Fig. 1.

CD8 reorders the agonist hierarchy of peptide ligands that induce the expression of CD69. MEL5 TCR⁺ CD8⁺ J.RT3-T3.5 cells were activated for 6 h with C1R cells expressing comparable levels of HLA-A2 D227K/T228A (KO), wild-type HLA-A2 (WT), or HLA-A2 A245V/K^b (VK^b) pulsed with various concentrations of 3T (blue), ELA (black), or FAT (red). Surface expression of CD69 was measured via flow cytometry. (A) Functional sensitivity (pEC₅₀) for each peptide ligand in the context of each MHCI. Four replicate experiments are shown. The value for 3T in the context of HLA-A2 D227K/T228A was set to zero for graphical purposes and treated as missing data for statistical purposes. P < 0.0001 for the ligand effect and P < 0.0001 for the MHCI effect (two-way ANOVA with Tukey’s post hoc test). (B) The agonist potency of FAT relative to the agonist potency of ELA expressed as pEC₅₀^FAT − pEC₅₀^ELA, which is equivalent to the logarithm of the fold difference in functional sensitivity. Four replicate experiments are shown. Horizontal bars indicate median values. *P < 0.05 and **P < 0.01 (one-way ANOVA with Tukey’s post hoc test). (C–E) Representative peptide titration experiment used to calculate the parameters in A and B. Curves were fitted in Mathematica. All four replicate experiments are shown in SI Appendix, Figs. S1 and S2.