Polymorphisms within HLA gene loci are strongly associated with susceptibility to autoimmune disorders; however, it is not clear how genetic variations in these loci confer a disease risk. Here, we devised a cell-surface MHC expression assay to detect allelic differences in the intrinsic stability of HLA-DQ proteins. We found extreme variation in cell-surface MHC density among HLA-DQ alleles, indicating a dynamic allelic hierarchy in the intrinsic stability of HLA-DQ proteins. Using the case-control data for type 1 diabetes (T1D) for the Swedish and Japanese populations, we determined that T1D risk–associated HLA-DQ haplotypes, which also increase risk for autoimmune endocrinopathies and other autoimmune disorders, encode unstable proteins, whereas the T1D–protective haplotypes encode the most stable HLA-DQ proteins. Among the amino acid variants of HLA-DQ, alterations in 47α, the residue that is located on the outside of the peptide-binding groove and acts as a key stability regulator, showed strong association with T1D. Evolutionary analysis suggested that 47α variants have been the target of positive diversifying selection. Our study demonstrates a steep allelic hierarchy in the intrinsic stability of HLA-DQ that is associated with T1D risk and protection, suggesting that HLA instability mediates the development of autoimmune disorders.
Authors
Hiroko Miyadera, Jun Ohashi, Åke Lernmark, Toshio Kitamura, Katsushi Tokunaga
(A) Structure of MHC II (DQ0602 [pdb: 1uvq]) (70). MHC II is a heterodimeric transmembrane glycoprotein that is composed of one α and one β subunit. The α1 and β1 domains constitute the peptide-binding groove, and the α2 and β2 domains form the constant domain. In the case of HLA-DQ, HLA-DQA1 and -DQB1 encode the α and β subunits, respectively. (B) Outline of the ΔMHC assay. The ΔMHC assay measures cell-surface MHC expression levels normalized to the internal control GFP. The HLA-DQB1–stable cells (i) were transduced with the retroviral vector pMXs-IG/DQA1 (52) (ii). HLA-DQ was expressed on the cell surface in the presence of both the HLA-DQA1 and -DQB1. Cell-surface HLA-DQ expression and cytosolic GFP expression were measured by flow cytometry (iii). (C) Representative data from the ΔMHC assay for DQ0602 (left panels) and DQ2.5 (right panels). To quantify cell-surface MHC expression, the HLA-DQB1–stable cell line was transduced with a graded concentration of retrovirus containing pMXs-IG/DQA1. Expression levels of both HLA-DQ and GFP increased with the concentration of the retrovirus. Numbers indicate the MFI for GFP-negative and -positive cells and for the isotype control and anti–HLA II β (WR18). Dashed lines indicate the highest MFI in each sample set. GFP (green), anti–HLA II β (WR18) (magenta), and isotype control (black). (D) The increase in cell-surface MHC expression relative to GFP (ΔMHC) was calculated by plotting the MFI (GFP) and MFI (MHC) (Supplemental Figure 3A). The ΔMHC for each HLA-DQ allelic pair was normalized to the ΔMHC of DQ0602, which was measured on the same day. The ΔMHC for DQ2.5 and its normalized value are shown. See also Supplemental Figures 3–5 and Methods.