Phenotype-Based Isolation of Antigen-Specific CD4⁺ T Cells in Autoimmunity: A Study of Celiac Disease

The pathogenic immune response in celiac disease (CeD) is orchestrated by phenotypically distinct CD4⁺ T cells that recognize gluten epitopes in the context of disease-associated HLA-DQ allotypes. Cells with the same distinct phenotype, but with elusive specificities, are increased across multiple autoimmune conditions. Here, whether sorting of T cells based on their distinct phenotype (Tphe cells) yields gluten-reactive cells in CeD is tested. The method's efficiency is benchmarked by parallel isolation of gluten-reactive T cells (Ttet cells), using HLA-DQ:gluten peptide tetramers. From gut biopsies of 12 untreated HLA-DQ2.5⁺ CeD patients, Ttet⁺ /Tphe⁺ , Ttet^- /Tphe⁺ , and Ttet^- /Tphe^- cells are sorted for single-cell T-cell receptor (TCR)-sequencing (n = 8) and T-cell clone (TCC)-generation (n = 5). The generated TCCs are TCR sequenced and tested for their reactivity against deamidated gluten. Gluten-reactivity is observed in 91.2% of Ttet⁺ /Tphe⁺ TCCs, 65.3% of Ttet^- /Tphe⁺ TCCs and 0% of Ttet^- /Tphe^- TCCs. TCR sequencing reveals clonal expansion and sequence sharing across patients, features reflecting antigen-driven responses. The feasibility to isolate antigen-specific CD4⁺ T cells by the sole use of phenotypic markers in CeD outlines a potential avenue for characterizing disease-driving CD4⁺ T cells in autoimmune conditions.