Predominant occupation of the class I MHC molecule H-2Kwm7 with a single self-peptide suggests a mechanism for its diabetes-protective effect

Abstract

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic beta cells. In both humans and the non-obese diabetic (NOD) mouse model of T1D, class II MHC alleles are the primary determinant of disease susceptibility. However, class I MHC genes also influence risk. These findings are consistent with the requirement for both CD4(+) and CD8(+) T cells in the pathogenesis of T1D. Although a large body of work has permitted the identification of multiple mechanisms to explain the diabetes-protective effect of particular class II MHC alleles, studies examining the protective influence of class I alleles are lacking. Here, we explored this question by performing biochemical and structural analyses of the murine class I MHC molecule H-2K(wm7), which exerts a diabetes-protective effect in NOD mice. We have found that H-2K(wm7) molecules are predominantly occupied by the single self-peptide VNDIFERI, derived from the ubiquitous protein histone H2B. This unexpected finding suggests that the inability of H-2K(wm7) to support T1D development could be due, at least in part, to the failure of peptides from critical beta-cell antigens to adequately compete for binding and be presented to T cells. Predominant presentation of a single peptide would also be expected to influence T-cell selection, potentially leading to a reduced ability to select a diabetogenic CD8(+) T-cell repertoire. The report that one of the predominant peptides bound by T1D-protective HLA-A*31 is histone derived suggests the potential translation of our findings to human diabetes-protective class I MHC molecules.

Fig. 1.

Characterization of H-2K^wm7-associated peptides. (A) Mass spectrometric sequence determination of peptide VNDXFERX. From H-2K^wm7-eluted peptides, the doubly charged ion at 503.27 m/z was selected for CAD fragmentation, and a CAD mass spectrum was obtained (upper panel). Calculated masses for type b and type y ions are shown above and below the deduced sequence, respectively. The b ions originate from the N-terminus of the peptide and the y ions originate from the C-terminus. Ions observed in the spectrum are underlined. X represents I or L, which were later assigned based on a database match to peptide VNDIFERI derived from human histone H2B. The CAD mass spectrum of the corresponding synthetic peptide (lower panel) confirms the predicted sequence. (B) Synthetic versions of three of the H-2K^wm7-eluted peptides were tested for binding to H-2K^wm7 using citric acid-stripped C1R-A2.1/K^wm7 cells. Stripped cells were incubated in serum-free medium with human β2m and peptide at 4°C overnight. Surface H-2K^wm7 expression was monitored by flow cytometry using the H-2K^wm7-specific mAb HD25 and FITC-conjugated goat anti-mouse IgG + IgM. The irrelevant peptide used as a negative control was the H-2K^d-binding peptide NRP-V7 (KYNKANVFL) (36). (C) An aliquot of a synthetic peptide was added to the extracted H-2K^wm7-associated peptides and analyzed by MS so that a correlation between ion current signal and peptide quantity could be defined. Peptide abundances are plotted as copies per cell. The peptides are numbered in the order in which they appear in Table 1. Peptide 1 corresponds to VNDIFERI. (D) A sequence logo (http://www.cbs.dtu.dk/∼gorodkin/appl/plogo.html) (37) was generated using the sequences of the twelve 8mer H-2K^wm7-eluted peptides (Table 1). At each position of the peptide, the sequence logo arranges the amino acids in order of predominance from top to bottom (i.e. the amino acid found most frequently is at the top). The relative heights of the amino acids at a given position reflect their relative frequencies (i.e. a large letter indicates a high-frequency amino acid).

Fig. 2.

Evolutionary relatedness and sequence comparison of H-2K^wm7, H-2K^b and H-2K^d. (A) Phylogenetic tree based on the amino acid sequence alignment of the extracellular domains of the indicated murine class I MHC molecules. (B) Amino acid sequence alignment of the extracellular domains of H-2K^wm7, H-2K^b and H-2K^d. Red boxes indicate conserved residues. Red lettering in white boxes indicates residues having similar properties. Asterisks indicate residues contacting the peptide in H-2K^wm7–VNDIFERI, as calculated by COOT (32) using a 4 Å distance cutoff.

Fig. 3.

Crystallographic analysis of H-2K^wm7. (A) The three-dimensional structure of H-2K^wm7–VNDIFERI. The structure shows a typical MHC fold and a peptide-binding groove composed of the α1 and α2 domains. The peptide is depicted in blue. An IgC α3 domain and β2m (blue green) provide further structure. (B) Superimposition of peptides VNDIFERI (magenta) and IQQSIERL (green) from the crystal structures of H-2K^wm7–VNDIFERI and H-2K^wm7/IQQSIERL. The upper panel is a side view of the peptides in which the β sheet floor of the peptide-binding groove is perpendicular to the plane of the page and below the peptides. The lower panel is a top view in which the β sheet floor is parallel to the plane of the page and below the peptides. (C) Ribbon diagrams of the α1 and α2 domains of H-2K^wm7–VNDIFERI or (D) H-2K^wm7–VNDIFEAI. In (C) and (D), the upper panel illustrates a side view and the lower panel a top view. In (C), arrows indicate the side chain of the Arg present at P7 of the peptide.

Fig. 4.

HD25 and HD24 do not recognize H-2K^wm7 molecules presenting R→A P7 peptide variants. Citric acid-stripped C1R-A2.1/K^wm7 cells were incubated in serum-free medium with human β2m and peptide at 4°C overnight. Surface H-2K^wm7 expression was monitored by flow cytometry using the H-2K^wm7-specific mAbs HD25, HD24 or HD42 and FITC-conjugated goat anti-mouse IgG + IgM. The irrelevant peptide used as a negative control was the HLA-A2.1-binding peptide HCV Core 132–140 (DLMGYIPLV).

Fig. 5.

Both peptide-dependent and peptide-independent anti-H-2K^wm7 antibodies show robust binding to C1R-A2.1/K^wm7 cells. Surface H-2K^wm7 expression in C1R-A2.1/K^wm7 cells was monitored by flow cytometry using the mAbs HD25 (IgG_2b), HD24 (IgG_2a) or HD42 (IgM) and FITC-conjugated rat anti-mouse IgG_2b, IgG_2a or IgM, respectively. C1R-A2.1 cells were used as a negative control.