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Generation of Lymphokine-Activated Killers on the Background of a Decresed Content of T-regulatory Cells In Vitro

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Abstract

In this work we studied the effect of different concentrations of interleukin-2 (IL-2) and interferon-γ (IFN-γ) on the proliferation and immunophenotype of lymphocytes obtained from patients with locally advanced breast cancer (stage II-III) after immunomagnetic depletion of regulatory T cells (Tregs) from the general pool of lymphocytes in vitro. The peripheral blood of 11 patients was used as a material. Peripheral blood mononuclear cells (MNCs) were isolated and regulatory T lymphocytes were removed by immunomagnetic separation. After separation, the cells were cultured in a nutrient medium RPMI-1640 with 10% fetal bovine serum (FBS) for 7 days. Lymphocytes were activated on the first day of cultivation with one of the following cytokines: IFN-γ (10 IU/mL), IL-2 (0.1 or 1 μg/mL); IL-2 (0.1 or 1 μg/mL) and IFN-γ together. Lymphocytes without cytokine addition was used as a control. Cells were counted using an automatic counter before addition cytokines and after 2, 4, and 7 days of cultivation with cytokines, and their phenotype was examined. The results showed some phenotypic differences in some elements of cellular immunity between control and experimental samples. Particular attention is paid to the description of changes in the expression of surface markers in the natural killer (NK) subpopulation. It was noted that the proportion of Tregs, despite their preliminary depletion, increased after exposure to cytokines. As a result, the preliminary decrease in the proportion of Treg cells before the stimulation of lymphocytes did not produce the expected effect, therefore the use of depletion in such a methodological mode did not lead to significant results. However, one should not exclude the possibility of inhibiting Tregs by other methods.

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Funding

The work was carried out according to the state assignment “Development and application of new methods of cellular technologies for tumor immunotherapy” (no. АААА-А18-118072790017-9).

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  1. National Medical Research Centre for Oncology, Ministry of Health of Russia, 344037, Rostov-on-Don, Russia

    A. O. Sitkovskaya, E. Yu. Zlatnik, T. V. Shamova, E. S. Bondarenko, I. A. Novikova, L. N. Vaschenko, E. E. Kechedzhieva, I. R. Dashkova, T. V. Ausheva & O. I. Kit

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  1. A. O. Sitkovskaya
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Correspondence to A. O. Sitkovskaya.

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Statement of compliance with standards of research involving humans as subjects. The work was approved by the Ethics Committee of the National Medical Research Centre for Oncology of the Ministry of Health of Russia (Rostov-on-Don), Protocol no. 33/1 dated November 29, 2018. Informed consent was signed with all patients to participate in the study.

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Sitkovskaya, A.O., Zlatnik, E.Y., Shamova, T.V. et al. Generation of Lymphokine-Activated Killers on the Background of a Decresed Content of T-regulatory Cells In Vitro. Cell Tiss. Biol. 15, 455–464 (2021). https://doi.org/10.1134/S1990519X21050102

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