Role of high-affinity HLA-DP specific CLIP-derived peptides in beryllium binding to the HLA-DPGlu69 berylliosis-associated molecules and presentation to beryllium-sensitized T cells

Abstract

Berylliosis is driven by the accumulation in the lung of beryllium-specific T helper type 1 (Th1) cells recognizing beryllium as antigen when presented principally by human leucocyte antigen DP molecules carrying a glutamate at position beta69 (HLA-DPGlu69). This study was designed to clarify the precise role of peptides in beryllium binding to the HLA-DP groove's pocket 4 and to identify peptides with higher affinity for pocket 4 that might prevent beryllium presentation and T-cell stimulation. Beryllium/HLA-DP interactions were analysed by the ability of beryllium to compete with CLIP and CLIP-derived peptides to HLA-DPGlu69 soluble molecule. The CLIP-derived low-affinity peptide CLIP-AA, could not outcompete beryllium; while the CLIP-derived high-affinity peptides CLIP-YY, CLIP-QY and CLIP-RF were only marginally influenced by the presence of beryllium in the competition assay. The effect of these CLIP-derived high-affinity peptides on beryllium presentation was determined by measuring interferon-gamma (IFN-gamma) release upon beryllium stimulation of peripheral blood mononuclear cells obtained from beryllium-hypersensitive subjects. CLIP-YY did inhibit beryllium presentation and T-cell activation, while CLIP-QY and CLIP-RF markedly enhanced the IFN-gamma response to beryllium. Anti-HLA-DP monoclonal antibody blocked the beryllium-induced IFN-gamma release in the presence of CLIP-QY (88%) and CLIP-RF (76%). A similar effect was observed for CLIP-YY capability to block IFN-gamma release by beryllium stimulation in the presence of CLIP-QY (79%) and CLIP-RF (76%). Overall, these data support the proposal that HLA-DP high-affinity peptides might be used as a model for specific berylliosis therapy.

Figure 1

Competition assay between biotinylated CLIP and CLIP-derived peptides (CLIP-AA, CLIP-YY, CLIP-QY and CLIP-RF) for the HLA-DPGlu69 soluble molecules. The IC₅₀ values are shown for each peptide as μm values on the abscissa of each panel. The ordinate is drawn through the point of equimolar competition with the biotinylated CLIP peptide (1 μm). The bars extending to the left of the vertical axis therefore represent CLIP mutated peptides that bind with higher affinity than biotinylated CLIP; bars extending to the right represent CLIP mutated peptides that bind with lower affinity than biotinylated CLIP. The IC₅₀ values obtained in the competition assays tests at pH 5·0 with an increased amount of CLIP-derived peptides (CLIP-AA, CLIP-YY, CLIP-QY, CLIP-RF) with respect to a fixed amount of biotinylated-CLIP peptide on HLA-DP2-soluble molecule are shown.

Figure 2

Analysis of the ability of CLIP derived high-affinity peptides to compete for soluble HLA-DPGlu69 with beryllium in vitro. Peptide and BeSO₄ competition curves generated in the presence of fixed amounts of biotinylated CLIP, CLIP-AA, CLIP-YY, CLIP-QY and CLIP-RF are shown (a–e). Data points represent the mean (± SD) of three separate experiments. In each graph, BeSO₄ competitions are represented using triangles and a continuous line, while CLIP (or CLIP-derived) peptide competitions are represented using circles and a dashed line.

Figure 3

Analysis of the ability of CLIP-derived peptides to interfere with T-cell stimulation by beryllium presented in the context of the HLA-DPGlu69 molecule using fresh blood mononuclear cells from individuals with berylliosis. The interference [inhibition or augmentation of beryllium-stimulated interferon-γ (IFN-γ)release] curves obtained with the CLIP, CLIP-AA, CLIP-YY, CLIP-QY and CLIP-RF peptides are shown. Each bar represents the mean (± SD) of triplicates generated from each of five study subjects, using the individual’s optimal BeSO₄ concentration.

Figure 4

Analysis of the HLA-DP restriction of beryllium-stimulated T-cell release of interferon-γ (IFN-γ) in the presence of the stimulation-augmenting peptides CLIP-QY and CLIP-RF. Cells were stimulated with CLIP-QY and CLIP-RF peptides at an optimal concentration of beryllium and peptide in the presence of monoclonal antibodies specifically reacting against HLA-DR, HLA-DP, HLA-DQ, HLA-class I and the Mycobacterium tuberculosis (Mtb) 19 000 molecular weight protein. Inhibition of IFN-γ release from beryllium-stimulated T cells in the absence of monoclonal antibody is shown.

Figure 5

Analysis of the ability of the CLIP-YY peptide to block the augmentation of beryllium-stimulated T-cell release of interferon-γ (IFN-γ) in the presence of the high-affinity peptides CLIP-QY and CLIP-RF. The graph shows the inhibition curves of IFN-γ release from T cells stimulated with BeSO₄ (10 μm), CLIP-QY and CLIP-RF (10 μm) in the presence of different concentrations of CLIP-YY peptide. Each bar represents the mean (± SD) of triplicates generated from each study subject at the individual’s optimal concentration of BeSO₄.

Figure 6

(a) Analysis of the HLA-DPGlu69 molecule and beryllium interaction in pocket 4 of HLA-DP2 molecule. The figure shown is the most stable HLA-DPGlu69/beryllium complex model among all the possible interaction models evaluated between HLA-DP groove electron donor groups and beryllium as ion. Specifically, the peptide-binding pocket 4 of HLA-DP2 is capable of co-ordinating beryllium by using its residues Glnβ13, Gluβ14, Argβ27 and Gluβ69. The HLA-DP α chain backbone is reported in grey coloured ribbon style, while the HLA-DP β-chain backbone is reported in light blue coloured ribbon. Amino acids are reported in stick style and coloured by atom (C: grey; O: red; N: blue; H: white). Beryllium is shown as van der Walls radius and coloured green. (b) Analysis of the HLA-DPGlu69 molecule and CLIP-RF interaction in pocket 4 of the HLA-DP2 molecule. The figure shows the Arg94 of the CLIP-RF peptide buried in pocket 4 of HLA-DP2. This amino acid does not form an H-bond with the Gluβ14 or Glnβ13 and together with Argβ27 and Gluβ69, which are interacting with H-bonds, are representing an electron donor environment that could determine beryllium co-ordination. The CLIP-RF backbone is shown in green. The HLA-DP surface is shown as semi-transparent and the electron donor amino acids in pocket 4 are shown in stick style coloured by atom (C: grey; O: red; N: blue; H: white). The H-bonds are shown in green. (c) Analysis of the HLA-DPGlu69 molecule and CLIP-YY interaction in pocket 4 of the HLA-DP2 molecule. The figure shows the Tyr94 of the CLIP-YY peptide deeply buried in pocket 4 of HLA-DP2 interacting by H-bond with the Gluβ14. In this way both these electron donor amino acids are unavailable for interacting with beryllium and the Glnβ13 is too far from Argβ27 and Gluβ69 to co-ordinate beryllium. Argβ27 and Gluβ69 are interacting with H-bond. The CLIP-YY backbone is shown in green. The HLA-DP surface is shown as semi-transparent and electron donor amino acids in pocket 4 are shown in stick style coloured by atom (C: grey; O: red; N: blue; H: white). H-bonds are shown in green.