Fig. 1

Schematic illustration of approach. a An array of overlapping peptides covering the entire candidate autoantigen HCRT is tested for HLA-DQ6 binding. b DQ6-HCRT_peptide tetramers are synthesized based on the DQ6-binding cores determined in a. c Ex vivo CD4⁺ T cells are isolated from PBMCs of DQ6⁺ patients and controls using negative selection by magnetic-activated cell sorting (MACS); purified CD4⁺ T cells are co-stained with candidate tetramers and antibodies distinguishing cell types (i.e., anti-CD4, anti-CD19). DQ6-HCRT_peptide tetramer⁺/CD4⁺ cells are sorted by single cell index sorting (iFACS). Given the discordance between tetramer positivity and true autoreactivity, the iFACS-sorted cells likely include tetramer⁺ clones with various tetramer-binding ranks, clone size, and disease-relevance: T1N-relevant (darker clones expressing public/related TCRs or unique/private TCRs) or irrelevant (lighter clones); expansion is indicated using doublets. Clones expressing TCR risk gene alleles may also be observed in DQ6⁺ controls, as DQ6-restricted selection may occur similarly in patients and controls and T1N development is thought to rely largely on antigen-driven clonal expansion. d Deep sequencing of TCR and phenotypic transcripts in sorted single cells allows further assessment of T1N-associated gene signatures of tetramer⁺ clones including both in vivo clonal expansion and expression of the TCR risk gene. e–h The DQ6-restricted TCR sequences are validated for ability to generate an expressed α/β TCR that binds relevant tetramers and signals after stimulation with relevant peptide epitopes. The first author created this figure