Single-cell transcriptomics of Treg reveals hallmarks and trajectories of immunological aging.

Age-related immunosenescense is characterized by progressive dysfunction of adaptive immune response and increased autoimmunity. Nevertheless, the impact of aging on CD4+ regulatory T cells (Treg) that are master regulators of the immune system remains largely unclear. Here, we report cellular and molecular hallmarks of Treg derived from murine lymphoid and adipose tissues at 3, 18 and 24 months of age, respectively, by analysing their heterogeneity that displays dynamic changes in transcriptomic effector signatures at a single-cell resolution. Although the proportion of Treg among total Cd4+ T cells, as well as their expression levels of Foxp3 did not show any global change with time, we have identified six transcriptomically distinct clusters of Treg with cross-tissue conserved hallmarks of aging including increased numbers of pro-inflammatory Treg, reduced precursor cells, increased immature and mature T follicular regulatory cells (Tfr) potentially supported by a metabolic switch from oxidative phosphorylation to glycolysis, a gradual loss of CD150hi Treg that support hematopoiesis and increased adipose tissue-specific Treg that are associated with metabolic disease. To dissect the impact of immunosenescense on humoral immunity, we propose some potential mechanisms underlying Tfr-mediated dysfunction by interactome analysis on Tfr, T follicular helper cells and B cells during aging. Lastly, spatiotemporal analysis further revealed trajectories of Treg aging that demonstrate the most significant changes in marrow and adipose tissues that might contribute to the development of age-related immunosenescense and type-2 diabetes. Taken together, our findings could provide a better understanding of age-associated Treg heterogeneity in lymphoid and adipose tissues, as well as Treg hallmarks during progressive adaptation to aging that could be therapeutically targeted for rejuvenating the aging immune system in future.