Program DHC-Virtual
20 - 21 January 2021
Abstracts Lymphoid session 4
A roadmap for human T cell development
21 January
11:54 12:06
K. Cante Barrett

Towards a roadmap for human T cell development using single-cell RNA-sequencing and multi-color spectral flow cytometry.

Martijn Cordes (1,2), Kirsten Canté-Barrett (1), Federico Morretti (1), Karin Pike-Overzet (1), Szymon Kiełbasa (3), Erik van den Akker (2), Marcel Reinders (2), Frank Staal (1)
(1) Leiden University Medical Center, Immunology, Leiden, (2) Leiden University Medical Center, Leiden Computational Biology Center, Leiden, (3) Leiden University Medical Center, Department of Biomedical Data Sciences, Leiden
No potential conflicts of interest

The development of T lymphocytes in the thymus has been extensively studied in the mouse via genetic models, transcriptome and flow cytometric analyses. For human T cell development our knowledge is less complete.

Current models indicate similarities but also some marked differences between mouse and human T cell development, especially in the earliest stages where T cell lineage commitment occurs with the help of important transcriptional regulators.


To resolve the transcriptional landscape of the initial stages of human thymopoiesis, we performed single cell RNA-Sequencing (scRNA-Seq) on 12000 thymocytes of 8 flow-sorted human thymocyte subpopulations obtained from thymi of 6 donors representing consecutive stages of early T cell development.

By combining transcriptomic data with γδ and αβ T cell receptor (TCR) enriched data, obtained through single-cell sequencing of various TCR genes from the same samples, we can combine expression profiles with rearrangements of the TCR from the same cell.

We complement our scRNA-seq data with multi-color flow cytometry to verify the presence of small populations.


We identified 19 different clusters within the earliest and highly heterogeneous CD34+ thymocyte population including a cluster of thymic Plasmacytoid Dendritic Cells (pDCs) based on the expression of known pDC transcription factors IRF8, SPIB and TCF4. Integration with publicly available bone marrow data sets indicate an overlap of differentiation paths of pDCs and other non-T cells within the thymus.

While the gene expression data showed a gradual increased expression of T cell commitment genes together with a decrease of multi-lineage genes, we could already detect cells expressing fully rearranged TCRβ and TCRγδ with our TCR enriched data. Strikingly, TCRγδ rearrangement continues into CD4/CD8 double-positive thymocytes that is also the path in which TCRαβ is rearranged.

Within a cluster of the earliest CD34+CD1a- progenitors, we could identify multiple subpopulations of putative thymus seeding progenitor cells (TSPs). These rare cells are not yet T-lineage primed and express stem cell related genes as well as migration markers such as CCR9 and L-selectin, suggesting that the human thymus is seeded by multiple different cell populations.





We have generated full gene expression profiles of 12000 cells from sorted human thymocytes together with TCR rearrangement data from the same cell.

Our data reveals a large heterogeneity within early CD34+ DN thymocytes, with novel TSP subsets. Full length V(D)J enriched data resulted in new insights with respect to the timing and duration of TCR rearrangements. While rare, our data indicates the presence and potential differentiation of small non-T cell populations such as pDCs in the thymus.

Collectively, these results stipulate a redefinition of the stages underlying human T cell development in the thymus.