Combined deep and single cell sequencing of autoantigen-specific CD4+ T cell receptor beta clonotypes shows unexpected publicity and disease-related differentiation

BACKGROUND: Analysis of autoantigen-specific CD4+ T cells is key for the understanding of T1D pathogenesis. To meet the dual challenge of understanding the frequency of such cells in different cellular compartments and their antigen experience, we developed an approach that combines T cell receptor (TCR) high throughput (HTS) sequencing and single-cell (SC) clonotyping of autoantigen-specific cells isolated after short-term in vitro activation.

METHODS: Peripheral blood of 6 newly diagnosed T1D patients and 6 healthy donors (HDs) was divided and (i) stained for true naïve (TN), central memory (CM), regulatory T cells (Treg) and stem-cell like memory (Tscm) CD4+ cells subset sorting for TCRB repertoire profiling by HTS; and (ii) stimulated for 18 hours in vitro with GAD65 (or CMV), followed by index sorting of activated (CD154+CD69+). TCRB chains were identified by SC-PCR.

RESULTS: We obtained 360 productive unique TCRB GAD-specific clonotypes, 22.5% of which were found by HTS in TN, CM, Treg and Tscm cells and were mainly private. In T1D patients, GAD-specific TCRB clonotypes were found preferentially in the CM pool, while in HD these clonotypes were also found in Tregs, suggesting an ongoing regulatory response. Interestingly when we performed the same analysis at the amino acid level the proportion of trackable cells was 50%; inter-subject sharing (publicity) was also higher, suggesting autoantigen-driven TCRB convergence. Over half (54%) of the GAD clonotypes, both in T1D and HD, tracked back into the TN pool, indicating that inter-subject GAD clonotype sharing has a thymic origin. In summary, a combined HTS and SC approach reveals a potentially public repertoire that becomes expanded in different cell compartments during disease.