Validation of Treg clusters in separate scRNA-seq cohorts and prognostic significance by imputation of Treg subset frequencies in bulk RNA-seq cohorts. (A) Heat map of top 30 DEGs across the 3 Tregs subsets; actTregs, restTregs, and LAG3⁺ Tregs were plotted after normalization using scTransform. (B) Unique gene signature matrices were developed for CD4⁺ T-cell clusters including actTregs, restTregs, and LAG3⁺ Tregs, using the computational framework of CIBERSORTx. The matrices were then used to reannotate cell clusters in our scRNA-seq data sets and are shown as histograms of the frequency of Treg subsets in healthy donor tonsils (n = 3), FL (n = 3), and DLBCL (n = 3) (left); and in external scRNA-seq data sets (right), healthy donor tonsils (n = 8), FL (n = 7), and DLBCL (n = 6). (C) Pearson correlation of Treg subsets distribution in the external scRNA-seq data sets with the distribution in the scRNA-seq data generated in this study. (D) Abundance of the Treg subsets (left) and Kaplan-Meier survival curve for progression-free survival of patients with FL in the bulk RNA-seq cohort, stratified above the 85th percentile for the actTreg population (right).³⁹ (E) Abundance of the Treg subsets (left) and Kaplan-Meier survival curve for failure-free survival of patients with FL in the external bulk RNA-seq cohort (right), in which patients were stratified using the same actTreg abundance threshold as in the discovery cohort (shown in panel D).⁴⁰ ∗∗∗P < .001.