SA-β-galactosidase activity in effector and regulatory T cells.

The aging of the immune system is accompanied by a significant increase in the risk of developing age-related pathologies, including inflammatory, autoimmune diseases and oncology. Age-dependent decline in immune function is accompanied by a gradual accumulation of senescent (aged) cells that are the source of chronic inflammation. The main producers of proinflammatory cytokines in the body are activated effector T cells. With age, along with an increase in chronic inflammation (InflamAging), the ability of the immune system to suppress and/or remove activated and senescent effector cells decreases. With age, there is also a decrease in the function of CD3⁺CD4⁺CD25^hiCD127^lo/- T-regulatory cells (Treg), which unbalances the immune system and increases the risk of autoimmune pathologies. In ex vivo analysis, senescence and effector function in CD4⁺ and CD8⁺, activated (CD25⁺ and HLA-DR+) and non-activated (CD25^- and HLA-DR^-) T cells, as well as the suppressor activity of senescent Tregs were assessed by functional tests and using SA-β-galactosidase assay. Additional analysis, p16^INK4A, p21^WAF1/CIP1, phospho-histone H2A.X and HMGB1 was applied to confirm the senescent status of these cells. PBMCs irradiated with UV or reaching the replication limit were used as a positive control of senescent cells. UV radiation increased SA-β-Gal activity and correlated with an increase in the level of phospho-histone H2A.X, confirming their senescent status and the relevance of SA-β-Gal as a marker of senescence. In middle-aged donors in activated (CD25⁺ and HLA-DR⁺) both CD4⁺ and CD8⁺ lymphocytes, SA-β-Gal activity was higher compared to non-activated lymphocytes. Whereas, SA-β-Gal activity was lowest in Tregs, suggesting their specific metabolic patterns. Among middle-aged donors, there were no significant differences between the populations of CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes in SA-β-Gal activity. Thus, the activity of SA-β-Gal marker of senescent and activated effector T cells can be used to analyze the aging of the immune system in further functional tests. Using this approach, the phenotypic features and functional activity of senescent T cells among young (<30 years old) and older (>60 years old) donors will be studied in detail. The development of this model in non-human primates of different age groups will facilitate future preclinical trials for senolytic and senomorphic drugs, and substances that enhance the repair of double-strand breaks.

This work was supported by the Russian Science Foundation, No. 23-15-00443.