Figure 3

Impact of peptide presented by HLA-C*06:02 on KIR2DS1 and KIR2DL1 binding. (a) Quantification of HLA-C*06:02 stabilization of 721.221-TAP1KO-C*06:02 pulsed with 19 different synthetic peptides and 568 HIV-1 clade B peptides. HLA-C*06:02 surface levels were determined by flow cytometry using an anti-pan-HLA class I antibody (clone W6/32). Peptides were added at a saturating concentration of 200 μM. Relative fluorescence intensity (RFI) was calculated as the pan-HLA MdFI of the sample divided by the pan-HLA MdFI of 721.221-TAP1KO-C*06:02 cells stained in the absence of peptide. Binding peptides were determined as (Mean + S.D.)_{MFI sample} > (Mean + 2*S.D.)_{MFI LIY}. The dotted line represents the cut-off set to determine the peptides binding to HLA-C*06:02. The screening was performed one time and the selected peptides were tested in three independent experiments. (b) Bar graphs showing fold increase in CD69 expression for KIR2DS1ζ⁺ (black bar) and KIR2DL1ζ⁺ (grey bar) Jurkat cells when co-incubated with 721.221-TAP1KO-C*06:02 pulsed with different peptides (MdFI of the sample divided by the MdFI of KIR2DS1ζ+ or KIR2DL1ζ+ Jurkat cells co-incubated with 721.221-TAP1KO-C*06:02 in the absence of peptide). (c) Bar graphs showing fold increase in CD69 expression for KIR2DS1ζ⁺ (black bar) and KIR2DL1ζ⁺ (grey bar) Jurkat cells when co-incubated with 721.221-C*06:02 pulsed with different peptides (MdFI of the sample divided by the MdFI of KIR2DS1ζ⁺ or KIR2DL1ζ⁺ Jurkat cells alone). (d) HLA-C blocking antibody (6A4) abrogated CD69 activation of the KIR2DS1ζ⁺ and KIR2DL1ζ⁺ Jurkat cells after pre-incubation with the indicated target cells and subsequent incubation with the KIR2DS1ζ⁺ or KIR2DL1ζ⁺ Jurkat cells. The experiments were repeated three times independently and a representative example is shown with the mean of the CD69 FI. (e) HLA-C*06:02- SRGPVHHLL -PE tetramer staining of Jurkat- β2mKO, KIR2DS1ζ⁺ or KIR2DL1ζ⁺ Jurkat cells. Results are shown as fold increase in PE MdFI (MdFI of the reporter cell line stained with the tetramer divided by the MdFI of the reporter cell line alone). For each bar graphs, the results are shown as median of three independent experiments +/− interquartile range.