Fig. 1.

Surface proteomics reveals both well-established and novel activation-induced changes in surface protein levels.A, top, schematic depicting expansion and SILAC labeling workflow. Primary human CD8⁺ T cells were isolated and expanded using anti-CD3/anti-CD28 stimulation in media supplemented with IL-2 and either heavy or light arginine and lysine. After expansion, cells were stimulated in varying conditions before surface protein enrichment and protein identification with LC-MS/MS. A, bottom, strategy for assessing the effect of immunosuppressive stimuli on the activated CD8⁺ T-cell surfaceome. First, the surfaceomic changes associated with CD8⁺ activation in monoculture under normoxic conditions were analyzed. These changes then served as a baseline for later experiments examining the surfaceomic consequences of activating CD8⁺ T cells in co-culture with primary Tregs or in hypoxic culture. B, Volcano plot of surface protein changes following stimulation of CD8⁺ T cells with anti-CD3/anti-CD28 beads. Data represent 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. C, Log₂(Enrichment Ratio) of indicated proteins. Each dot represents data from an individual donor. Line represents the mean and error bars are standard deviation. SILAC, stable isotopic labeling with amino acids in cell culture.