Fig. 2.

Tregs and hypoxia induce surfaceomic changes representative of immunosuppression.A, CD8⁺ T cells were stimulated with anti-CD3/anti-CD28 beads either in the absence or presence of Tregs. Following culture, CD8⁺ cells were isolated for cell surface proteomics. Volcano plot shows compiled results from N = 4 donors. Proteins with a −/+1.5-fold change and p < 0.05 were included in downstream analysis. Proteins significantly down- (blue) or upregulated (red) are indicated. B, Log₂(Enrichment Ratio) of indicated proteins. Each dot represents data from an individual donor. Line represents mean and error bars are standard deviation. C, CD8⁺ T cells were stimulated with anti-CD3/anti-CD28 beads in either normoxic (20% O₂) or hypoxic (1% O₂) conditions for 3 days. Volcano plot shows compiled results from N = 3 donors. Proteins with a −/+1.5-fold change and p < 0.05 were included in downstream analysis. Proteins significantly down- (blue) or upregulated (red) are indicated. D, Log₂(Enrichment Ratio) of indicated proteins. Each dot represents data from an individual donor. Line represents mean and error bars are standard deviation. E, Top 20 enriched gene sets identified by GSEA of all proteins identified in the three datasets. Negative (downregulation) and positive (upregulation) normalized effect sizes are shown in blue and red, respectively. SILAC peptide ratio was used to prerank proteins, then GSEA was performed with the MySigDB C5 GO gene set collection. F, several proteins responsible for L-amino acid transport (GO:0015807) are upregulated in activated CD8⁺ T cells, but downregulated by both Tregs and hypoxia. Treg, regulatory T cell.