Soto-Heredero, GonzaloGabandé-Rodríguez, EnriqueCarrasco, ElisaEscrig-Larena, José IgnacioGómez de Las Heras, Manuel MDelgado-Pulido, SandraFrancos-Quijorna, IsaacBlanco, Eva MFernández-Almeida, ÁlvaroAbia, DavidRodríguez, María JosefaFernández-Díaz, Cristina MÁlvarez-Flores, María BeatrizRamírez de Molina, AnaJung, SaschaDel Sol, AntonioZorita, VirginiaSánchez-Cabo, FátimaTorroja, CarlosMittelbrunn, María2026-03-252026-03-252025-05Nat Aging. 2025 May;5(5):799-815.https://hdl.handle.net/20.500.12105/27349Recent studies using single-cell RNA sequencing technology have uncovered several subpopulations of CD4 T cells that accumulate with aging. These age-associated T cells are emerging as relevant players in the onset of inflammaging and tissue senescence. Here, based on information provided by single-cell RNA sequencing data, we present a flow cytometry panel that allows the identification of age-associated T cell subsets in systematic larger analysis in mice. We use this panel to evaluate at the single-cell level mitochondrial and senescence marks in the different age-associated CD4 T cell subpopulations. Our analysis identifies a subpopulation of regulatory T (T) cells that is characterized by the extracellular expression of the co-inhibitory molecule killer cell lectin-like receptor subfamily G member 1 (KLRG1) and accumulates with aging in humans and mice. KLRG1-expressing T cells display senescence features such as mitochondrial alterations, increased expression of cell-cycle regulators and genomic DNA damage. Functionally, KLRG1 T cells show a reduced suppressive activity in vivo accompanied by a pro-inflammatory phenotype.Funded by the European Union. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. This study was supported by European Research Council grant ERC-2021-CoG 101044248-LetTBe, and the Y2020/BIO-6350 NutriSION-CM synergy grant from Comunidad de Madrid, Ministerio de Ciencia e Innovacion, Spain (grants PID2022-141169OB-I00 and PID2022-138295OB-I00). GENYAL Clinical Trials Platform of IMDEA Alimentacion led by A. Ramirez de Molina and R. Ramos Ruiz conducted the volunteer recruitment and human clinical trial. Flow cytometry of human samples was conducted by Flow Cytometry Unit, CNIC, being the cytometer part of the grant EQC2018-005009-P funded by MCIN/AEI/10.13039/501100011033 and by 'ERDF A way of making Europe'. G.S.-H. is supported by a FPI-UAM grant (Universidad Autonoma de Madrid). E.G.-R. was funded by a Juan de la Cierva grant (IJC2018-036850-I; Universidad Autonoma de Madrid). M.G. and J.I.E.-L. are supported by FPU grants (FPU19/02576 and FPU20/04066, respectively), both from Ministerio de Ciencia, Innovacion y Universidades (Spain). E.C. is supported by SI4/PJI/2024-00166 from Comunidad de Madrid and UAM (Spain). S.D.-P. was funded by a PIPF grant (PIPF-2022/SAL-GL-25208), from Comunidad de Madrid (Spain). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.engVoR40307497Nature Agingopen access