Stimulation of CD8⁺T cells with agonist MHCp dimer but not with partial agonist MHCp dimer exposes the APA1/1 epitope. (A) Induction of CD69 expression by agonist and partial agonist H-2K^b/Ig dimers. Spleen cells from OT-IxRAG2^-/- mice were stimulated with 500 ng/mL of H-2K^b/Ig dimer unloaded (H-2K^b; gray shaded curve) or loaded with the agonist OVAp peptide (SIINFEKL) or with the partial agonist peptide E1 (EIINFEKL). Expression of CD69 in gated CD8⁺ T cells was analyzed 24 hours later. (B) Time course of APA1/1 epitope exposure. Spleen cells from OT-I mice were stimulated at room temperature for the times indicated with 500 ng/mL of the H-2K^b/Ig dimer loaded with the indicated peptides. APA1/1 immunofluorescence (expressed as MFI) was recorded on the H-2K^b/Ig dimer⁺ cells. (C) Binding of OVAp- and E1-loaded H-2K^b/Ig dimer to OT-I CD8⁺ T cells. Spleen cells from OT-I mice were incubated for 5 minutes at room temperature with 500 ng/mL of the H-2K^b/Ig dimers loaded with the indicated peptides and triple stained for Thy1, the H-2K^b/Ig dimer, and APA1/1. The Thy1⁺ population was analyzed for peptide H-2K^b/Ig binding (left). The brightest population for OVAp-H-2K^b/Ig binding (22%) and E1-H-2K^b/Ig binding (28%) was gated as indicated and reanalyzed for APA1/1 expression (right).