Fig. 1. Single-cell transcriptomic profiling of T_reg in treatment-naïve and neoadjuvant anti–PD-1–treated NSCLCs.

Coupled scRNA-seq/TCR-seq was performed on T cells isolated from resected tumor (n = 15), adjacent NL (n = 12), TDLN (n = 3), and a resected brain metastasis (n = 1) from patients with NSCLC treated with two doses of neoadjuvant anti–PD-1 as well as resected tumor (n = 10) and paired adjacent NL (n = 8) from treatment-naïve patients with NSCLC. (A) Two-dimensional (2D) UMAP projection of the expression profiles of the 73,882 T_reg that passed QC. T_reg subsets, defined by 10 unique clusters, are annotated and marked by color code. (B) Relative expression [average log₂(fold change)] for top differential genes for each cluster is visualized on a heatmap. Three thousand cells (or all cells in the cluster if cluster size <3000 cells) were randomly sampled from each cluster for visualization. Differential expression tests for T_reg cell subsets were performed with Wilcoxon rank sum test. Genes with >0.25 log₂ fold changes, at least 25% expressed in tested groups, and genes with Bonferroni-corrected P values < 0.05 were regarded as differentially expressed. (C) The expression of canonical T_reg subset marker genes and cell subset selective genes was visualized in red scale using UMAP projection. (D) PCA and canonical correlation of pseudobulk gene expression for individual tumor (yellow, n = 25) and adjacent NL (dark blue, n = 20) samples. Canonical correlation with tissue type = 0.56, P < 0.001. (E) PCA and canonical correlation of pseudobulk gene expression for individual nonresponder (red, n = 9) and responder (blue, n = 6) tumors. Canonical correlation with response status = 0.36, P = 0.50.