Moleirinho Beatriz, Paulo-Pedro Margarida, Martins Nicole C, Jelagat Emily, Conti Eller, Velho Tiago R, Abecasis Miguel, Anjos Rui, Almeida Afonso R M, Sousa Ana E
GIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, Portugal.
Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
Front Immunol. 2025 Mar 3;16:1553535. doi: 10.3389/fimmu.2025.1553535. eCollection 2025.
Thymus-committed regulatory T cells (Tregs) are essential for immune homeostasis. Recent findings stress their heterogeneity, suggesting possible alternate routes for thymic Treg development with unique features in humans, namely the clear evidence of Treg commitment at the double-positive (DP) stage and the presence of a significant population of CD8 single-positive (SP) FOXP3 Tregs. Here, we present a dedicated analysis strategy to a spectral flow cytometry-based study of thymus from children and aged adults (≥ 74-years-old), to further elucidate Treg development and heterogeneity in the human thymus. We applied an unsupervised analysis pipeline to data generated from 6 high-dimensional panels, taking advantage of a common backbone of 11 markers, and we were able to map thymocytes along T cell maturation stages. Generating UMAP and FlowSOM cluster coordinates from the backbone, we projected all other markers onto these, characterizing clusters with the information of all markers. Focusing this analysis on events inside a putative total Treg gate, we could portray rarer subsets of human thymic Tregs and investigate their trajectories using pseudotime analysis. We uncover clusters within human DP thymocytes uniquely expressing FOXP3 or CD25, a DP-branching trajectory towards a CD103CD8SP Tregs endpoint, and define trajectories towards CD4SP Tregs, including towards a cluster of CXCR3CD4SP Tregs, that may consist of thymic resident or recirculating Tregs, and do not expand in the elderly. Our flow cytometry approach separates Treg populations with likely distinct functions and facilitates the design of future studies to unravel the complexity of human regulatory T cells.
胸腺定向调节性T细胞（Tregs）对免疫稳态至关重要。最近的研究结果强调了它们的异质性，提示人类胸腺Treg发育可能存在具有独特特征的替代途径，即双阳性（DP）阶段Treg定向的明确证据以及大量CD8单阳性（SP）FOXP3 Tregs的存在。在此，我们提出了一种专门的分析策略，用于基于光谱流式细胞术对儿童和老年人（≥74岁）胸腺进行研究，以进一步阐明人类胸腺中Treg的发育和异质性。我们将无监督分析流程应用于从6个高维面板生成的数据，利用11个标志物的共同主干，能够将胸腺细胞沿着T细胞成熟阶段进行映射。从主干生成UMAP和FlowSOM聚类坐标后，我们将所有其他标志物投影到这些坐标上，利用所有标志物的信息对聚类进行表征。将此分析聚焦于假定的总Treg门内的事件，我们可以描绘人类胸腺Tregs中较罕见的亚群，并使用伪时间分析研究它们的轨迹。我们在人类DP胸腺细胞中发现了独特表达FOXP3或CD25的聚类、一条朝向CD103CD8SP Tregs终点的DP分支轨迹，并定义了朝向CD4SP Tregs的轨迹，包括朝向CXCR3CD4SP Tregs聚类的轨迹，该聚类可能由胸腺驻留或再循环Tregs组成，且在老年人中不扩增。我们的流式细胞术方法分离出可能具有不同功能的Treg群体，并有助于设计未来的研究以揭示人类调节性T细胞的复杂性。
