Identification of HIV-specific CD8⁺ T-cell clones with distinct antigen sensitivities. (A) Half-maximal effective concentrations (EC₅₀) of HLA B*2705-restricted KK10-specific CD8⁺ T-cell clones were determined in standard chromium release assays using peptide titrations and a common antigen-presenting HLA B*2705⁺ B-cell line. The tested CD8⁺ T-cell clones are classified according to increasing antigen sensitivity from bottom to top. TCRBV usage, CDR3 amino acid sequence, and TCRBJ usage are shown for each clone (ND, not done). Same colors indicate identical clonotypes. The clone reference is indicated on the y-axis (the code last letter corresponds to the patient from which the clone was obtained: A and F for patient 1, B and G for patient 2, and C and H for patient 3). (B) Representative flow cytometric data showing combined αCD8 mAb and KK10/HLA B*2705 tetramer staining of KK10-specific CD8⁺ T-cell clones with higher or lower levels of antigen sensitivity; staining of a HLA A*0201-restricted CMV pp65-specific clone is shown for comparison. (C) Representative intracellular stainings for perforin and granzyme B in the same CD8⁺ T-cell clones.