The thymus is the critical organ for T cell development and T cell receptor (TCR) repertoire formation, which shapes the landscape of adaptive immunity. T cell development in the thymus is spatially coordinated, and this process is orchestrated by diverse cell types constituting the thymic microenvironment. Although the thymus has been extensively studied using diverse animal models, human immunity cannot be understood without a detailed atlas of the human thymus.
To provide a comprehensive atlas of thymic cells across human life, a team led by researchers at the Wellcome Sanger Institute performed single-cell RNA sequencing (scRNA-seq) using dissociated cells from human thymus during development, childhood, and adult life. They sampled 15 embryonic and fetal thymi spanning thymic developmental stages between 7 and 17 post-conception weeks, as well as nine postnatal thymi from pediatric and adult individuals. Diverse sorting schemes were applied to increase the coverage on underrepresented cell populations. Using the marker genes obtained from single-cell transcriptomes, the researchers spatially localized cell states by single-molecule fluorescence in situ hybridization (smFISH). To provide a systematic comparison between human and mouse, they also generated single-cell data on postnatal mouse thymi and combined this with preexisting mouse datasets. Finally, to investigate the bias in the recombination and selection of human TCR repertoires, the researchers enriched the TCR sequences for single-cell library generation.
Constructing the human thymus cell atlas
The researchers analyzed human thymic cells across development and postnatal life using scRNA-seq and spatial methods to delineate the diversity of thymic-derived T cells and the localization of cells constituting the thymus microenvironment. With T cell development trajectory reconstituted at single-cell resolution combined with TCR sequence, they investigated the bias in the VDJ recombination and selection of human TCR repertoires. Finally, they provide a systematic comparison between human and mouse thymic cell atlases.
The researchers identified more than 50 different cell states in the human thymus. Human thymus cell states dynamically change in abundance and gene expression profiles across development and during pediatric and adult life. They identified novel subpopulations of human thymic fibroblasts and epithelial cells and located them in situ. They computationally predicted the trajectory of human T cell development from early progenitors in the hematopoietic fetal liver into diverse mature T cell types. Using this trajectory, the researchers constructed a framework of putative transcription factors driving T cell fate determination. Among thymic unconventional T cells, they noted a distinct subset of CD8αα+ T cells, which is marked by GNG4 expression and located in the perimedullary region of the thymus. This subset expressed high levels of XCL1and colocalized with XCR1+ dendritic cells. Comparison of human and mouse thymic cells revealed divergent gene expression profiles of these unconventional T cell types. Finally, the researchers identified a strong bias in human VDJ usage shaped by recombination and multiple rounds of selection, including a TCRα V-J bias for CD8+ T cells.
This single-cell transcriptome profile of the thymus across the human lifetime and across species provides a high-resolution census of T cell development within the native tissue microenvironment. Systematic comparison between the human and mouse thymus highlights human-specific cell states and gene expression signatures. The detailed cellular network of the thymic niche for T cell development will aid the establishment of in vitro organoid culture models that faithfully recapitulate human in vivo thymic tissue.