PD-1 levels and ERK1/2 activation during deletion and immunity. A total of 2 × 10⁶ CFSE-labeled OT-I cells were injected intravenously into either RIP-OVA^hi mice or OCS/LPS primed B6 mice (OCS/LPS). Sixty hours after transfer, the proliferating cells within the sacral and pancreatic lymph nodes (RIP-OVA^hi) or spleen (OCS/LPS) were stained and analyzed by flow cytometry. (A) PD-1 staining showing dot plots (left) and quantitated mean fluorescence intensity (MFI; right). MFI graph shows PD-1 MFI values minus background, which was determined independently for each cell division. Error bars represent SEM. Representative data are shown from 1 of 3 independent experiments, with 3 mice per group per experiment. (B) Levels of activated (ie, phosphorylated) ERK (ppERK1/2) on in vitro peptide restimulation of T cells during immunity and deletion. Cell suspensions were either left unstimulated or restimulated with OVA_257-264 peptide or PMA. Cells were then fixed, permeabilized, and stained intracellularly for ppERK1/2. Dot plots are shown for all conditions (left panels), and histograms showing ppERK1/2 levels on divided OT-I cells during both immunity (open histograms) and deletion (gray shaded histograms) are included (right panels). Representative data are shown from 1 of 2 independent experiments, with 3 mice per group per experiment.