Ageing compromises mouse thymus function and remodels epithelial cell differentiation
- MRC Human Genetics Unit, University of Edinburgh, United Kingdom
- Wellcome Sanger Institute, Wellcome Genome Campus, United Kingdom
- Cancer Research United Kingdom - Cambridge Institute, Li Ka Shing Centre, University of Cambridge, United Kingdom
- Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom
- Department of Paediatrics, University of Oxford, Cancer Research, United Kingdom
- Department of Biomedicine, University of Basel, and University Children’s Hospital, Switzerland
- Chan Zuckerberg Biohub, United States
- EMBL-EBI, Wellcome Genome Campus, United Kingdom
- Department of Biosystems Science and Engineering, ETH Zurich, Switzerland
Figures
Figure 1 with 3 supplements
The decline of thymic cellularity and immune function with age.
(a) Age-dependent changes in thymic architecture, as shown by representative H and E staining of thymic sections. Scale bars represent 150 µm. Medullary islands stain as light purple while cortical …
Figure 1—figure supplement 1
Alterations in TEC composition throughout ageing.
(a) Thymocyte cellularity fluctuations over age. (b) Ratio of %TEC:%thymocytes over age. (c) TEC cellularity over age for mTEC and cTEC FAC-sorted populations. Error bars represent mean +/- standard …
Figure 1—figure supplement 2
Changes in T-cell populations throughout ageing.
(a) FACS gating strategy to separate different T-cell subtypes. (b) Maturation trajectory and negative selection checkpoints for TCR-positive thymocytes. (c) Frequency of different thymocyte …
Figure 1—figure supplement 3
T cell receptor repertoire simulations.
(a) A schematic of T cell receptor rearrangements used to design simulations. (b) Proportions of productive TCRs (y-axis) simulated at different sample sizes (x-axis). (c–f) Proportions of TCR alpha …
Figure 2 with 7 supplements
Thymic stromal composition remodelling during ageing.
(a) A schematic showing the experimental design and FACS phenotypes of sorted cells for single-cell RNA-sequencing. Right panel shows cell composition fluctuations as a relative fraction of all …
Figure 2—figure supplement 1
Experimental investigation of the ageing thymus.
(a) FACS gating strategy for TEC isolation. Identical gating strategies were used for all time points. (b) Filtering strategy to identify high-quality TEC libraries. (c–f) Fractions of libraries …
Figure 2—figure supplement 2
Comparison of Bornstein et al., 2018, Park et al., 2020 single-cell transcriptomes to the TEC subtypes defined in this study.
This figure depicts the assignment of single TEC from the Bornstein, the Park or our Ageing dataset to our nine TEC subtypes (left side) or the Bornstein/Park mTECI-IV nomenclature.
Figure 2—figure supplement 3
Relationship between classical FAC sorted subpopulations and transcriptionally defined single-cell subtypes.
(a) Observed percentages (%) of TEC based on pre-scoring into FAC sorted subpopulations. (b) Estimated contributions of each FACS sort type to each single cell subtype through age. Each coloured dot …
Figure 2—figure supplement 4
Expression of selected marker genes overlaid on SPRING plot.
Each subplot is a SPRING-layout of the shared nearest-neighbour graph of single TEC, derived from scRNA-seq transcriptional profiles as in Figure 2. Graph nodes are coloured according to the …
Figure 2—figure supplement 5
MSigDB pathways enriched for expression of marker genes for each single-cell subtype.
The X-axis shows the fraction of marker genes that overlap the specified pathway, the size of the dot represents the number of marker genes in the enriched pathway, and the colour of the dot …
Figure 2—figure supplement 6
Reactome pathways enriched for expression of marker genes for each single cell subtype.
The X-axis shows the fraction of marker genes that overlap the specified pathway, the size of the dot represents the number of marker genes in the enriched pathway, and the colour of the dot …
Figure 2—figure supplement 7
Consensus clustering of ageing single-cell TEC libraries.
The heatmap shows the fraction of times that the libraries are co-clustered based on a variety of transformations, clustering methods and the number of features (Materials and methods). The heatmap …
Figure 3 with 1 supplement
Thymic stromal remodelling during ageing.
(a) Enrichment of MSigDB biological pathways with age in mature cTEC, intertypical TEC and mature mTEC, annotated as in (Figure 2b). Bars denote normalised enrichment score (NES) for significant …
Figure 3—figure supplement 1
Differential expression of genes throughout ageing.
Each panel shows a heatmap and a MA-plot depicting the expression of the differentially expressed genes within the (a) mature cTEC, (b) intertypical TEC, or (c) mature mTEC subtypes. The heatmaps …
Figure 4
Tissue-restricted gene expression in mTEC.
(a) TRA expression enriched in Mature mTEC and Post-Aire mTEC. Shown is the percentage of expressed genes that are classed as TRAs (see Materials and methods), for each TEC subtype across mouse …
Figure 5 with 2 supplements
Intertypical TEC and medullary TEC are derived from a β−5t+ progenitor.
(a) A schematic representing the transgenic Dox-inducible ZsGreen (ZsG) lineage tracing of β−5t-expressing mTEC precursors (top), and lineage tracing experiment in 1-week-old thymi (bottom). The …
Figure 5—figure supplement 1
Details of the ZsGreen labelling and sorting strategy.
