Analyzing Genes for Autoimmune Thyroiditis and Diabetes: Translation to Therapy

PROJECT SUMMARY Autoimmune thyroid diseases (AITD) frequently develop together with type 1 diabetes (T1D), a phenotype considered a variant of the Autoimmune Polyglandular Syndrome type 3 (APS3v). The overall goal of our work is to map and sequence the joint susceptibility genes for AITD and T1D, to dissect the mechanisms by which they cause disease, and to use this knowledge to design new therapies. In the last grant period, we made substantial progress toward this goal: (1) we completed targeted next generation sequencing of the APS3v linked chromosomal loci and for the first time we mapped rare variants associated with APS3v risk. We identified a functional variant within the BTN3A1 gene that altered the activation of Vγ9Vδ2 T cells, uncovering a previously unknown role of these T cells in AITD and T1D autoimmunity; (2) we identified 3 pathogenic thyroid and islets peptides that bind to the APS3v-specific HLA-DR3 pocket we discovered and activate T cells. We used these peptides to generate a mouse model for APS3v, APS3v NOD-DR3 mice; (3) we identified several small molecule inhibitors that block pathogenic peptide binding to the APS3v HLA-DR3 pocket. Among these, Cepharanthine, showed strong inhibition of autoreactive T cells in our APS3v mouse model and in peripheric blood mononuclear cells (PBMC) from AITD patients; (4) we have identified 2 D-amino acid peptides that blocked the APS3v HLA- DR3 in our APS3v mouse model. Building on these findings we propose to: (1) Dissect the mechanisms by which the BTN3A1 variant we identified triggers APS3v; (2) Test the hypothesis that extracellular vesicles (EVs) carrying unique miRNAs modulate APS3v susceptibility genes to trigger disease; and (3) To translate our discovery of the major APS3v antigens into novel immune therapies. Our hypothesis is that complex interactions between APS3v gene variants and environmental triggers lead to activation of shared pathways that facilitate both thyroid and islet autoimmunity. In Aim 1 we will investigate the mechanism by which BTN3A1 variant triggers APS3v and the role of Vγ9Vδ2 T cells in AITD and T1D shared autoimmunity using in silico conformational analyses, BTN3A1-specific cell lines, as well as our novel APSv3 mouse model and PBMCs from APS3v patients. In Aim 2 we will test the hypothesis that EVs secreted by pancreatic β cells during inflammation regulate FOXP3 and CTLA-4 expression, triggering APS3v. We will use ex vivo cellular models as well as NOD- Foxp3-GFP and APS3v mice to investigate the mechanisms by which β cells inflammatory EVs regulate T cell expression of FOXP3 and CTLA-4 to trigger autoimmunity. In Aim 3 we will develop a novel therapeutic platform, termed ImmunoSTAT, to specifically block activation of T-cells targeting the major APS3v autoantigens we identified. Our multidisciplinary translational project builds on the knowledge gained in the previous grant period. Our goals are to explore the function and regulation of the APS3v genes we identified and to pursue pre-clinical development of novel APS3v ICT therapies based on selective blocking of the T cells. Our multidisciplinary team has the capacity and expertise to achieve these goals.