1. Institute of Biomedical Chemistry, Moscow, Russia 2. Research Center of Neurology, Moscow, Russia 3. Department of Biochemistry, Peoples Friendship University of Russia (RUDN University), Moscow, Russia 4. Institute of Biomedical Chemistry, Moscow, Russia; Department of Biochemistry, Peoples Friendship University of Russia (RUDN University), Moscow, Russia
Regulatory T-cells CD4⁺CD25⁺FoxP3⁺CD127low (Tregs) play a key role in the maintenance of tolerance to auto antigens, inhibit function of effector T and B lymphocytes, and provide a balance between effector and regulatory arms of immunity. Patients with autoimmune diseases have decreased Treg numbers and impaired suppressive activity. Transformed ex vivo autologous Tregs could restore destroyed balance of the immune system. We developed a method for Treg precursor cell cultivation. Following the method, we were able to grown up 300-400 million of Tregs cells from 50 ml of peripheral blood during a week. Transformed ex vivo Tregs are 90-95% CD4⁺CD25⁺FoxP3⁺CD127low and have increased expression of transcription genes FoxP3 and Helios. Transformed ex vivo Tregs have increased demethylation of FoxP3 promoter and activated genes of proliferation markers Cycline B1, Ki67 and LGALS 1. Transformed ex vivo Tregs have increased suppressive activity and up to 80-90% these cells secrete cytokines TNFα и IFNγ. Our data suggest transformed ex vivo autologous Tregs have genetic, immunophenotypic and functional characteristics for regulatory T-cells and further can be used for adoptive immunotherapy autoimmune diseases and inhibition of transplantation immunity.
Blinova V.G., Gladilina Y.A., Eliseeva D.D., Lobaeva T.A., Zhdanov D.D. (2022) Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor. Biomeditsinskaya Khimiya, 68(1), 55-67.
Blinova V.G. et al. Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor // Biomeditsinskaya Khimiya. - 2022. - V. 68. -N 1. - P. 55-67.
Blinova V.G. et al., "Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor." Biomeditsinskaya Khimiya 68.1 (2022): 55-67.
Blinova, V. G., Gladilina, Y. A., Eliseeva, D. D., Lobaeva, T. A., Zhdanov, D. D. (2022). Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor. Biomeditsinskaya Khimiya, 68(1), 55-67.
Hartigan-O'Connor D.J., Poon C., Sinclair E., McCune J.M. (2007) Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorting of live cells. J. Immunol. Methods, 319, 41-52. CrossRef Scholar google search
Gambineri E., Torgerson T.R., Ochs H.D. (2003) Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis. Curr. Opin. Rheumatol., 15, 430-435. CrossRef Scholar google search
Rossetti M., Spreafico R., Saidin S., Chua C., Moshref M., Leong J.Y., Tan Y.K., Thumboo J., van Loosdregt J., Albani S. (2015) Ex vivo-expanded but not in vitro-induced human regulatory T cells are candidates for cell therapy in autoimmune diseases thanks to stable demethylation of the FOXP3 regulatory T cell-specific demethylated region. J. Immunol., 194, 113-124. CrossRef Scholar google search
Fontenot J.D., Gavin M.A., Rudensky A.Y. (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol., 4, 330-336. CrossRef Scholar google search
Thornton A.M., Korty P.E., Tran D.Q., Wohlfert E.A., Murray P.E., Belkaid Y., Shevach E.M. (2010) Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J. Immunol., 184, 3433-3441. CrossRef Scholar google search
Callahan M.K., Postow M.A., Wolchok J.D. (2014) CTLA-4 and PD-1 pathway blockade: combinations in the clinic. Front. Oncol., 4, 385. CrossRef Scholar google search
