Impact of I133T mutation on the structures of pHLA and responding T cell subsets. (A) Top; Side view of structures of the Nef126-10(8I10F) (colored purple) and Nef126-10(8T10F) (colored green) is shown. Superimposition is based on the HLA-A*24:02 peptide binding domain. Water molecules are represented in blue and yellow spheres for the Nef126-10(8I10F) and Nef126-10(8T10F) epitopes, respectively. The number of peptide residues is labeled in black through P1 to P10. The HLA-A*24:02 is represented as a dark grey (Nef126-10(8T10F) and light grey cartoon. (B) The presence of the responses to each peptide was examined by ex vivo ELISpot assay. A pair of sticks, Nef126-10(8I10F) (purple) and Nef126-10(8T10F) (green), represents the average SFU of duplicate wells to each peptide in one patient. Four individuals on the left side harbored 133I/135F mutant and 10 individuals on the right side harbored 133T/135F mutant. (C) Functional avidity curve was drawn for 9 subjects who harbored Nef133T/135F sequence. From the curve, SD50 was calculated and projected in this chart. Nef126-10(8I10F)-specific CTL responses (purple) showed significantly higher functional avidity compared to Nef126-10(8T10F) (green). Each point represents functional avidity for specific peptide in one individual. The significance between two groups was calculated by Mann–Whitney U-test; *, p < 0.05.