J.OT1 activation assays were performed similarly to primary CD8+ OT1 T cell cocultures. J.OT1 cells (5 × 104) were combined with T2-Kb cells (APCs) that had been incubated with peptide antigen for 1 hour at a ratio of 3:1 (APC:J.OT1). Cells were cultured for 16 hours and then placed on ice and stained for CD69 (1:200 in FACS buffer). CD69 up-regulation was assessed by flow cytometry using a BD Fortessa and quantified using FlowJo software.

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