Defining the T cell transcriptional landscape in pediatric liver transplant rejection at single cell resolution

Acute cellular rejection (ACR) affects >80% of pediatric liver transplant recipients within 5 years, and late ACR is associated with graft failure. Traditional anti-rejection therapy for late ACR is ineffective and has remained unchanged for six decades. Although CD8+ T cells promote late ACR, little has been done to define their specificity and gene expression. Here, we used single-cell sequencing and immune repertoire profiling (10X Genomics) on 30 cryopreserved 16G liver biopsies from 14 patients (5 pre-transplant or with no ACR, 9 with ACR). We identified expanded intragraft CD8+ T cell clonotypes (CD8_EXP) and their gene expression profiles in response to anti-rejection treatment. Notably, we found that expanded CD8⁺ clonotypes (CD8_EXP) bore markers of effector and CD56^hiCD161^- 'NK-like' T cells, retaining their clonotype identity and phenotype in subsequent biopsies from the same patients despite histologic ACR resolution. CD8_EXP clonotypes localized to portal infiltrates during active ACR, and persisted in the lobule after histologic ACR resolution. CellPhoneDB analysis revealed differential crosstalk between KC and CD8_EXP during late ACR, with activation of the LTB-LTBR pathway and downregulation of TGFß signaling. Therefore, persistently-detected intragraft CD8_EXP clones remain active despite ACR treatment and may contribute to long-term allograft fibrosis and failure of operational tolerance.