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Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
Published on: February 27, 2019
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A backbone-based flow cytometry approach to decipher regulatory T cell trajectories in the human thymus
Beatriz Moleirinho1,2, Margarida Paulo-Pedro1,2, Nicole C Martins1,2
1GIMM- Gulbenkian Institute for Molecular Medicine, Lisbon, Portugal.
Frontiers in Immunology
|March 18, 2025
Summary
Regulatory T cells (Tregs) are crucial for immune balance. This study reveals novel human thymic Treg developmental pathways and distinct subsets using advanced flow cytometry, offering insights into immune homeostasis.
Area of Science:
- Immunology
- Cell Biology
Background:
- Regulatory T cells (Tregs) are vital for maintaining immune homeostasis.
- Human thymic Treg development is heterogeneous, with unique features like double-positive (DP) stage commitment and CD8 single-positive (SP) FOXP3+ Tregs.
- Understanding these developmental routes is key to immune regulation.
Purpose of the Study:
- To elucidate Treg development and heterogeneity in the human thymus.
- To identify and characterize distinct human thymic Treg subsets and their developmental trajectories.
- To analyze Treg populations in both children and elderly adults.
Main Methods:
- Spectral flow cytometry analysis of human thymus from children and aged adults.
- Application of an unsupervised analysis pipeline with 6 high-dimensional panels and 11 common markers.
- Utilized UMAP, FlowSOM, and pseudotime analysis to map thymocytes and characterize Treg subsets.
Main Results:
- Identified unique clusters within DP thymocytes expressing FOXP3 or CD25.
- Revealed a DP-branching trajectory towards CD103+CD8SP Tregs.
- Defined trajectories towards CD4SP Tregs, including CXCR3+CD4SP Tregs, which do not expand in the elderly.
Conclusions:
- Advanced flow cytometry successfully separated human thymic Treg populations with potentially distinct functions.
- Uncovered novel Treg developmental pathways and subsets in the human thymus.
- Provides a foundation for future studies on the complexity of human regulatory T cells and immune aging.
