Fig. 2: Pseudotime inference captures continuous maturation trajectory and clarifies intermediate thymocyte stages.

a, UMAP plot of the totalVI latent space from positively selected thymocytes with cells colored by Slingshot pseudotime and smoothed curves representing the CD4⁺ and CD8⁺ T cell lineages. b, Heatmap of RNA (top) and protein (bottom) markers of thymocyte development over pseudotime in the CD4⁺ and CD8⁺ T cell lineages. Features are colored by totalVI denoised expression, scaled per row, and sorted by peak expression in the CD4⁺ T cell lineage. Pseudotime axis is the same as in a. c, Expression of features in the CD4⁺ and CD8⁺ T cell lineages that vary over pseudotime. Features are totalVI denoised expression values scaled per feature and smoothed by loess curves. d, Heatmap of all RNA differentially expressed over pseudotime in any lineage. Features are scaled and ordered as in b. Labeled genes are highly differentially expressed over time (Methods). e, In silico flow cytometry plots of log(totalVI denoised expression) of CD8a and CD4 from positively selected thymocytes (left) and the same cells separated by lineage (right). Cells are colored by pseudotime. Gates were determined based on contours of cell density. WT, wild-type. f, In silico flow cytometry plot of data as in e separated by lineage and pseudotime. g, UMAP plot of the totalVI latent space from positively selected thymocytes with cells colored by gate. Cells were computationally grouped into eight gates using CD4, CD8a, CD69, CD127(IL-7Ra), and TCRβ. h, Histograms of cells separated by lineage and gate with cells colored by gate as in g. i, Stacked histograms of gated populations in CD4-fated (top) and CD8-fated (bottom) thymocytes, with thresholds classifying gated populations over pseudotime (Methods). j, Schematic timeline aligns pseudotime with gated populations, with population timing determined as in i.