ThymoSpheres culture: A model to study human polyclonal unconventional T cells

Area of Science:

• Immunology
• Developmental Biology
• Cell Biology

Background:

• In vitro T-cell development studies are vital for understanding human T-cell differentiation.
• Current methods require thymic epithelial cells, limiting scalability and manipulation.

Purpose of the Study:

• To introduce a novel in vitro cultivation system, ThymoSpheres (TS), for T-cell development.
• To characterize the T cells generated by TS cultures and their potential lineage.

Main Methods:

• Development of ThymoSpheres (TS) using DLL4-expressing stromal cells and human hematopoietic precursor cells.
• Culture of TS at the air-liquid interphase.
• Characterization of mature T cells using single-cell RNA and T-cell receptor (TCR) sequencing.

Main Results:

• TS cultures efficiently generate large numbers of mature T cells, primarily CD8α T cells.
• Generated T cells exhibit transcriptional and TCR CDR3 features of human polyclonal αβ unconventional T cells (UTCs).
• UTCs express hallmark agonist selection genes (e.g., IKZF2/Helios) and NK receptors, with a polyclonal TCR repertoire enriched for autoreactive features.

Conclusions:

• ThymoSpheres (TS) provide a scalable and manipulable platform for studying human T-cell development.
• TS cultures are suitable for investigating the development and selection mechanisms of the human polyclonal unconventional T cell (UTC) lineage.