Figure 2. Naive cells diverge into effector and memory states early after activation.
(A) Naive CD8+ T cells were activated with αCD3 and αCD28 and 0.05 ng/mL IL-12, as well as IL-2, IL-7, and IL-15. After 1, 2, and 4 days, cells were treated with 4sU for 2 h to label new transcripts, then harvested for time-resolved transcriptomics using sci-fate.
(B) Heatmap showing the absolute Pearson’s correlation coefficient between the activities of pairs of TFs. Key TFs in each module are labeled at right. T cell differentiation module used for subsequent analysis is boxed.
(C) UMAP visualization of cells based on the activity of T cell differentiation-related TF module, using newly synthesized mRNA, colored by cluster ID (top). Percentage of cells in each T cell differentiation state cluster after indicated days (bottom).
(D) Aggregated expression (scaled, log10 normalized) of top 400 differentially expressed (DE) genes between clusters (adj. p < 3 × 10−45 for all genes except for Ifng, adj. p = 7.3 × 10−29).
(E) DE genes between E1 (early) and MP (early) at day 2 only; log2FC > 0.5 and adj. p < 0.05.
(F) UMAP visualization as in (C), characterized by labeling-based RNA velocity analysis. Streamlines indicate the integration paths that connect local projections from the observed state to the extrapolated future state.25
(G) Pseudotemporal ordering of top 200 DE genes and additional genes of interest (adj. p < 1.4 × 10−17) between trajectories. Gene labels correspond to all DE TFs in the T cell differentiation TF module (left text) and DE target genes linked to Tbx21, Egr1, Eomes, and Tcf7 (right text).
(H and I) (H) RNA velocity magnitude and (I) loess smoothed TF activity over pseudotime for four of the most DE genes between trajectories. TF activity is calculated as the normalized aggregation of newly synthesized mRNA for all TF target genes, scaled across all cells. Cells in the undifferentiated (U) cluster are set to pseudotime = 0 for each trajectory.