Usage of IFNγ or TNFα-T in protocols for obtaining cytokine-induced killer cells for adoptive immunotherapy

Methods of immunocorrection based on injection of in vitro activated autologous leukocytes are becoming widespread. Our previous studies suggest that implementation of immunotherapy with in vitro activated lymphocytes using cytokines is effective for the treatment of cancer patients, and that this therapy is well tolerated. This article discusses the use of biological drugs, i.e., recombinant interferon-gamma (IFNγ) and recombinant tumor necrosis factor-alpha-thymosin-alpha-1 (TNFα-T) for the in vitro production of human cytokine-induced killer cells (CIC). The purpose of our study was to assess the possibility of IFNγ and TNFα-T usage within protocols for in vitro production of human CIC. Isolated mononuclear blood cells from 15 donors were cultured in a CO2 incubator for 12 days and the morpho-functional characteristics of lymphocytes were studied. To assess the in vitro toxicity of IFNγ and TNFα-T towards lymphocytes, the drug concentrations have been adjusted experimentally, with respect to control of cell viability. The levels of cytokines in the supernatants at the stages of cell culture were determined by enzyme-linked immunosorbent assay (ELISA). Before and after cultivation, the presence of adhesion and granularity of the studied lymphocytes was assessed, the phenotype of cultured cells was studied by flow cytometry, and the level of viability of activated lymphocytes was assessed in a haemocytometer chamber. We carried out a comparison of methods for culturing lymphocytes under different conditions. We studied culture media containing the following supplements: only IFNγ (group 1); IFNγ, interleukin-2 (IL-2) and interleukin-15 (IL-15) (group 2); TNFα-T only (group 3); TNFα-T, IL-2 and IL-15 (group 4), and only IL-2 and IL-15 (control group). When mononuclear cells are activated in a medium containing IFNγ, IL-2 and IL-15, or in a medium containing TNFα-T, IL-2 and IL-15, we observed a general trend towards significant increase in T-cytotoxic cells (CD3 ⁺CD8⁺), NKT cells (CD3⁺CD16⁺CD56⁺), activated lymphocytes (HLA-DR⁺), activated T lymphocytes (CD3⁺HLA-DR⁺), and activation markers of all lymphocytes (CD38⁺) and on T cells ( CD3⁺CD38⁺). We assessed the possibility of using IFNγ and TNFα-T to obtain human CIK in vitro. Activated cytotoxic lymphocytes obtained by this approach may be used for fundamental research, in order to identify new patterns and morpho-functional characteristics of activated human lymphocytes, as well as for adoptive immunotherapy for cancer patients.

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