Integrative epigenomic and transcriptomic profiling reveals dysregulated T cell regulatory networks in Stage 3 Type 1 diabetes

Aims/hypothesis. Type 1 diabetes (T1D) is driven by destruction of the pancreatic beta cells by autoreactive T cells, which occurs as a result of failed immune tolerance. The disruptions to the molecular mechanisms that maintain this tolerance are complex, and the balance between conventional CD4⁺ T cells (Tconv) and regulatory T cells (Treg) in T1D remain poorly defined. We hypothesised that by integrating chromatin accessibility, 3D chromatin organisation, transcriptomes and functional perturbation we can reveal the key T cell-centred networks altered in T1D. Methods. We performed parallel ATAC-seq and RNA-seq on sorted stimulated Tconv and Treg from children with T1D and age-matched autoantibody-negative controls. We mapped differentially accessible (DA) regions to putative target genes in human Treg and activated CD4⁺ T cells using Hi-C and asked whether 3D contacts assigned enhancers to distal genes not captured by nearest-gene annotation. To interrogate rare T cell subsets and age effects, we analysed single-cell RNA-seq (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) of adults with T1D and controls. Finally, we used CRISPR–Cas13d to perform multiplex knockdown of 7 candidate transcription factors (TFs) from a TNFα/NF-κB–linked module (FOS, FOSL1, FOSL2, MAFF, EGR1, EGR2 and NR4A3) in primary human Treg, followed by RNA-seq to functionally test the impacts. Results. Hundreds of differentially accessible regions and expressed genes were detected in paediatric Treg and Tconv cells in T1D, with changes enriched for TNFα signalling via NF-κB, interferon responses and IL-2/STAT signalling. TF footprinting highlighted altered occupancy at AP-1 motifs and other immune regulators, consistent with subtle rewiring of regulatory circuits. Integration with T cell Hi-C revealed that a large fraction of T1D-altered enhancers contacts genes other than the nearest transcription start site and uncovered new altered enhancer-gene pairs. Cas13d-mediated 7-TF knockdown induced transcriptional changes strongly overlapping those seen in paediatric T1D Treg. Conclusions. By combining paediatric case–control T-cell ATAC-seq and RNA-seq with T cell Hi-C, adult single-cell transcriptomes and CRISPR–Cas13d perturbation, we describe a multi-layered, Treg-centred network in T1D. This integrative framework provides a blueprint for moving from non-coding association signals to mechanistic models of T-cell dysregulation in T1D and suggests candidate pathways for therapeutic intervention.