Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2K(d) that stimulates CD8 T cells in insulin-dependent diabetes mellitus - PubMed (original) (raw)

Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2K(d) that stimulates CD8 T cells in insulin-dependent diabetes mellitus

F Susan Wong et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The recognition of MHC-peptide complexes by T cells is governed by structural considerations that are determined by the sequences of the individual components and their interaction with each other. We have studied the function of a highly diabetogenic CD8 T cell clone that is specific for insulin B15-23:H-2K(d). We have then related this to modeled MHC-peptide structures. The native peptide binds poorly to H-2K(d), because of the small glycine residue at peptide position p9 that is incapable of productive interactions with the hydrophobic residues of pocket F. In addition, electrostatic repulsions between the peptide glutamate residue at position 7 and 152D of the MHC molecule heavy chain contribute to the poor binding. However, B chain peptide 15-23 bound to K(d) shows excellent T cell stimulation and the induction of CD8 cytotoxic T cells. Peptide substitution has also shown that p6G is likely to be a T cell antigen receptor interaction site. Our studies have shown that the predictions seen in the models correlate closely with the observed effects in functional assays and provide insight into how this peptide, which would not be predicted to stimulate these cells on H-2K(d) binding studies alone, could activate such highly pathogenic T cells.

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Figures

Figure 1

Figure 1

H-2Kd binding of peptide is less sensitive than cytotoxicity or induction of IFN-γ secretion. (A) H-2Kd binding assay. Stabilization of H-2Kd by increasing concentrations of peptide is shown by the mean channel fluorescence of binding with anti-H-2Kd Ab SF1-1.1 on RMAS-Kd cells. The figure shows the mean staining by using anti-H-2Kd mAb in the presence of different concentrations of the insulin B chain peptide 15-23. The figure shows very poor binding as staining increases very little over background levels in the absence of peptide shown by the horizontal line. The scale has been chosen because it covers the range of the scale used for peptides that bind well. (B) Proliferation and IFN-γ production by the CD8 T cell clone G9C8 with increasing concentrations of peptide B15-23. The background level of proliferation on this occasion was 1,927 cpm and for IFN-γ production was 0.1 units/ml. (C) 51Cr release cytotoxicity assay showing lysis of P815 targets by the CD8 T cell clone G9C8, with increasing concentrations of peptide B15-23 (closed circles) and peptide B15-24(open circles). (D) 51Cr release cytotoxicity assay using peptides substituted at each position by alanine. Nomenclature used indicates the amino acid in the wild-type B15-23 peptide followed by the position of the amino acid, followed by the substituted amino acid, in this case, alanine (A). Wild-type peptide (closed circles), C5A (open squares), E7A (open triangles), and G9A (open circles) stimulate cytotoxicity. Substitutions L1A (*), Y2A (open diamond), L3A (©), V4A (X), G6A (+), and R8A (closed squares) abolish cytotoxicity.

Figure 2

Figure 2

The modeled structure of H-2Kd binding the insulin B chain peptide 15-23. (A) View of the modeled structure of the α1α2 region of the H-2Kd molecule in complex with the murine insulin B15-23 peptide, as seen from above (TCR view). Several ways of depiction are shown simultaneously to appreciate how the peptide fits into the groove. The antigenic peptide is in space-filling form with its carbon atoms shown in green, nitrogen in blue, oxygen in red, hydrogen in white, and sulfur in orange. The α1α2 domain of the molecule is depicted according to its secondary structure in different regions: α-helix in red, β-pleated sheet in turquoise, and random coil in gray. The solvent-accessible surface of the α1α2 domain is shown in gray with colorings according to the electrostatic surface potential (blue for positive, red for negative, and intermediate hues for neutral). Depressions in the surface within the antigen-binding groove (not shown) are potential pockets. The surface of the heavy chain is made transparent so that peptide residue p2Y, which is buried in pocket B (see also Fig. 4), as well as the heavy chain residues making contact with the insulin peptide can be seen, albeit in a lighter color. These residues are shown with their carbon atoms in orange. (B) View of pocket B (position p2) containing the insulin B16Y residue and surrounding heavy chain amino acids, in the complex of H-2Kd and insulin B15-23, as seen from above (TCR view). The p2Y residue, nearly completely buried in this pocket, is in space-filling form, whereas the residues forming the p2 pocket are in stick form with van der Waals surface representation. Unlike HLA-A2, pocket B in H-2Kd can accommodate an aromatic residue because of F9V and V67A mutations. In both cases, the smaller residues are found in H-2Kd, hence the specific difference in the p2 motif. The several aromatic residues from the heavy chain are favorably situated to interact with p2Y/F. Color, surface electrostatic, and transparency conventions are as in_A_.

Figure 3

Figure 3

Binding to H-2Kd as determined by surface stabilization of H-2Kd in RMAS-Kd cells, compared with51Cr release cytotoxicity assays. (A) H-2Kd binding assay for native peptide and mutants at position 9. Stabilization of H-2Kd by increasing concentrations of peptide is shown by the mean channel fluorescence of binding with anti-Kd Ab SF1-1.1. The wild-type peptide containing glycine at position 9 (G9G), shown as closed circles, binds very poorly to the MHC, and increasing concentrations of peptide do not increase the staining with the Ab. A similar result is found with the substitution using tyrosine (G9Y), shown as open triangles. By contrast, substitution of the glycine by valine (G9V), shown as open circles, improves the binding to the H-2Kd molecule. (B) 51Cr release cytotoxicity assay for same mutants as in A, showing that the mutants that bind better to the H-2Kd molecule also stimulate improved cytotoxicity by the G9C8 T cell clone, the best binding found with G9V (open circles) also stimulating cytotoxicity at a lower concentration of peptide. However, even the poorly binding wild-type peptide (closed circles) or the tyrosine-substituted peptide, G9Y (open triangles), will still stimulate cytotoxicity at levels as low as 0.01 μg/ml.

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