ISSN Print 2500–1094
ISSN Online 2542–1204
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
ORIGINAL RESEARCH
T-cell receptor chain centricity in the primarily activated effectors and re-stimulated memory cells
About authors
1 Blokhin National Medical Research Center of Oncology, Moscow, Russia
2 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Moscow, Russia
Correspondence should be addressed: Dmitry B. Kazansky
Kashirskoye Shosse, 24, bld. 15, Moscow, 115478, Russia; ur.xednay@1yksnazak
About paper
Funding: the study was supported by the RSF grant No. 22-15-00342 (2022–2024).
Acknowledgements: the authors would like to express their gratitude to O. Britanova (Institute of Bioorganic Chemistry RAS) and K. Lupyr (Skolkovo Institute of Science and Technology) for generation and bioinformatics analysis of the mouse TCR α-chain libraries.
Author contribution: Kalinina AA — study planning, literature review, experimental procedure, data analysis and interpretation, manuscript writing; Kubekina MV — cloning; Persiyantseva NA — transfection; Bruter AV — selection of oligonucleotides, cloning; Khromykh LM — study planning, literature review, data analysis and interpretation, manuscript editing; Kazansky DB — study planning, literature review, data analysis and interpretation, manuscript editing.
Compliance with ethical standards: the study was approved by the Ethics Committee of the Blokhin National Medical Research Center of Oncology (protocol No. 3P-10.06.2022 dated 10 June 2022), it was conducted in strict compliance with the provisions of the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Received: 2024-10-09
Accepted: 2024-10-30
Published online: 2024-11-30
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Fig. 1. Analysis of mouse splenic T cells after in vivo immunization with the allogeneic tumor cells. C57BL/6 mice (n = 9) were immunized intraperitoneally with allogeneic mastocytoma Р815 cells. Spleen cells were analyzed by flow cytometry 2 months after immunization. Splenocytes of intact (not immunized with P815) C57BL/6 mice (n = 6) were used as a control. А) The relative count (%) of T cells (CD3+). B) The percentage (%) of cytotoxic CD8+ Т cells. C) The percentage (%) of CD8+ Т cells with the phenotype of naïve cells (CD44–CD62L+), central memory cells (CD44+CD62L+), and effector memory cells (CD44+CD62L–). The data are presented as m ± SEM (n = 6–9). *р ≤ 0.05 compared to the intact control (Student's t-test)
Fig. 2. Levels of antigen-induced proliferation in vitro of T-cells in the primary and secondary immune responses to the allogeneic tumor cells. Splenic cells of intact (not immunized with P815) and Р815-immunized C57BL/6 mice were cultured in the presence of allogeneic mastocytoma Р815 for 72 h. Splenocytes similarly cultured without antigenic stimulation were used to assess baseline proliferation. The index of antigen-induced proliferative response to alloantigens was calculated as described in the Methods section. The data are presented as m ± SEM (n = 6–9). * *р ≤ 0.01 (Student's t-test)
Fig. 3. T-cell receptor α-chain variants selected for in vitro screening. Frequency of the T-cell receptor α-chain clonotype in the repertoire of effectors involved in the primary immune response (А) and of re-stimulated memory T cells (B)
Fig. 4. In vitro antigen-induced proliferation levels of T cells transduced with individual T-cell receptor (TCR) α-chain variants. Activated T cells of an intact C57BL/6 mouse were transduced with the TCR α-chains from the repertoire of primarily activated effectors (А) or re-stimulated memory cells (B). Modified lymphocytes were placed in the culture with syngeneic stimulators (EL-4 lymphoma cells) or allogeneic specific stimulators (Р815 mastocytoma cells) for 72 h. To assess baseline proliferation levels, transduced T cells were cultured without tumor cells (baseline). Non-transduced activated lymphocytes (NTLs) and GFP-modified T cells were used as controls. The data of one representative experiment out of two are presented as m ± SEM for three technical replicates. *р ≤ 0.05 (Student's t-test)
