A spatial in situ hybridization approach to T cell clonotype analysis using T cell receptor variable gene probes

Resolving T cell clonality at single-cell spatial resolution remains a major challenge. Here, we developed an in situ hybridization panel comprising probes for immune and tissue cell types alongside comprehensive coverage of TRAV, TRBV, TRGV, and TRDV gene segments, enabling unbiased spatial mapping of T cell clones in situ. As a proof of principle, we applied this approach to human kidney biopsies from patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and controls. Combinatorial T cell receptor variable (TRV) gene expression allowed identification of T cell clones and their spatial organization. Confined clusters of clonally related αβ T cells were found in proximity to increased numbers of antigen-presenting cells, consistent with local immune activation, while γδ T cells occupied distinct peripheral niches. Although paired αβ chain detection is currently limited, this method establishes a scalable framework for spatially resolved clonotype analysis and provides a broadly applicable tool to investigate tissue-level immune organization across diseases.