Type 1 diabetes-associated HLA-DQ8 transdimer accommodates a unique peptide repertoire

Abstract

HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared with HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*05:01) and the β-chain of HLA-DQ8 (DQB1*03:02). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized its peptide binding repertoire, and defined a unique transdimer-specific peptide binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes.

FIGURE 1.

Expression of HLA-DQ2/8 transdimers on viral-transduced HEK293 cells. Expression of the DQ transdimer molecules on HEK293 cells was examined by FACS analysis using a phosphatidylethanolamine-conjugated pan-HLA-DQ antibody (clone 1a3, Leinco Technologies). Shown is HLA-DQ expression on the surface of transduced cells (dark gray) compared with non-transduced HEK293 cells (dashed line), which do not naturally express MHC class II molecules. An isotype control antibody for SPV-L3 was included in the FACS experiments (light gray). Cells were single cell-sorted to obtain clones with high DQ expression. Clones obtained by this strategy displayed >95% DQ expression, which was stable in time (not shown).

FIGURE 2.

Binding of a selected panel of HLA-DQ2trans and HLA-DQ8trans peptides representing the identified binding cores. Randomly selected panels of 8 DQ2trans- and 8 DQ8trans-eluted peptides containing the identified binding cores were synthesized as 13-mers containing two extra alanines, both N- and C-terminally necessary for proper DQ binding. Binding of a titration range (0–300 μm) of peptides was tested in a direct binding assay or in a competitive binding assay using a fixed concentration (0.6 μm) of biotinylated indicator peptide. A and B, shown is direct binding of a panel of 8 DQ2trans- and 8 DQ8trans- eluted peptides to DQ2trans (A) and DQ8trans (B). C and D, shown is competitive binding of the biotinylated DQ2trans indicator peptide AAEAALEAEEWAA (▼) and DQ8trans indicator peptide AAPHTTQPAVEAA (■) versus the same non-biotinylated peptide to DQ2trans (C) and DQ8trans (D). Data represent mean ± S.E. (n = 3).

FIGURE 3.

Binding of selected panels of HLA-DQ2trans and HLA-DQ8trans peptides in a competitive peptide binding assay. The two panels of 8 DQ2trans and DQ8trans peptides were tested in a titration range (0–300 μm) for binding in a competitive binding assay using a fixed concentration (0.6 μm) of the selected biotinylated indicator peptides AAEAALEAEEWAA (DQ2trans) and AAPHTTQPAVEAA (DQ8trans). A and B show competitive binding of the peptide panels to DQ2trans and DQ8trans, respectively. C and D show EC₅₀ values were calculated based on the observed competition between the biotinylated indicator peptides and the test peptides for DQ2trans and DQ8trans, respectively. EC₅₀ represents the concentration of the test peptide that is required for 50% inhibition of the binding of the indicator peptide. Data represent the mean ± S.E. (n = 3). Shown on the x axes is 1/EC₅₀, thereby illustrating that large bars represent better binding.

FIGURE 4.

Amino acid substitutions at anchor and non-anchor positions within the HLA-DQ2trans and HLA-DQ8trans indicator peptides. Binding of p1/p4/p6/p7/p9 (anchor) Lys-substituted DQ2trans indicator peptide AAEAALEAEEWAA and DQ8trans indicator peptide AAPHTTQPAVEAA to their respective transdimer is shown. Also, binding of p2/p3/p5/p8 Lys-substituted DQ2trans indicator peptide and DQ8trans indicator peptide was tested. Binding of A-substituted indicator peptides to DQ2trans and DQ8trans is shown. EC₅₀ values were calculated on the basis of competition between the biotinylated indicator peptide and the non-biotinylated Lys-substituted test peptides. EC₅₀ represents the concentration of the test peptide that is required for 50% inhibition of the binding of the indicator peptide. Data represent the mean ± S.E. (n = 3). Shown on the x axes is % relative binding of peptide without any substitutions (dashed line).

FIGURE 5.

Binding of predicted HLA-DQ8trans candidate epitopes with double mismatches to all four HLA-DQ2/8 molecules. Prediction of potential DQ8trans epitopes within the top known diabetogenic proteins was performed with double mismatches at the anchor positions of the DQ8trans binding motif using the algorithm program MOTIFS. A selection of 46 potential DQ8trans epitopes was tested for binding all four DQ2/8 cis and trans molecules. EC₅₀ values were calculated on the basis of competition between the biotinylated indicator peptide and the test peptides. Data represent the mean ± S.E. (n = 3). Shown on the x axes is 1/EC₅₀, thereby illustrating that large bars represent better binding. Red bars represent HLA-DQ8trans-specific peptides. PPI, preproinsulin; IGRP, glucose-6-phosphatase catalytic subunit-related protein.

FIGURE 6.

Proposed concept for the high risk association of HLA-DQ2/8 heterozygosity with development of T1D. HLA-DQ8 homozygous individuals only express a single HLA-DQ dimer on the surface of APCs, resulting in the presentation of a select number of epitopes and induction of a small repertoire of autoreactive CD4 T cells. Individuals heterozygous for the high risk-associated HLA-DQ2/8 haplotype can express multiple HLA-DQ dimers on APCs, consequently resulting in the presentation of a diversity of auto-antigen-derived epitopes and induction of a broader repertoire of autoreactive CD4 T cells. This may underlie the association of HLA-DQ2/8 with development of T1D.