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Related Experiment Video

Updated: Nov 11, 2025

Preparation and Applications of Organotypic Thymic Slice Cultures
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Development of double-positive thymocytes at single-cell resolution.

Young Li1, Kun Li1, Lianbang Zhu1

  • 1Department of oncology, The First Affiliated Hospital of USTC, Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medicine, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230021, Anhui, China.

Genome Medicine
|March 27, 2021
PubMed
Summary

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This study classifies double-positive (DP) thymocyte subtypes, revealing distinct proliferation patterns and immune synapse formation during T cell development. Findings advance understanding of thymopoiesis and T cell immunity.

Area of Science:

  • Immunology
  • Developmental Biology
  • Genetics

Background:

  • T cells are crucial for immunity, with thymopoiesis generating these cells in the thymus.
  • Single-cell studies have illuminated thymocyte development genetically and epigenetically.
  • The development of double-positive (DP) T cells, the most abundant thymocytes, remains less understood.

Purpose of the Study:

  • To investigate the developmental subtypes of double-positive (DP) thymocytes using single-cell resolution.
  • To characterize the proliferation dynamics and cell-cell interactions of DP thymocyte subtypes.
  • To identify conserved and species-specific regulatory mechanisms in thymopoiesis.

Main Methods:

  • Single-cell RNA sequencing of mouse thymocytes analyzed with Seurat for unsupervised clustering.
Keywords:
Cell cycleSingle-cell sequencingThymic selectionThymocyte

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Last Updated: Nov 11, 2025

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  • Flow cytometry validation of DP thymocyte subtypes using surface markers CD2 and Ly6d.
  • Analysis of cell cycle phases, immune synapse formation via immunofluorescence and ImageStream flow cytometry.
  • Integration of human thymocyte data and cross-species comparisons.

    • Main Results:

    • DP thymocytes classified into blast, rearrangement, and selection subtypes based on CD2 and Ly6d expression.
    • Blast DP cells exhibit unique proliferation patterns, exiting the cell cycle after one round.
    • CD8-associated immune synapses observed during DP selection, suggesting intrathymocyte interactions.
    • Cross-species comparison identified species-specific transcription factors influencing thymocyte transcriptional differences.

    Conclusions:

    • This study provides a novel classification of DP thymocyte subtypes at different developmental stages.
    • New insights into DP thymocyte development at single-cell resolution were generated.
    • The findings enhance fundamental knowledge of thymopoiesis and T cell development.