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. 2020 Oct 5;205(10):2861–2872. doi: 10.4049/jimmunol.2000810

FIGURE 1.

FIGURE 1.

Peptide binding to HLA-E*01:01, HLA-E*01:03, Mamu-E, and Qa-1b quantified with an UV-mediated peptide exchange assay and a competition-based HPLC assay. (A) Schematic representation of the UV-mediated peptide exchange assay to measure peptide binding to HLA-E*01:01 and HLA-E*01:03 by capturing rescued complexes with plates coated with a specific HLA-E Ab. (B) Schematic representation of a modified UV-mediated peptide exchange assay to measure peptide binding to Mamu-E and Qa-1b in which the capture of previously biotinylated complexes is done with streptavidin-coated plates. In both (A and B), rescued complexes are detected with an HRP-coupled β2m Ab. (C–E) Previously described MHC-E binding peptides in humans: pCMV (VLAPRTLLL, red bar) and VL9, canonical HLA-E binders; p34-p68, M. tuberculosis–derived peptides previously predicted to bind HLA-E (7); in mice, Qdm-FL9, Qa-1b binders (12); and in NHP, Gag120-Gag69, SIV-derived peptides known to bind Mamu-E (15). The sequences of these peptides are available in Supplemental Table I. Peptides are ranked according to their capacity to bind HLA-E*01:01 to facilitate comparison between alleles. (C) Absorbance represents the number of MHC-E complexes that are rescued in the presence of test peptide, the value being higher for peptides with better binding capacity. Bars show the average and SD of a representative experiment with two biological repeats and two technical replicates. (D) The s/p ratios were calculated by normalizing values to positive control (pCMV) after subtraction of background obtained in the absence of test peptide (no rescue): (value − no rescue)/(pCMV − no rescue). Bars represent calculations from one representative experiment containing two biological repeats and two technical replicates. (E) IC50 were obtained by HPLC size exclusion chromatography using recombinant MHC-E and competition of a fluorescently labeled peptide. Binding was calculated as the concentration (micromolar) of peptide required to reduce fluorescence intensity of the standard peptide by 50% (IC50), and 100 μM was the highest concentration tested. Bars represent one representative experiment.