(a) Representative FACS plots depicting the labelling efficiency of cTEC and mTEC of 1-week-old mice, 48 hr after treatment with 0.3 mg of doxycycline per mouse. (b) FACS gating strategy employed …
Figure 5—figure supplement 2
Random Forest training using 1-week-old single TEC.
(a) The top genes that discriminate between TEC subtypes trained using single TEC from 1-week-old mice. Shown is the mean decrease in accuracy when dropping each gene in the random forest (left) and …
Figure 6 with 6 supplements
Lineage tracing and scRNA-seq reveal the dynamics of TEC precursor - progeny relationship across ages.
(a) The mean percentage of cTEC (left) and mTEC (right) labelled either 2 days (blue points) or 28 days (orange points) after doxycycline treatment of 3xtgβ5t mice. Error bars are the mean +/- the …
Figure 6—figure supplement 1
Representative FACS plots depicting the gating strategy employed to determine the labelling efficiency of cTEC and mTEC of 1-week-old, 4-week-old and 16-week-old mice, 2 or 28 days after treatment with 0.004 mg (newborns) or 2 mg (adult mice) of doxycycline per mouse.
(a) cTEC/mTEC distribution as traditionally defined by flow cytometry using the surface markers Ly51 and reactivity to UEA1. (b) Proportion of ZsGreen-labelled total TEC detected at each time-point, …
Figure 6—figure supplement 2
Summary of multiplexed single-cell droplet RNA sequencing.
(a) FAC-Sorting gating strategy for the isolation of ZsGreen +/- TEC subpopulations. (b) Multiplet detection using multiplexed hashtag oligos (HTO). Coloured bars denote the number of cells in each …
Figure 6—figure supplement 3
Quality control of multiplexed single-cell droplet RNA sequencing.
(a) Boxplots showing the distributions of the estimated deconvolution size factors (left) and number of detected genes for each single-cell cluster (right). (b) Uniform manifold approximation and …
Figure 6—figure supplement 4
Droplet single-cell RNA-sequencing cluster annotation and comparison with TEC subtypes.
(a) A mapping of single-cell clusters onto TEC subtype phenotypes. (b) A confusion matrix showing the proportions of single-cells in each TEC cluster (columns) and their classification into Ageing …
Figure 6—figure supplement 5
Marker gene expression profiles across TEC clusters from β−5t lineage-traced single cells.
Boxplots showing the distribution of marker gene expression (y-axis) for TEC subtypes across TEC clusters (x-axis). Boxes are coloured by the inferred TEC subtype to which they belong.
Figure 6—figure supplement 6
UMAP visualisation of TEC sub-clusters across all single-cells from lineage-traced thymi.
Each panel is coloured according to the TEC subtype annotation and corresponds to Figure 6c. Arrows point to the positions of the relevant clusters in the UMAP.
Figure 7 with 3 supplements
Ageing restricts the differentiation of intertypical TEC into mature mTEC.
(a) UMAP as in (Figure 6c) coloured by diffusion pseudotime (DPT) distance in the medullary lineage (see Figure 7—figure supplement 1 for details). (b) Changes in ZsGreen+ labelled TEC …
Figure 7—figure supplement 1
Pseudotime trajectory inference across medullary lineages.
(a–b) Diffusion maps of single mTEC and Intertypical TEC, showing two medulla branches on DC1 vs DC2 (a) and DC2 vs DC3 (b). (c) A UMAP subset to mTEC and Intertypical TEC coloured by DPT on both …
Figure 7—figure supplement 2
Pseudotime trajectory inference in the cortical lineage.
(a–b) Diffusion maps of single cTEC and Intertypical TEC showing diffusion components (DC) (a) 1 vs. 2 and (b) 2 vs. 3. (c) A rug plot showing the positions of TEC sub-clusters (y-axis) along the …
Figure 7—figure supplement 3
Percentage of cells in each subtype with expression of key TEC genes.
Boxplot showing the proportion of TEC in each subtype cluster which express key genes linked to thymic involution and TEC identity: (a) Psmb11 (β5-t), (b) Ly6a (Sca1), (c) Bmp4, (d) Inhba (Activin …
Author response image 1
Author response image 2
Co-expression of Psmb11 and Ccl21a in single TEC.
Expression in single TEC of (a) Psmb11 , (b) Ccl21a , (c) both (Psmb11+/Ccl21a+; blue), and (d) expression levels of Psmb11 vs. Ccl21a, with TEC subtype indicated by colour. TEC with no detected …
Author response image 3
Additional files
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Supplementary file 1
Tables listing relevant experiment related information.
(Table 1) Numbers of TEC isolated in the single-cell experiment. (Table 2) Marker genes for each subtype of TEC. (Table 3) Tests conducted for marker proteins in TEC. (Table 4) Single-cell defined TEC subtypes and known concordant phenotypes. (Table 5) Details of antibodies used in flow cytometry staining panels to identify TEC and thymocytes undergoing negative selection. (Table 6) ADT primers for Droplet sequencing. (Table 7) HTO primers for Droplet sequencing. (Table 8) Tissue-specific gene classification via FANTOM5 Cage-Seq:Tissue samples were grouped into 27 broad groups based on the annotation data.
- https://cdn.elifesciences.org/articles/56221/elife-56221-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56221/elife-56221-transrepform-v2.docx
