Enhanced homeostatic proliferation of t lymphocytes after cyclophosphamide injection in c57bl/6 mice

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Abstract

Chemotherapeutic agents are used in medicine to treat cancer. They can damage immune system and lead to the secondary immunodeficiency. T cells are most severely affected during chemotherapy. Restoration of the T lymphocytes is an important topic in research to understand pathogenesis of damaging effects caused by cytostatics and searching ways to correct the resulting disorders. The aim of our study was to follow the process of T cell recovery, and to understand the role of its homeostatic proliferation. 33 female C57BL/6 mice were included into the experiment. The experimental group (25 mice) received a single injection of cyclophosphamide (Cy) at a dose of 125 mg/kg; the control group did not receive the drug. Biomaterials for the study were splenocytes isolated on days 5, 10, 20, 30 and 60 after the drug administration. Flow cytometry was used to measure the recovery of T helpers (CD3+CD4+) and cytotoxic T lymphocytes (CTL, CD3+CD8+), as well as their “age-related” phenotype assayed for naive (Tnaive) and central memory (Tcm) T cells. The level of homeostatic proliferation was determined by the Tnaive/Tcm ratio. The total amount of splenocytes, T helpers, CTLs and the CD4+/CD8+ ratio showed a statistically significant increase at the early terms after Cy administration (day 5). Further, a decrease in splenocytes and their subpopulations was observed. We found that the CTL subpopulation didn’t recover even 2 months after the drug administration and was more sensitive to the action of cyclophosphamide than the T helper subpopulation. We have also revealed that naive T helpers and naive CTLs are most susceptible to the Cy action; these subpopulations also failed to recover 60 days after the drug administration. At the same time, the amount of central memory T cells predominated by the end of the experiment, showing conversion of the T cell phenotype. Thus, we have shown an increase in homeostatic proliferation, along with conversion of naive T cell phenotype to the central memory T cells after Cy administration accompanied by deficiency of naive T cells. Such changes cause skewing of TCR repertoire. This shift may cause premature aging of immune system and increases the risk of autoimmune diseases.

About the authors

E. K. Grinko

National Research Center – Institute of Immunology; K. Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology

Email: info@nrcii.ru

Junior Research Associate

Russian Federation, 115522, Moscow, Kashirskoye highway, 24; Moscow

Saida N. Marzanova

K. Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology

Email: info@nrcii.ru

PhD (Biology), Associate Professor

Russian Federation, 115522, Moscow, Kashirskoye highway, 24

Almira D. Donetskova

National Research Center – Institute of Immunology; N. Pirogov Russian National Research Medical University (Pirogov Medical University); Peoples’ Friendship University of Russia

