Single-cell transcriptional landscape of liver transplant rejection reveals tissue persistence of clonally expanded, treatment-resistant T cells.

T cell-mediated rejection (TCMR) affects ∼50% of pediatric liver transplant recipients within 5 years, and late TCMR is associated with graft failure due to ineffective treatment. Although CD8+ T cells promote late TCMR, their clonal expansion, intragraft persistence, localization, and gene expression remain largely undefined. Here, single-cell RNA sequencing and immune repertoire profiling of 30 cryopreserved liver biopsies from rejecting and nonrejecting pediatric patients identified expanded intragraft CD8+ T cell clonotypes (CD8EXP) and their gene expression profiles. Expanded CD8+ clonotypes (CD8EXP) bore markers of effector and CD56hiCD161- "natural killer-like" T cells, retaining their clonotype identity and phenotype in serial biopsies despite histologic TCMR resolution. CD8EXP clonotypes localized to portal infiltrates during active TCMR and persisted in the lobule after histologic TCMR resolution. MultiNicheNet analysis revealed differential crosstalk between Kupffer cells and CD8EXP, with activation of transforming growth factor ß and downregulation of CD47 immune checkpoint signaling. Therefore, persisting intragraft CD8EXP clones remain active despite TCMR treatment and may contribute to long-term allograft fibrosis and failure of operational tolerance.