Tracking GAD-specific T cell expansions in type 1 diabetes by intradermal GAD-alum challenge

Identifying and monitoring autoreactive T cells that drive β cell destruction remains a major obstacle to developing effective immunotherapies for type 1 diabetes (T1D). These cells are extremely rare in peripheral blood and cannot be accessed directly from the pancreas. We used intradermal injection of glutamic acid decarboxylase (GAD)-alum to recruit GAD-specific T cells to accessible sites in the skin and skin-draining lymph nodes (LNs), sampled by skin suction blisters and ultrasound-guided LN aspiration. Peripheral blood samples obtained before GAD injection were restimulated with GAD in vitro to detect reactive CD4+ T cells. Single-cell RNA sequencing (scRNA-seq) followed by re-expression of selected T cell receptors (TCRs) confirmed antigen specificity. Up to 70% of T cells at the skin injection site were clonally expanded and 4 of 14 (28%) re-expressed TCRs were GAD reactive. In LNs, 1 of 14 (4%) clonally expanded TCRs was GAD reactive, representing approximately 0.08% of all T cells. GAD-reactive cells across compartments displayed Th1- and Th17-associated transcription signatures. These results demonstrate that intradermal autoantigen challenge and scRNA-seq enable direct identification and molecular profiling of autoreactive T cells in vivo. This minimally invasive approach provides a powerful platform for tracking antigen-specific T cells to monitor disease activity and evaluate immune interventions in T1D.