Distinct localization, transcriptional profiles, and functionality in early life tonsil regulatory T cells
CD4+ regulatory T cells (Tregs) are key orchestrators of the immune system, fostering the establishment of protective immunity while preventing deleterious responses. Infancy and childhood are crucial periods of rapid immunologic development but how Tregs mediate immune responses at these earliest timepoints of human life is poorly understood. Here we compare blood and tissue (tonsil) Tregs across pediatric and adult subjects to investigate age related differences in Treg biology. We observed increased FoxP3 expression and proportions of Tregs in tonsil compared to paired blood samples in children. Within tonsil, early life Tregs accumulated in extrafollicular regions with cellular interactions biased towards CD8+ T cells. Tonsil Tregs in both children and adults expressed transcriptional profiles enriched for lineage defining signatures and canonical functionality compared to blood, suggesting tissue as the primary site of Treg activity. Early life tonsil Tregs transcriptional profiles were further defined by pathways associated with activation, proliferation, and polyfunctionality. Observed differences in pediatric tonsil Treg transcriptional signatures were associated with phenotypic differences, high proliferative capacity and robust production of IL-10 compared to adult Tregs. These results identify tissue as a major driver of Treg identity, provide new insights into developmental differences in Treg biology across the human lifespan and demonstrate unique functional properties of early life Tregs. Overall design: In this study, we applied  high dimensional flow cytometry, RNAsequencing, and immunofluorescent imaging to  blood and tonsil Tregs obtained from pediatric and adult donors to reveal distinct properties of early life Tregs. Sequencing data was generated by sorting conventional CD4 T cells and regulatory (CD25+/CD127-) T cells with comparisons made across tissue (tonsil/blood) and age (infant vs adult) to define transcriptional differences in regaulatory T cells. These results were then validated using high dimensional flow cytometry with additional insights generated by the use of imaging of tonsil tissue.