1 . 2025

Determination of the phenotype and cytokine production of TCR-like CAR/CAR/TCR T cells upon contact with tumor cell spheroids in vitro

Abstract

Introduction. Over the last decade, technologies have emerged in cancer therapy that use T cells that have a genetically modified T cell receptor or have a chimeric T cell receptor that allows them to recognize various antigens. Some types of such cells have been shown to be highly effective in treating malignant neoplasms. The use of 3D-cultures to determine the mechanisms of action of such T cells provides additional insight into the action of transduced T cells when in contact with tumors in the form of 3D-cultures.

Aim of the study – determination of the phenotype and cytokine production of TCR-like CAR/CAR/TCR T cells upon contact with tumors in the form of 3D-cultures.

Material and methods. TCR-like CAR/CAR/TCR T cells were generated by transduction of healthy donor peripheral blood cells with γ-retroviral vectors. The phenotype of the transduced TCR-like CAR/CAR/TCR T cells was assessed by flow cytometry. 3D-cultures of the SK-MEL-37 melanoma tumor cell line were generated using the hanging drop method. Cytokine production in the culture of TCR-like CAR/CAR/TCR T cells and a 3D-spheroid of the SK-MEL-37 melanoma line was evaluated using multiplex analysis.

Results. Phenotype analysis showed that after transduction of TCR-like NY-ESO-1, TCR-like CAR MAGE-A4 and CAR GD2 specific T cells, dominant populations of CD4⁺ and CD8⁺ effector T cells are formed. Evaluation of cytokine production showed that in the conditioned medium from co-cultures of TCR-like CAR/CAR/TCR T cells and SK-MEL-37 3D-spheroid, a large number of antitumor cytokines are observed.

Conclusion. Transduction with γ-retroviral vectors targeting specific antigens leads to the formation of effector T cells, which, when co-cultured in 3D-cultures, produce a large number of antitumor cytokines, making the use of 3D-models promising for the development of clinical models of solid tumor therapy.

Keywords: TCR-like/CAR/TCR T-cells; SK-Mel-37; 3D-spheroid; flow cytometry; cytokines

For citation: Bulygin A.S., Philippova J.G., Alsalloum A., Alrhmoun S., Kireev F.D., Shevchenko J.A., Fisher M.S., Perik-Zavodskii R.Yu., Perik-Zavodskaia O.Yu., Nazarov K.V., Shiku H., Golikova E.A., Obleuhova I.A., Silkov A.N., Sennikov S.V. Determination of the phenotype and cytokine production of TCR-like CAR/CAR/TCR T cells upon contact with tumor cell spheroids in vitro. Immunologiya. 2025; 46 (1): 51–61. DOI: https://doi.org/10.33029/1816-2134-2025-46-1-51-61 (in Russian)

Funding. The study was supported by the grant of Russian Science Foundation No. 21-65-00004.

Conflicts of interests. Authors declare no conflict of interests.

Authors’ contributions. The concept and design of the study – Bulygin A.S., Filippova J.G., Shevchenko J.A., Shiku H. and Sennikov S.V.; collection and processing of material – Fisher M.S., Shevchenko J.A., Philippova J.G., Kireev F.D., Nazarov K.V., Obleuhova I.A., Bulygin A.S.; statistical processing – Bulygin A.S., Alrhmoun S., Perik–Zavodskii R.Yu., Perik–Zavodskaia O.Yu.; writing the text – Bulygin A.S., Filippova J.G., Kireev F.D., Alsalloum A.; editing – Sennikov S.V., Silkov A.N. Golikova E.A.

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