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1 . 2024
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Effect of detoxified Shigella sonnei lipopolysaccharide on the expression of tumor-associated antigen gp100 and MHC I antigens by B16 melanoma cells

Abstract

Introduction. In recent decades, active researches have been conducted on the suitability immunotherapeutic agents based on drugs that stimulate the innate immune response, in particular TLRs ligands (agonists) (Toll-like receptors, TLRs), in complex therapy for malignant melanoma. TLR agonists are considered as promising drugs due to their ability to stimulate the antitumor immune response. It has been established that TLRs are present both on cells of the immune system and on malignant melanocytes, therefore the effect of drugs based on TLRs agonists on the progression of malignant melanoma will depend on both the effect on tumor cells and on cells of the immune system.

Aims of the study – to investigate the effect of detoxified lipopolysaccharide (Ac3-S-LPS) of Shigella sonnei, phase I, on the expression of tumor-associated gp100 antigen and MHC I antigens by B16 melanoma cells in vitro and on the infiltration of primary tumor nodes by CD8+-T lymphocytes after administration of Ac3-S-LPS in vivo.

Material and methods. To study the effect of Ac3-S-LPS on B16 melanoma cells, we used two experimental models: B16 melanoma cell culture (provided by the Cellular Immunity Laboratory, Research Institute of Experimental Diagnostics and Tumor Therapy, N.N. Blokhin NMRCO, MOH of Russia) and primary tumor nodes (PTN), obtained after inoculation of melanoma cells into experimental animals. When assessing the effect of Ac3-S-LPS on melanoma cells in culture, melanocytes were cultured with the drug for 48 hours. To study the effect of Ac3-S-LPS on subpopulations of cells in the PTN, the drug was injected into the area of the formed nodes.

The expression of TLR4 and gp100 antigens (AGs) was determined using fluorochrome-labeled polyclonal antibody (Abs) to melanoma cells grown on coverslips, followed by analysis of the preparations with a fluorescence microscope.

The expression of the AGs CD8a, CD45, MHC I, CD F4/80 was determined using monoclonal Abs labeled with fluorochromes in cell suspensions obtained after culturing cells in vitro and in cell suspensions of PTN, and subsequent analysis of the resulting suspensions with a flow cytometer.

To assess the effect of Ac3-S-LPS on the proliferation of tumor cells in culture, a test with the VPD450 dye was used.

Results. B16 melanoma cells have been shown to express TLR4. The presence of this receptor on the surface of malignant melanocytes suggests that the effects that were noted when culturing cells with Ac3-S-LPS are due to its interaction with this receptor, since it has been established that lipopolysaccharides of gram-negative microorganisms (including Shigella sonnei cells) are ligands of this receptor. Cultivation of melanoma cells with Ac3-S-LPS leads to a change in the pattern of expression of surface AGs – gp100 and MHC I. The level of gp100 expression decreases, while the number of cells bearing MHC I increases. The proliferative activity of tumor cells decreases. When Ac3-S-LPS is administered directly into the area of PTN, in the tumor node the relative amount of tumor cells expressing MHC I and T lymphocytes carrying the CD8a antigen increases.

Conclusion. The effect of detoxified lipopolysaccharide (Ac3-S-LPS) of Shigella sonnei, phase I on B16 malignant melanoma cells was studied using experimental models in vitro and in vivo. It was found that the use of Ac3-S-LPS reduces the expression of melanoma-associated antigen gp100 on tumor cells and increases the expression of MHC I antigens, which is accompanied by increase the amount of T lymphocytes expressing the antigen CD8a in PTN.

Keywords:B16 melanoma; malignant cells; detoxified lipopolysaccharide; TLR4; gp100; MHC I; VPD450 test

For citation: Novikova E.M., Chukhina E.S., Razvalyayeva N.A., Kozyreva O.V., Golovina M.E., L’vov V.L., Aparin P.G., Stepanenko R.N. Effect of detoxified Shigella sonnei lipopolysaccharide on the expression of tumor-associated antigen gp100 and MHC I antigens by B16 melanoma cells. Immunologiya. 2024; 45 (1): 68–81. DOI: https://doi.org/10.33029/1816-2134-2024-45-1-68-81 (in Russian)

Funding. Study was performed within a state task under the agreement between FMBA of Russia and NRC Institute of Immunology, FMBA of Russia, No 388-03-2023-041 of 19.01.2023. Open publication of the research results is allowed.

Conflict of interests. The authors declare no conflict of interests.

Authors’ contribution. Study conception and design – Stepanenko R.N.; material collection and processing – Novikova E.M., Kozyreva O.V., Razvalyaeva N.A., Chukhina E.S., Golovina M.E., L’vov V.L., Aparin P.G., Stepanenko R.N.; statistical processing – Kozyreva O.V., Chukhina E.S., Stepanenko R.N.; article writing – Novikova E.M., Kozyreva O.V., Stepanenko R.N.; article editing – Chukhina E.S., L’vov V.L., Aparin P.G., Stepanenko R.N.

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