Single-Cell Transcriptomics Reveals Discrete Steps in Regulatory T Cell Development in the Human Thymus

Area of Science:

• Immunology
• Cell Biology
• Developmental Biology

Background:

• CD4+CD25+FOXP3+ regulatory T (Treg) cells are crucial for maintaining immunological tolerance.
• Treg cells develop either in the thymus (tTreg) or periphery, with tTregs preferred for adoptive cell therapy (ACT) due to lineage fidelity.
• The developmental pathways of human tTreg cells remain incompletely understood.

Purpose of the Study:

• To delineate the developmental stages of human thymus-derived Treg cells.
• To identify surface markers for distinguishing these developmental stages and enabling viable cell isolation.
• To inform the selection of stable tTreg cells for ACT and guide future mechanistic studies.

Main Methods:

• Single-cell transcriptomics and flow cytometry were employed to analyze human thymic Treg cell development.
• Cells were characterized based on gene expression and surface marker profiles.
• Developmental stages were defined by the sequential expression of specific transcription factors and surface markers.

Main Results:

• Three major Treg developmental stages in the human thymus were identified: pre-Treg I, pre-Treg II, and mature recent thymic emigrant (RTE) Treg cells.
• Pre-Treg I cells show signs of TCR signaling and express lineage-inappropriate genes.
• Pre-Treg II cells are characterized by FOXO1, KLF2, CCR7, CD31, and GPA33 expression, preparing for thymic exit. Mature RTE Treg cells express CD45RA.
• Mature effector Treg cells were found in the thymus, distinct from RTEs, with unclear origins and potential implications for ACT.

Conclusions:

• Human tTreg development proceeds through distinct stages, identifiable by specific molecular and surface markers.
• Surface markers CD1a, CD27, CCR7, and CD39 can distinguish major developmental stages for viable isolation.
• Understanding these stages is critical for selecting optimal tTreg cells for ACT and for further research into tTreg biology.