RBC autoreactive RTE tolerization involves checkpoint molecules and Tregs. CD4⁺ T cells from 10- to 12-week-old HODxOTII F1 mice were sorted for RNAseq. (A) Heat map representing differences of transcript expression in CD4⁺ T cells from autoreactive HOD⁺OTII⁺ mice (blue) and littermate control HOD^NEGOTII⁺ mice (green; n = 4 per genotype). (B) Dot plot showing pathway enrichment from differential gene expression analysis. Dot size represents the number of genes in each pathway; red to blue scale of P values indicates red < purple < blue. (C) Plot representing upregulated transcripts in CD4⁺ T cells from autoreactive HOD⁺OTII⁺, compared with littermate control HOD^NEGOTII⁺ mice. Transcripts are grouped in colors: those associated with anergy and/or exhaustion (red), immunosuppressive cytokines (green), Tregs (blue), and purinergic signaling (black). Data are combined from 4 mice of each genotype. (D) Representative histograms of checkpoint molecules PD1, Lag-3, and CTLA-4 expression on CD4⁺ T cells from HOD⁺OTII.Rag2p-GFP (red) and littermate control HOD^NEGOTII.Rag2p-GFP (blue) mice. CD4⁺ T cells from HOD⁺OTII.Rag2p-GFP and HOD^NEGOTII.Rag2p-GFP mice were sorted based on GFP expression, an indirect measure of T-cell age: GFP^HI (ie, RTEs) on the left, GFP^INT in the middle, and GFP^LO (ie, mature naïve T cells) on the right. (E-F) Expression of CD73 and CD39 on (E) total CD4⁺Vα2⁺Vβ5⁺ OTII T cells and on (F) CD4⁺FoxP3⁺CD25⁺Vα2⁺Vβ5⁺ Tregs from HOD⁺OTII.Rag2p-GFP (top row) and HOD^NEGOTII.Rag2p-GFP (bottom row). Data in panels D-E are representative of 3 mice per group and 3 independent experiments.