Recent 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 (Treg) 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 Treg cells display senescence features such as mitochondrial alterations, increased expression of cell-cycle regulators and genomic DNA damage. Functionally, KLRG1+ Treg cells show a reduced suppressive activity in vivo accompanied by a pro-inflammatory phenotype.
© 2025. The Author(s).

We previously found that activated CD8+ T-cells increase expression of PD-1, which can be attenuated in the presence of specific Toll-like receptor (TLR) agonists, mediated by IL-12 secreted by professional antigen-presenting cells. While these CD8+ T-cells had greater anti-tumor activity, T-cells stimulated by different TLR had different gene expression profiles. Consequently, we sought to determine whether combinations of TLR agonists might further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity. Activation of CD8+ T-cells in the presence of specific TLR ligands resulted in decreased expression of PD-1, LAG-3, and CD160, notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of E.G7-OVA or TRAMP-C1 tumor-bearing mice with peptide or DNA vaccines, co-administered with combination of TLR3 and TLR9 agonists, showed greater suppression of tumor growth. The anti-tumor effect of TLR1/2 and/or TLR9, but not TLR3, was abrogated in IL-12KO mice. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and type 1 IFN pathway activation following TLR3 stimulation. Our results provide a mechanistic rationale for the choice of optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.
© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.