Phenotype and effector functions of GD2-specific CAR-T lymphocytes in vitro

Abstract

Introduction. The application of T lymphocytes with a chimeric antigen receptor (CAR) underlies new promising technologies for tumor immunotherapy, the main idea of which is associated with the adoptive transfer of modified T cells that are capable of cytotoxicity, this does not require additional costimulation and recognize surface antigens in an MHC-independent manner due to their difference from TCR to receptor structure. This paper presents characterization of CAR-T cells specific for disialoganglioside GD2 ex vivo, which expression is restricted to malignant neoplasms, making this molecule a promising target. The addition of costimulatory domains, such as 4-1BB or CD28 to bone CAR cells increases survival and proliferation of the resulting CAR-T cells, and the insertion of the GITR ligand (GITRL) promotes self-maintenance of the resulting populations and improves antitumor activity.

Aim of the study – to research the phenotype and effector functions of GD2-specific CAR-T lymphocytes in vitro using various functional tests.

Material and methods. Transduced and non-transduced GD2-specific CAR lymphocytes from healthy adult donors were examined for the content of cell memory subpopulations by CD45RA and CD62L markers and cell exhaustion by PD-1 and TIM-3 markers using flow cytometry. Cytotoxicity was studied by the multiplex analysis of conditioned media after cocultivation of the studied genetically modified cells and tumor cell lines on a flow cytometer and by the colorimetric method by the content of lactate dehydrogenase. The effector functions of the resulting CAR-T cells were assessed after cocultivation with target cells for cell activation markers 4-1BB, CD40L, CD69 and FasL using flow cytometry.

Results. Transduced CD4+ and CD8+ lymphocytes represented the least differentiated population of CD45RA+CD62L+ effector cells with high viability and proliferative activity, also, a proportion of exhausted PD-1+TIM-3+ cells was low. The resulting transduced cells exhibited antigen-specific properties for tumor cell lines carrying the target antigen. In addition, the cytotoxic function of transduced T lymphocytes carrying cellular activation markers 4-1BB, CD69 and CD40L, as well as their cytokine-producing activity in the presence of GD2-expressing tumor cells, was observed.

Conclusion. The obtained GD2-specific CAR-T cells exhibit antigen-specific antitumor properties in vitro, which is due to their cytokine-producing activity, increased expression of cell activation markers 4-1BB, CD69 and CD40L, and a low proportion of exhausted PD-1+TIM-3+ cells.

Keywords:CAR-T cells; GD2; GITRL; T cell exhaustion; activation markers; antigen-specific cytotoxicity

For citation: Philippova J.G., Kuznetsova M.S., Shevchenko J.A., Tereshchenko V.P., Fisher M.S., Kurilin V.V., Pashkina E.A., Akahori Ya., Shiku H., Sennikov S.V. Phenotype and effector functions of GD2-specific CAR-T lymphocytes in vitro. Immunologiya. 2022; 43 (5): 525–35. DOI: https://doi.org/10.33029/0206-4952-2022-43-5-525-535 (in Russian)

Funding. The study was supported by the grant of the Russian Science Foundation (project No. 21-65-00004). URL: https://rscf.ru/project/21-65-00004/

Conflict of interests. Authors declare no conflict of interests.

Authors’ contributions. The concept and design of the study – Kuznetsova M.S., Shevchenko J.A., Tereshchenko V.P., Kurilin V.V., Akahori Ya., Shiku H., Sennikov S.V.; data collection and processing – Kuznetsova M.S., Shevchenko J.A., Tereshchenko V.P., Philippova J.G., Fisher M.S., Pashkina E.A.; statistical data processing – Kuznetsova M.S., Shevchenko J.A., Pashkina E.A.; text writing – Philippova J.G.; editing – Philippova J.G., 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 – Philippova J.G.



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