Figure 5.

Proliferating CD4⁺ T Cells Contain Regulatory and Cytotoxic Cell States

(A) Heatmap showing expression of select cytotoxic, regulatory, and proliferating marker genes (rows) for individual single cells (columns) within the CD4_PROLIF cluster. Samples were hierarchically clustered. Log₂-transformed expression of each gene was row scaled.

(B) Representative flow cytometry staining from a bladder tumor showing expression of CD25, GZMB, GZMK, and Ki67.

(C) Single cells expressing the top 3 most expanded clonotypes found in the CD4_PROLIF T cell population are shown in red in the same UMAP space as in Figure 2A. The regions composed of proliferating, regulatory, and cytotoxic T cells are outlined and superimposed on the UMAP projection for visualization.

(D) Left panel: pseudotime trajectories derived from all tumors (N = 7 samples) and non-malignant samples (N = 6 samples). Cells with expanded TCRs from the proliferating (CD4_PROLIF, green), regulatory (CD4_IL2RAHI and CD4_IL2RALO, shades of red), and cytotoxic (CD4_GZMB and CD4_GZMK, shades of purple) states were used for this analysis. Specific branches corresponding to proliferating cytotoxic cells (top right), non-proliferating cytotoxic cells (bottom right), proliferating regulatory cells (top left), and non-proliferating regulatory cells (bottom left) are labeled. Right panel: branches are color-coded according to the above proliferating or non-proliferating identities. Also labeled are branch points that discriminate proliferating and non-proliferating cytotoxic CD4⁺ T cells (branch point 1) and proliferating and non-proliferating regulatory T cells (branch point 2).

(E) Heatmap showing all differentially expressed genes (columns) between branches for branch point 1 across cells in the pseudotime analysis (rows). Cells are grouped by their proliferating or non-proliferating branch assignments, color-coded at the right of the heatmap and corresponding to colors in (D). Genes are grouped by color-coded clusters (1–8) shown at the top of the plot, which result from hierarchical clustering based on co-regulation in specific branches.

(F) Cytotoxic CD4⁺ T cell gene signature scores were plotted in clinical responders (complete response or partial response) versus non-responders (stable disease or progressive disease) from baseline metastatic biopsies from bladder cancer patients with inflamed tumors on the IMvigor210 clinical trial (N = 62 tumors). The signature score was obtained from the IMvigor210 bulk RNA-seq dataset for the cytotoxic CD4⁺ T cell-specific genes derived from non-proliferating (cluster 4) and proliferating (cluster 7) cytotoxic CD4⁺ clusters from the pseudotime analysis shown below the heatmap in (E). Median ± SEM is shown; ^∗p = 0.037 by two-tailed t test.