РОЛЬ РЕГУЛЯТОРНЫХ Т-КЛЕТОК В РАЗВИТИИ АУТОИММУННЫХ НАРУШЕНИЙ ПРИ РАССЕЯННОМ СКЛЕРОЗЕ

In the maintenance of immunological tolerance important role belongs to the recently discovered population of regulatory T-cells CD4 + CD25 + FoxP3 +. These cells have potential in suppressing pathologic immune responses observed at various autoimmune diseases including multiple sclerosis. We have shown a reduction in the number and functional activity of T-reg in peripheral blood of patients with multiple sclerosis in the acute stage, the increase in their number during remission, duration of the relationship of the autoimmune process and the degree of disability of patients with the contents of T-reg. The possibility of using the grown ex vivo T-reg for the correction of immunopathological process in multiple sclerosis. В процессах поддержания иммунологической толерантности важная роль принадлежит недавно открытой популяции регуляторных Т-клеток CD4+CD25+FoxP3+ (Т-reg).  Эти клетки обладают огромным потенциалом в подавлении патологического иммунного ответа, наблюдающегося при различных аутоиммунных заболеваниях, в том числе при рассеянном склерозе. Продемонстрировано  снижение числа и функциональной активности Т-reg в периферической крови  больных рассеянным склерозом в стадии обострения, увеличение их количества при ремиссии заболевания, связь длительности аутоиммунного процесса и степени инвалидизации больных с количеством  Т-reg.  Показана возможность использования выращенных ex vivo Т-reg для коррекции иммунопатологических нарушений при рассеянном склерозе

Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor [Povyshennaia supressornaia aktivnost' transformirovannykh ex vivo reguliatornykh T-kletok v sravnenii s nestimulirovannymi kletkami togo zhe donora]

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.Reguliatornye T-kletki CD4⁺CD25⁺FoxP3⁺SD127low (Treg) igraiut kliuchevuiu rol' v podderzhanii tolerantnosti k autoantigenam, podavliaiut funktsiiu éffektornykh T- i V-limfotsitov i obespechivaiut balans mezhdu éffektornym i reguliatornym zvenom immuniteta. U bol'nykh s autoimmunnymi zabolevaniiami snizheno soderzhanie Treg i narushena funktsiia étikh kletok. Zamestitel'naia terapiia autologichnymi kletkami patsienta, vyrashchennymi ex vivo, mozhet vosstanovit' narushennyĭ balans immunnoĭ sistemy. Nami razrabotana metodika kul'tivirovaniia kletok-predshestvennikov Treg vne organizma cheloveka, kotoraia pozvoliaet iz 50 ml perifericheskoĭ krovi vyrastit' 300-400 mln Treg v techenie odnoĭ nedeli. V nastoiashchem issledovanii pokazano, chto po sravneniiu s Treg perifericheskoĭ krovi, vyrashchennye ex vivo kletki na 90-95% imeiut fenotip CD4⁺CD25⁺FoxP3⁺SD127low Treg i povyshennuiu ékspressiiu kliuchevykh genov transkriptsii FoxP3 i Helios. V transformirovannykh Treg povyshena stepen' demetilirovaniia v promotornom uchastke gena FoxP3 i aktivirovany geny-markery proliferatsii tsiklina V1, Ki67 i LGALS 1. Transformirovannye ex vivo Treg obladaiut povyshennoĭ supressornoĭ aktivnost'iu, i do 80-90% kletok v populiatsii sekretiruiut tsitokiny TNFα i IFNγ. Nashi dannye pokazyvaiut, chto vyrashchennye ex vivo autologichnye Treg kletki imeiut geneticheskie, markernye i funktsional'nye kharakteristiki reguliatornykh T-kletok, kotorye v budushchem mogut byt' ispol'zovany dlia adaptivnoĭ immunoterapii bol'nykh s autoimmunnymi zabolevaniiami, a takzhe dlia podavleniia transplantatsionnogo immuniteta, v chastnosti, reaktsii transplantata protiv khoziaina

Phenotypical and functional characteristics of human regulatory t cells during ex vivo maturation from cd4+ t lymphocytes

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells. © 2021 by the authors. Licensee MDPI, Basel, Switzerland