Cells were isolated from the spleen and lymph node. CD8+ OT1 T cells were purified by negative selection using biotinylated antibodies and magnetic beads as previously described (68). Splenocytes were used as APCs and were isolated from T cell–deficient mice (Cα−/− or Zap70−/−). Before culture, red blood cells removed using ACK lysis. Splenocytes were incubated with peptide antigens for 1 hour at 37°C. Small-molecule inhibitors (3-IB-PP1 or PP2) or DMSO control were then added followed immediately by OT1 T cells at a ratio of 5:1 (APC:T cell). Cells were cultured overnight at 37°C for a total of 16 hours and placed on ice before staining for CD69 and other surface markers (CD19/B220, CD8α, TCRβ, or Vα2) using an antibody dilution of 1:200 in FACS buffer and Fc blocking antibody (2.4G2) at 1:1000. Up-regulation of CD69 or Nur77-GFP was assessed by flow cytometry using a BD Fortessa and quantified using FlowJo software. The OVA peptide (SIINFEKL), its variants (Q4R7, T4, Q4H7, and G4), and the VSV peptide were synthesized by GenScript.

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