Bono M.R., Fernández D., Flores-Santibáñez F., Rosemblatt M., Sauma D. (2015) CD73 and CD39 ectonucleotidases in T cell differentiation: Beyond immunosuppression. FEBS Lett., 589(22), 3454-3460. CrossRef Scholar google search
Chen X., Oppenheim J.J. (2010) Oppenheim, TNF-alpha: an activator of CD4+FoxP3+TNFR2+ regulatory T cells. Curr. Dir. Autoimmun., 11, 119-134. CrossRef Scholar google search
Booth N.J., McQuaid A.J., Sobande T., Kissane S., Agius E., Jackson S.E., Salmon M., Falciani F., Yong K., Rustin M.H., Akbar A.N., Vukmanovic-Stejic M. (2010) Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO. J. Immunol., 184, 4317-4326. CrossRef Scholar google search
Yadav M., Stephan S., Bluestone J.A. (2013) Peripherally induced tregs — role in immune homeostasis and autoimmunity. Front. Immunol., 4, 232. CrossRef Scholar google search
Kukreja A., Cost G., Marker J., Zhang C., Sun Z., Lin-Su K., Ten S., Sanz M., Exley M., Wilson B., Porcelli S., Maclaren N. (2002) Multiple immuno-regulatory defects in type-1 diabetes. J. Clin. Invest., 109, 131-140. CrossRef Scholar google search
de Kleer I.M., Wedderburn L.R., Taams L.S., Patel A., Varsani H., Klein M., de Jager W., Pugayung G., Giannoni F., Rijkers G., Albani S., Kuis W., Prakken B. (2004)CD4+CD25 bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis. J. Immunol., 172, 6435-6443. CrossRef Scholar google search
Crispin J.C., Alcocer-Varela J., de Pablo P., Martinez A., Richaud-Patin Y., Alarcón-Segovia D. (2003) Immunoregulatory defects in patients with systemic lupus erythematosus in clinical remission. Lupus, 12, 386-393. CrossRef Scholar google search
Valencia X., Yarboro C., Illei G., Lipsky P.E. (2007) Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J. Immunol., 178, 2579-2588. CrossRef Scholar google search
Lyssuk E.Y., Torgashina A.V., Soloviev S.K., Nassonov E.L., Bykovskaia S.N. (2007) Reduced number and function of CD4+CD25highFoxP3+ regulatory T cells in patients with systemic lupus erythematosus. Adv. Exp. Med. Biol., 601, 113-119. CrossRef Scholar google search
Venken K., Hellings N., Thewissen M., Somers V., Hensen K., Rummens J.-L., Medaer R., Hupperts R., Stinissen P. (2008) Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level. Immunology, 123, 79-89. CrossRef Scholar google search
Bach J.F., Chatenoud L. (2001) Tolerance to islet autoantigens in type 1 diabetes. Annu. Rev. Immunol., 19, 131-161. CrossRef Scholar google search
Kohm A.P., Carpentier P.A., Anger H.A., Miller S.D. (2002) Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J. Immunol., 169, 4712-4716. CrossRef Scholar google search
Wu A.J., Hua H., Munson S.H., McDevitt H.O. (2002) Tumor necrosis factor-alpha regulation of CD4+CD25 +T cell levels in NOD mice. Proc. Natl. Acad. Sci. USA, 99, 12287-12292. CrossRef Scholar google search
Hoffmann P., Ermann J., Edinger M., Fathman C.G., Strober S. (2002) Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Exp. Med., 196, 389-399. CrossRef Scholar google search
Mekala D.J., Geiger T.L. (2005) Immunotherapy of autoimmune encephalomyelitis with redirected CD4+CD25+ T lymphocytes.Blood, 105, 2090-2092. CrossRef Scholar google search
Miyara M., Ito Y., Sakaguchi S. (2014) TREG-cell therapies for autoimmune rheumatic diseases. Nat. Rev. Rheumatol., 10, 543-551. CrossRef Scholar google search
Serr I., Weigmann B., Franke R.K., Daniel C. (2014) Treg vaccination in autoimmune type 1 diabetes. BioDrugs, 28, 7-16. CrossRef Scholar google search
Buc M. (2013) Role of regulatory T cells in pathogenesis and biological therapy of multiple sclerosis. Mediators Inflamm., 2013, 963748. CrossRef Scholar google search
Fessler J., Felber A., Duftner C., Dejaco C. (2013) Therapeutic potential of regulatory T cells in autoimmune disorders. BioDrugs, 27, 281-291. CrossRef Scholar google search