Author for correspondence.
Email: info@nrcii.ru

PhD, MD (Medicine), Professor, Leading Research Associate

Russian Federation, 115522, Moscow, Kashirskoye highway, 24; Moscow; Moscow

References

  1. Гринько Е.К., Вериго К.С., Мухина Е.А., Шарова Н.И., Комогорова В.В., Литвина М.М., Марзанова С.Н., Донецкова А.Д., Митин А.Н. Динамика восстановления субпопуляций Т-лимфоцитов после действия различных повреждающих агентов // Иммунология, 2021. Т. 42, № 4. С. 346-355. [Grinko E.K., Verigo K.S., Mukhina E.A., Sharova N.I., Komogorova V.V., Litvina M.M., Marzanova S.N., Donetskova A.D., Mitin A.N. Dynamics of T-lymphocyte subpopulations’ recovery after influence of various damaging agents. Immunologiya = Immunologiya, 2021, Vol. 42, no. 4, pp. 346-355. (In Russ.)]
  2. Козлов В.A. Гомеостатическая пролиферация как основа неизбежного формирования тотального иммунодефицита // Медицинская иммунология, 2014. T. 16, № 5. С. 403-408. Kozlov V.A. Homeostatic proliferation as a basis for the inevitable formation of total immunodeficiency. Meditsinskaya immunologiya = Medical Immunology (Russia), 2014, Vol. 16, no. 5, pp. 403-408. (In Russ.)] doi: 10.15789/1563-0625-2014-5-403-408.
  3. Шевырев Д.В., Терещенко В.П., Козлов В.А. Гомеостатическая пролиферация: от нормы к патологии // Российский иммунологический журнал, 2018. Т. 21, № 2. С. 91-105. [Shevyrev D.V., Tereshchenko V.P., Kozlov V.A. Нomeostatic proliferation: from health to pathology. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2018, Vol. 21, no. 2, pp. 91-105. (In Russ.)]
  4. Boyman O., Letourneau S., Kreig C., Sprent J. Homeostatic proliferation and survival of naive and memory T cells. Eur. J. Immunol., 2009, Vol. 39, no. 8, pp. 2088-2094.
  5. Farber D.L., Yudanin N.A., Restifo N.P. Human memory T cells: generation, compartmentalization and homeostasis. Nat. Rev. Immunol., 2014, Vol. 14, no. 1, pp. 24-35.
  6. Goldrath A.W., Bogatzki L.Y., Bevan M.J. Naive T cells transiently acquire a memory-like phenotype during homeostasis driven proliferation. J. Exp. Med., 2000, Vol. 192, no. 4, pp. 557-564.
  7. Gudmundsdottir H., Turka L.A. A closer look at homeostatic proliferation of CD4+ T cells: costimulatory requirements and role in memory formation. J. Immunol., 2001, Vol. 167, no. 7, pp. 3699-3707.
  8. Heninger A.K., Theil A., Petzold C., Huebel N., Kretschmer K., Bonifacio E., Monti P. IL-7 abrogates suppressive activity of human CD4+CD25+FoxP3+ regulatory T cells and allows expansion of alloreactive and autoreactive T cells. J. Immunol., 2012, Vol. 189, no. 12, pp. 5649-5658.
  9. Heylmann D., Bauer M., Becker H., van Gool S., Bacher N., Steinbrink K., Kaina B. Human CD4+CD25+ regulatory T cells are sensitive to low dose cyclophosphamide: implications for the immune response. PLoS One, 2013, Vol. 8, no. 12, e83384. doi: 10.1371/journal.pone0083384.
  10. Hong S.H., Yoon I.H., Kim Y.H., Yang S.H., Park M.J., Nam H.Y., Kim B., Kim Y., Park C.S., Park C.G. High-dose cyclophosphamide-mediated anti-tumor effects by the superior expansion of CD44(high) cells after their selective depletion. Immunobiology, 2010, Vol. 215, no. 3, pp. 182-193.
  11. Huyan X.H., Lin Y.P., Gao T., Chen R.Y., Fan Y.M. Immunosuppressive effect of cyclophosphamide on white blood cells and lymphocyte subpopulations from peripheral blood of Balb/c mice. Int. Immunopharmacol., 2011, Vol. 11, no. 9. pp. 1293-1297.
  12. Kieper W.C., Burghardt J.T., Surt C.D. A role for TCR affinity in regulating naive T cell homeostasis. J. Immunol., 2004, Vol. 172, no. 1, pp. 40-44.
  13. Kuwatani M., Ikarashi Y., Mineishi S., Asaka M., Wakasugi H. An irradiation-free nonmyeloablative bone marrow transplantation model – importance of the balance between donor T-cell number and the intensity of conditioning. Transplantation, 2005, Vol. 80, no. 9, pp. 1145-1152.
  14. Lin J.X., Leonard W.J. The common cytokine receptor γ chain family of cytokines. Cold Spring Harb Perspect. Biol., 2018, Vol. 10, no. 9, a028449. doi: 10.1101/cshperspect.a028449.
  15. Mackall C.L., Hakim F.T., Gress R.E. Restoration of T-cell homeostasis after T-cell depletion. Semin. Immunol., 1997, Vol. 9, no. 6, pp. 339-346.
  16. Motoyoshi Y., Kaminoda K., Saitoh O., Hamasaki K., Nakao K., Ishii N., Nagayama Y., Eguchi K. Different mechanisms for anti-tumor effects of low- and high-dose cyclophosphamide. Oncol. Rep., 2006, Vol. 16, no. 1, pp. 141-146.
  17. Nolz J.C., Richer M.J. Control of memory CD8+ T cell longevity and effector functions by IL-15. Mol. Immunol., 2020, Vol. 117, pp. 180-188.
  18. Prlic M., Blazar B.R., Khoruts A., Zell T., Jameson S.C. Homeostatic expansion occurs independently of costimulatory signals. J. Immunol., 2001, Vol. 167, no. 10, pp. 5664-5668.
  19. Seddon B., Tomlinson P., Zamoyska R. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells. Nat. Immunol., 2003, Vol. 4, no. 7, pp. 680-686.
  20. Tan J.T., Ernst B., Kieper W.C., LeRoy E., Sprent J., Surh C.D. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med., 2002, Vol. 195, no. 12, pp. 1523-1532.
  21. van Belle T.L., Dooms H., Boonefaes T., Wei X.Q., Leclercq G., Grooten J. IL-15 augments TCR-induced CD4+ T cell expansion in vitro by in-habiting the suppressive function of CD25high CD4+ T cells. PLoS One, 2012, Vol. 7, no. 9, e45299. doi: 10.1371/journal.pone.0045299
  22. Włodarczyk M., Ograczyk E., Kowalewicz-Kulbat M., Druszczyńska M., Rudnicka W., Fol M. Cyclophosphamide treatment on central and effector memory T Cells in Mice. Int. J. Toxicol., 2018, Vol. 37, no. 5, pp. 373-382.
  23. Yamaki S., Ine S., Kawabe T., Okuyama Y., Suzuki N., Soroosh P., Mousavi S.F., Nagashima H., Sun S.L., So T. OX40 and IL-7 play synergistic roles in the homeostatic proliferation of effector memory CD4+ T cells. Eur. J. Immunol., 2014, Vol. 44, no. 10, pp. 3015-3025.

Supplementary files

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2. Figure 1. Dynamics of changes in the number of splenocyte, T helper and CTL populations in C57BL/6 mice after a single injection of cyclophosphamide

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3. Figure 2. Algorithm for cytometric determination of naive and central memory T cell subpopulations using CTLs of the spleen of an intact C57BL/6 mouse as an example

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4. Figure 3. Dynamics of changes in the number of T helper “age” subpopulations of in C57BL/6 mice after a single injection of cyclophosphamide

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5. Figure 4. Dynamics of changes in the number of CTL “age” subpopulations in C57BL/6 mice after a single injection of cyclophosphamide.

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6. Figure 5. Changes in the ratio of naive and central memory T cells for T helpers (CD4+) and CTL (CD8+) in the spleen of C57BL/6 mice on day 60 after a single injection of cyclophosphamide

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Copyright (c) 2022 Grinko .K., Marzanova S.N., Donetskova A.D.

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This work is licensed under a Creative Commons Attribution 4.0 International License.

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