Study of MAGE-A4 specific TCR-like CAR-T lymphocytes in vitro


Introduction. Currently, one of the promising options for CAR-T technology is the development of TCR-like CAR-T lymphocytes that recognize epitopes of tumor-associated antigens in combination with MHC, which opens up a wide range of possible target antigens of any cellular localization. This work presents characterization in vitro of TCR-like CAR-T cells specific for MAGE-A4, a cancer-germline antigen, which expression is observed only in malignant neoplasms and immune-privileged organs, which makes it a promising target for effective CAR-T cellular therapy with reduced off-target toxicity.

Material and methods. Transduced and non-transduced lymphocytes were studied for the memory subsets (by markers CD45RA and CD62L) and cell activation and exhaustion mar- kers (CD69, CD95, PD-1, TIM3) by flow cytometry. The cytotoxicity of the obtained CAR-T cells was studied colorimetrically by the content of lactate dehydrogenase. The effector function of CAR-T cells was studied after cocultivation with target cells by cell activation markers (4-1BB, CD69, CD40L, FasL) detection using flow cytometry.

Results. Transduced CD4+ and CD8+ lymphocytes were characterized by an increased content of terminal effector cells, as well as activated (according to CD69 marker) and exhausted cells (PD-1+TIM3+ phenotype). However, predominantly, transduced cells were represented by low differentiated memory T cell subsets, and did not carry markers of activation and exhaustion. Transduced cultures exhibited antigen-specific cytotoxicity greater than those of non-transduced cells. At the same time, the cytotoxicity of transduced cells was accompanied by an increase in the number of cells carrying activation immunostimulating molecules 4-1BB, CD69, CD40L.

Conclusion. The MAGE-A4-specific TCR-like CAR-T cells exhibit antitumor response in vitro and can be recommended for further preclinical study in experimental models in vitro and in vivo.

Keywords:CAR; TCR-like CAR-T cells; MAGE-A4; GITR; memory T cell subsets; T cell activation; T cell exhaustion

For citation: Tereshchenko V.P., Kuznetsova M.S., Shevchenko J.A., Fisher M.S., Kurilin V.V., Alsalloum A., Akahori Y., Shiku H., Sennikov S.V. Study of MAGE-A4 specific TCR-like CAR-T lymphocytes in vitro. Immunologiya. 2022; 43 (4): 401–11. DOI: (in Russian)

Funding. The research was supported by the grant of the Russian Science Foundation (project No. 21-65-00004). URL:

Conflict of interests. Authors declare no conflict of interests.

Authors’ contributions. The concept and design of the study – Tereshchenko V.P., Kuznetsova M.S., Shevchenko J.A., Kurilin V.V., Akahori Y., Shiku H., Sennikov S.V.; data collection and processing – Tereshchenko V.P., Kuznetsova M.S., Shevchenko J.A., Fisher M.S., Alsallum A.; statistical data processing – Tereshchenko V.P.; text writing – Tereshchenko V.P.; editing – Tereshchenko V.P., Kuznetsova M.S., Sennikov S.V.; approval of the final version of the article – Sennikov S.V.; responsibility for the integrity of all parts of the article – Tereshchenko V.P.


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Musa R. Khaitov

Corresponding member of Russian Academy of Sciences, MD, Professor, Director of the NRC Institute of Immunology FMBA of Russia