Miyara M., Gorochov G., Ehrenstein M., Musset L., Sakaguchi S., Amoura Z. (2011) Human FoxP3+ regulatory T cells in systemic autoimmune diseases. Autoimmun. Rev., 10, 744-755. CrossRef Scholar google search
Xia G., Kovochich M., Truitt R.L., Johnson B.D. (2004) Tracking ex vivo-expanded CD4+CD25+ and CD8+CD25 +regulatory T cells after infusion to prevent donor lymphocyte infusion-induced lethal acute graft-versus-host disease. Biol. Blood Marrow Transplant., 10, 748-760. CrossRef Scholar google search
Trzonkowski P., Dukat-Mazurek A., Bieniaszewska M., Marek-Trzonkowska N., Dobyszuk A., Juścińska J., Dutka M., Myśliwska J., Hellmann A. (2013) Treatment of graft-versus-host disease with naturally occurring T regulatory cells. BioDrugs, 27, 605-614. CrossRef Scholar google search
di Ianni M., Falzetti F., Carotti A., Terenzi A., Castellino F., Bonifacio E., del Papa B., Zei T., Ostini R.I., Cecchini D., Aloisi T., Perruccio K., Ruggeri L., Balucani C., Pierini A., Sportoletti P., Aristei C., Falini B., Reisner Y., Velardi A., Aversa F., Martelli M.F. (2011) Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood, 117, 3921-3928. CrossRef Scholar google search
Edinger M., Hoffmann P. (2011) Regulatory T cells in stem cell transplantation: strategies and first clinical experiences. Curr. Opin. Immunol., 23, 679-684. CrossRef Scholar google search
Safinia N., Scotta C., Vaikunthanathan T., Lechler R.I., Lombardi G. (2015) Regulatory T cells: serious contenders in the promise for immunological tolerance in transplantation.Front. Immunol., 6, 438. CrossRef Scholar google search
Askar M. (2014) T helper subsets and regulatory T cells: rethinking the paradigm in the clinical context of solid organ transplantation. Int. J. Immunogenet., 41, 185-194. CrossRef Scholar google search
Hilbrands R., Howie D., Cobbold S., Waldmann H. (2013) Regulatory T cells and transplantation tolerance. Immunotherapy, 5, 717-731. CrossRef Scholar google search
Zhdanov D.D., Vasina D.A., Orlova V.S., Gotovtseva V.Y., Bibikova M.V., Pokrovsky V.S., Pokrovskaya M.V., Aleksandrova S.S., Sokolov N.N. (2016) Apoptotic endonuclease EndoG induces alternative splicing of telomerase catalytic subunit hTERT and death of tumor cells. Biomeditsinskaya Khimiya, 62(3), 239-250. CrossRef Scholar google search
Zhdanov D.D., Pokrovsky V.S., Orlova E.V., Orlova V.S., Pokrovskaya M.V., Aleksandrova S.S., Sokolov N.N. (2017) Intracellular localization of apoptotic endonuclease EndoG and splice-variants of telomerase catalytic subunit hTERT. Biochemistry (Moscow), 82, 894-905. CrossRef Scholar google search
Wieczorek G., Asemissen A., Model F., Turbachova I., Floess S., Liebenberg V., Baron U., Stauch D., Kotsch K., Pratschke J., Hamann A., Loddenkemper C., Stein H., Volk H.D., Hoffmüller U., Grützkau A., Mustea A., Huehn J., Scheibenbogen C., Olek S. (2009) Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. Cancer Res., 69, 599-608. CrossRef Scholar google search
Blinova V.G., Novachly N.S., Gippius S.N., Hilal A., Gladilina Y.A., Eliseeva D.D., Zhdanov D.D. (2021) Phenotypical and functional characteristics of human regulatory T cells during ex vivo maturation from CD4+ T lymphocytes. Appl. Sci., 11, 5776. CrossRef Scholar google search
Ukena S.N., Höpting M., Velaga S., Ivanyi P., Grosse J., Baron U., Ganser A., Franzke A. (2011) Isolation strategies of regulatory T cells for clinical trials: phenotype, function, stability, and expansion capacity. Exp. Hematol., 39, 1152-1160. CrossRef Scholar google search
