Figure 5: Single-Cell RNA-Sequencing Suggests Low-Dose Gemcitabine Depletes TI-Tregs.

Exhibiting high TI-Treg Master-Regulator Activity: (A) Schematic of experimental workflow. (B) UMAP plot and unsupervised clustering by VIPER-inferred protein activity of Tregs from untreated and gemcitabine-treated tumor and spleen. (C) Heatmap of cell-by-cell protein activity for each Tumor-Treg MR identified by scRNA-seq, grouped by cluster. (D) Distribution of the 17-gene TI-Treg MR signature normalized enrichment score by Gene Set Enrichment Analysis (GSEA), grouped by cluster, such that cluster TRC₃ is most enriched for the TI-Treg MR signature. (E) Distribution of IKZF2 (Helios) Normalized Gene Expression, grouped by cluster, such that the cluster TRC₃ has highest expression. (F) Barplot of cluster frequencies in each sample, such that cluster TRC₃ has a baseline frequency of 7.8% in spleen of vehicle-control sample and 30.1% in tumor (p = 1.78e-84), with frequency of only 14.9% in tumor of gemcitabine-treated sample (p = 1.51e-20). (G) Cox proportional hazard ratios of cluster TRC₃ frequencies in vehicle vs gemcitabine treated mice in tumor (OR = 0.407 [95% CI: 0.334-0.494]) and spleen (p = 0.242, OR = 1.063 [95% CI: 0.958-1.17]).

See also Figure S6.