Fig. 4. Murine TR-T_reg gene signature defines an orthologous subset among human NSCLC TIL-T_reg that is enriched in anti–PD-1 R.

(A) Red scale overlay of the TR-T_reg gene score, with red indicating higher expression and blue indicating low expression. Red dotted line represents the UMAP region with highest expression. Refined clustering was performed on the T_H1-like/cytotoxic subset, and 2D UMAP projection of six unique subclusters (SC0-SC5) is visualized by UMAP and marked by color code. (B) Relative expression of the top 10 most differential genes for each subcluster is visualized on a heatmap. (C) The expression of biologically relevant genes from the TR-T_reg score is visualized in red scale. (D) Boxplots showing differences in expression of FoxP3 (P = 6.8 × 10⁻⁹), IL2RA (CD25; P = 1.9 × 10⁻¹), and TBX21 (Tbet, P = 0.00024) by TIL-T_reg in SC0 versus all other T_reg. Each dot represents an individual tumor sample per patient (n = 14). Comparisons were performed at the individual patient level using paired t test. Patient-averaged log₂-transformed and library size–normalized expression values are shown. (E) Cell density plots of the T_H1-like/cytotoxic T_reg subclusters stratified by response/treatment status. The TR-T_reg score^hi population is indicated with a dotted line. Increased density is represented by red scale, and decreased density is represented by green/blue. (F) Boxplots showing the frequency of SC0 in the tumors (left) and adjacent NLs (right) of NR (red) and responders (blue). Comparisons were performed at the individual patient level using Wilcoxon rank test. (G) Diffusion map with RNA velocity between SC0, Activated (1)/OX40^hiGITR^hi, and LN-homing clusters for NR (left) and R (right). (H) Cross-cluster sharing of T_reg TCR clonotypes detected in the SC0 subcluster with non-SC0 T_reg and T_conv clusters (as shown in fig. S1C). Red indicates a higher frequency of the clone in the relevant subcluster. T_cm, central memory T cells; T_em, effector memory T cells.