Diabetes and other autoimmune endocrine diseases
The major genetic determinant in susceptibility or protection from many autoimmune diseases, including type 1 diabetes (T1D), resides in the human leukocyte antigen (HLA) region. Particular class II alleles (e.g., HLA-DQ8) increase the risk for developing T1D, whereas others (e.g., HLA-DQ6) lead to dominant protection from T1D. HLA class II genes encode major histocompatibility (MHC) molecules that present peptides to T cells. We hypothesized that the protective DQ6 (DQB*0602) allele presents insulin peptides to regulatory CD4 T cells (Tregs), resulting in downstream anti-inflammatory responses. We expanded insulin-specific Tregs from peripheral blood mononuclear cells of DQ6+ non-diabetic individuals (n=6) using an insulin B chain mimotope, known to be a strong T cell agonist in murine models and human T1D. After 7 days in culture, there were increased CD4+CD25+Foxp3+ cells compared to no antigen (5.9% vs. 4.0%, p=0.03). In separate assays, insulin-expanded Tregs were isolated as CD4+CD25hiCD127loCTVlo cells for T-cell receptor sequencing, performed separately by single-cell TCR sequencing and paired single-cell TCR/RNA-seq. There was good concordance between the two methodologies. Of the 122 expanded Tregs with paired αβTCR sequences from one individual, there were 61 TCRs (50%) that were present ≥2 times, indicating a skewed repertoire. The transcriptomes of the expanded Tregs were consistent with memory cells expressing Foxp3. Using a cytokine ELISPOT assay, insulin-proliferated Tregs secreted IL-10 upon repeat antigen stimulation. A TCR hybridoma was generated and confirmed response to native insulin peptides presented by DQ6. Our findings provide a mechanistic basis for understanding HLA-linked protection from autoimmune disease development.