Hippen K.L., Merkel S.C., Schirm D.K., Sieben C.M., Sumstad D., Kadidlo D.M., McKenna D.H., Bromberg J.S., Levine B.L., Riley J.L., June C.H., Scheinberg P., Douek D.C., Miller J.S., Wagner J.E., Blazar B.R. (2011) Massive ex vivo expansion of human natural regulatory T cells (T(regs)) with minimal loss of in vivo functional activity. Sci. Transl. Med., 3, 83ra41. CrossRef Scholar google search
Hoffmann P., Eder R., Kunz-Schughart L.A., Andreesen R., Edinger M. (2004) Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood, 104, 895-903. CrossRef Scholar google search
Lifshitz G.V., Zhdanov D.D., Lokhonina A.V., Eliseeva D.D., Lyssuck E.Y., Zavalishin I.A., Bykovskaia S.N. (2016) Ex vivo expanded regulatory T cells CD4 + CD25 + FoxP3 +CD127Low develop strong immunosuppressive activity in patients with remitting-relapsing multiple sclerosis. Autoimmunity, 49, 388-396. CrossRef Scholar google search
Feng Y., van der Veeken J., Shugay M., Putintseva E.V., Osmanbeyoglu H.U., Dikiy S., Hoyos B.E., Moltedo B., Hemmers S., Treuting P., Leslie C.S., Chudakov D.M., Rudensky A.Y. (2015) A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance. Nature, 528, 132-136. CrossRef Scholar google search
Miyazaki T., Arai S. (2007) Two distinct controls of mitotic cdk1/cyclin B1 activity requisite for cell growth prior to cell division. Cell Cycle, 6, 1419-1425. CrossRef Scholar google search
Thiemann S., Man J.H., Chang M.H., Lee B., Baum L.G. (2015) Galectin-1 regulates tissue exit of specific dendritic cell populations. J. Biol. Chem., 290, 22662-22677. CrossRef Scholar google search
Tegla C.A., Cudrici C.D., Nguyen V., Danoff J., Kruszewski A.M., Boodhoo D., Mekala A.P., Vlaicu S.I., Chen C., Rus V., Badea T.C., Rus H. (2015) RGC-32 is a novel regulator of the T-lymphocyte cell cycle. Exp. Mol. Pathol., 98, 328-337. CrossRef Scholar google search
Williams L.M., Rudensky A.Y. (2007) Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat. Immunol., 8, 277-284. CrossRef Scholar google search
Dieckmann D., Plottner H., Berchtold S., Berger T., Schuler G. (2001) Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood. J. Exp. Med., 193, 1303-1310. CrossRef Scholar google search
Ferber I.A., Brocke S., Taylor-Edwards C., Ridgway W., Dinisco C., Steinman L., Dalton D., Fathman C.G. (1996) Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol., 156, 5-7. Scholar google search
Dominguez-Villar M., Baecher-Allan C.M., Hafler D.A. (2011) Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease. Nat. Med., 17, 673-675. CrossRef Scholar google search
Chen X., Bäumel M., Männel D.N., Howard O.M.Z., Oppenheim J.J. (2007) Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J. Immunol., 179, 154-161. CrossRef Scholar google search
Grell M., Becke F.M., Wajant H., Männel D.N., Scheurich P. (1998) TNF receptor type 2 mediates thymocyte proliferation independently of TNF receptor type 1. Eur. J. Immunol., 28, 257-263. CrossRef Scholar google search
Zhdanov D.D., Gladilina Y.A., Pokrovsky V.S., Grishin D.V., Grachev V.A., Orlova V.S., Pokrovskaya M.V., Alexandrova S.S., Sokolov N.N. (2018) Murine regulatory T cells induce death of effector T, B, and NK lymphocytes through a contact-independent mechanism involving telomerase suppression and telomere-associated senescence. Cell. Immunol., 331, 146-160. CrossRef Scholar google search
Zhdanov D.D., Gladilina Y.A., Grishin D.V., Grachev V.A., Orlova V.S., Pokrovskaya M.V., Alexandrova S.S., Pokrovsky V.S., Sokolov N.N. (2018) Contact-independent suppressive activity of regulatory T cells is associated with telomerase inhibition, telomere shortening and target lymphocyte apoptosis. Mol. Immunol., 101, 229-244. CrossRef Scholar google search
