HLA Gene Editing of the Type I Diabetes Susceptible DQ8 Allele Inhibits Hybrid Insulin Peptide Binding and T Cell Activation 2310163

Abstract

Abstract Introduction The risk for developing T1D is strongly associated with HLA-DQ8 in humans and I-Ag7 in NOD mice, both of which have an alanine at position 57 and an asparagine in position 82. In the NOD mouse model, replacing alanine (A) with aspartic acid (D) renders the mice resistant to diabetes, suggesting that the edit should block binding of autoantigens. Here, we compared the T1D-specific gene edit (A57D) with a universal approach replacing the invariant asparagine (N) with leucine (L) at position 82 (N82L). Asparagine forms hydrogen bonds with the peptide backbone near the N-terminus of a peptide and appears to be critical for all high-affinity peptide binding. Methods DQ8, DQ8(A57D) and DQ8(N82L) edits were cloned individually into the K562 cell line. HIP-11, a T1D pathogenic peptide, was synthesized with a biotinylated polyethylene-3 linker on the N-terminus. K562 transfectants were cultured with biotinylated peptide overnight. Peptide binding was detected with streptavidin-PE and analyzed by flow cytometry. The T cell transductant (GSE.8E3), which was generated by cloning the HIP-11-specific T cell receptor from a T1D patient, was co-cultured with HLA-transfected K562 cells plus HIP-11 and T cell activation measured using an NFAT reporter. Results Replacing alanine at position 57 with aspartic acid in DQ8 reduced HIP-11 peptide binding by 37%. In contrast, replacing asparagine at position 82 with leucine reduced HIP-11 peptide binding by 87%. However, both edits completely blocked T cell activation of HIP-11-specific T cells. Conclusion The global (N82L) gene edit in DQ8 reduced binding of HIP-11 more effectively than the A57D edit. However, both gene edits blocked the activation of a HIP-11 pathogenic T1D T cell line. These findings support the potential development of HLA gene editing to treat T1D. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)