(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.