Sequential induction of functions with increasing antigen concentration. (A) Representative example of the assessment of individual functions for a KK10-specific CD8⁺ T-cell clone (G11C) upon activation with a gradient of peptide concentration. Cells are gated on CD8⁺ViViD⁻. (B) The MFI values for CD107a, IFN-γ, TNF-α, and MIP-1β are plotted for distinct KK10-specific CD8⁺ T-cell clones ordered according to their antigen sensitivity. Cells were stimulated with 10⁻⁶ M cognate peptide in the presence of antigen-presenting EBV-transformed HLA B*2705⁺ B cells; MFI values were determined after gating solely on cells that were positive for the relevant function, excluding responses less than 20% of the total clonal population in each case. (C) Representative peptide titration assays for the induction of individual functions in a KK10-specific CD8⁺ T-cell clone (G11C). (D) EC₅₀ values for each individual function obtained with different KK10-specific CD8⁺ T-cell clones are plotted to highlight the sequential induction of separate functions with antigen concentration. Horizontal bars indicate median values; ○ represent clones with lower levels of antigen sensitivity. *P < .05, **P < .01 calculated using the Wilcoxon signed rank test.