986.
13. Bonam S.R., Bayry J. For antigen-specific effector or Foxp3+ regulatory T cell fate, cyclin-dependent kinases hold the trump card. Cell Mol Immunol. 2020; 17 (4): 310–2. DOI: https://www.doi.org/10.1038/s41423-019-0349-3
14. Teh P.P., Vasanthakumar A., Kallies A. Development and function of effector regulatory T cells. Prog Mol Biol Transl Sci. 2015; 136: 155–74. DOI: https://www.doi.org/10.1016/bs.pmbts.2015.08.005
15. Koizumi S.I., Ishikawa H. Transcriptional regulation of differentiation and functions of effector T regulatory cells. Cells. 2019; 8 (8): 939. DOI: https://www.doi.org/10.3390/cells8080939
16. Qiu R., Zhou L., Ma Y., Zhou L., Liang T., Shi L., Long J., Yuan D. Regulatory T cell plasticity and stability and autoimmune diseases. Clin Rev Allergy Immunol. 2020; 58 (1): 52–70. DOI: https://www.doi.org/10.1007/s12016-018-8721-0
17. Shevyrev D., Tereshchenko V. Treg Heterogeneity, function, and homeostasis. Front Immunol. 2020 14; 10: 3100. DOI: https://www.doi.org/10.3389/fimmu.2019.03100
18. Hall B.M. T Cells: Soldiers and spies – the surveillance and control of effector T cells by regulatory T cells. Clin J Am Soc Nephrol. 2015; 10 (11): 2050–64. DOI: https://www.doi.org/10.2215/CJN.06620714
19. Vignali D.A., Collison L.W., Workman C.J. How regulatory T cells work. Nat Rev Immunol. 2008; 8 (7): 523–32. DOI: https://www.doi.org/10.1038/nri2343
20. Wardell C.M., MacDonald K.N., Levings M.K., Cook L. Cross talk between human regulatory T cells and antigen-presenting cells: Lessons for clinical applications. Eur J Immunol. 2021; 51 (1): 27–38. DOI: https://www.doi.org/10.1002/eji.202048746