TgRhoA CD4 populations have transcriptional and biochemical evidence of mTORc1 pathway activation. (A) Principal component analysis of biological replicates of WT and tgRhoA CD4⁺ naive and activated populations compared with WT, Tet2-deleted (Tet2) and tgRhoA; Tet2^fl/fl; Vav-Cre T_FH-cell populations 6 days after immunization with NP40-Ova/Alum. (B) Unsupervised hierarchical clustering of the same populations based on top 1000 genes. (C) Top 10 signatures enriched in tgRhoA vs WT naive or (D) tgRhoA; Tet2^fl/fl; Vav-Cre⁺ vs Tet2^fl/fl; Vav-Cre⁺ T_FH cells, using Hallmark GSEA gene sets. Gene sets are ranked by normalized enrichment score values. (E) Representative flow cytometric intracellular staining of phospho-S6 (pS6), phospho-4EBP1 (p4EBP1), and phospho-Akt (pAkt) in splenic WT or tgRhoA naive CD4 T cells after CD3 and CD28 cross-linking for the indicated durations. All histograms display frequency of events as percentage of cells within the population indicated.