Spatiotemporal Single-Cell Profiling Reveals T Cell Clonal Dynamics and Phenotypic Plasticity in Human Graft-versus-Host Disease

Allogeneic hematopoietic cell transplantation (alloHCT) cures hematologic disease but is limited by acute graft‑versus‑host disease (GVHD). How human T‑cell clones evolve across blood and gut to injure epithelium remains unclear. We prospectively fingerprinted T cell clones pre‑transplant and tracked them longitudinally in 20 recipients, integrating TCR repertoire profiling with scRNA/TCR‑seq and high‑resolution spatial transcriptomics (Visium HD). We developed DecompTCR to resolve temporal expansion programs in blood and adapted computational tools to map clonotype phenotypes and niches in gut tissue. Severe GVHD was marked by persistent expansion and diversification of alloreactive clones and by shared CD8⁺ clonotypes that acquired Hobit (ZNF683)⁺ tissue‑resident memory (TRM) programs migrating from lamina propria to epithelium. Spatial deconvolution identified CD8⁺ effector/Hobit+ TRM hubs that localized near intestinal stem‑cell (ISC)–rich crypt bases and concentrated in crypt‑loss regions. Post‑transplant cyclophosphamide selectively depletes alloreactive clones, whereas incomplete depletion and early expansion are associated with severe disease. This clonotype‑resolved framework links tissue‑instructed TRM remodeling to ISC‑proximal cytotoxic hubs, nominating early repertoire scores and spatial hub burden as biomarker candidates.