Fig. 2

Gene expression dynamics along the pseudotime of T cell exhaustion. a Single-cell trajectories across three distinct states of CD8⁺ T cells derived from human melanoma (GSE72056). The cells were classified into different T cell types using Monocle 2 based on the following criteria: effector (CD62L⁻, CD127⁻, PDCD1⁻), exhausted (PDCD1⁺), memory (either CD62L⁺ or CD127⁺), ambiguous (classified into multiple cell types), and unknown (classified into none of the cell types). The ambiguous cells and unknown cells were not visualized in the t-stochastic neighbor embedding (tSNE) plot. Based on the enriched cell type, the cells were classified into three states (of CD8⁺ T cell): effector, exhausted, and memory states (P < 2.2e−16 for exhausted state and memory state, P = 7.07e−07 for effector state by the binomial test). b Distribution of CD8⁺ T cells from each patient of the dataset across three branches of single-cell trajectories. c, d The expression dynamics of immune checkpoint (IC) genes and TOX along the pseudotime of CD8⁺ T cells in two alternative trajectories from the effector state to the memory state or to the exhausted state were summarized using BEAM analysis (c) and scatter plots with regression curves (right column for the trajectory toward exhausted state and left column for the trajectory toward memory state) (d). The significance of upregulated expression in the exhausted T cells (or memory T cells) relative to the effector T cells was tested by one-tailed Mann-Whitney U test