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.

CD4+调节性T细胞（CD4+ regulatory T cells, Tregs）是免疫系统的核心调控因子，既可促进保护性免疫的建立，同时抑制有害免疫应答。婴幼儿时期是免疫系统快速发育的关键阶段，但目前学界对Tregs在人类生命早期阶段如何介导免疫应答的认知仍较为匮乏。本研究对比了儿童与成人受试者的血液及扁桃体（tonsil）组织中的Tregs，以探究Treg生物学特性的年龄相关差异。研究发现，与配对血液样本相比，儿童扁桃体中的叉头框P3蛋白（FoxP3）表达水平及Tregs占比均显著升高。在扁桃体组织内，生命早期的Tregs聚集于滤泡外区域，其细胞相互作用偏向CD8+ T细胞（CD8+ T cells）。与血液中的Tregs相比，儿童及成人扁桃体Tregs均呈现富集谱系特异性特征及经典功能相关的转录谱，提示组织是Treg发挥活性的主要位点。生命早期扁桃体Tregs的转录谱进一步以与活化、增殖及多功能特性相关的通路为核心特征。与成人Tregs相比，儿童扁桃体Tregs的转录特征差异还与表型差异、高增殖能力及高水平分泌白细胞介素10（IL-10）相关。本研究结果明确了组织是决定Treg身份的主要因素，为阐明人类生命周期中Treg生物学特性的发育差异提供了新见解，并揭示了生命早期Tregs独特的功能特性。实验设计概述：本研究针对从儿童及成人供体来源的血液与扁桃体Tregs，采用高维流式细胞术（high dimensional flow cytometry）、RNA测序（RNA sequencing）及免疫荧光成像（immunofluorescent imaging），以揭示生命早期Tregs的独特特性。研究通过分选常规CD4 T细胞及CD25+/CD127- 调节性T细胞生成测序数据，并对比不同组织（扁桃体/血液）与不同年龄组（婴儿与成人）的样本，以明确调节性T细胞的转录差异。随后通过高维流式细胞术验证上述研究结果，并结合扁桃体组织成像进一步获取更多研究见解。