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 hi CD161 - ‘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. Graphical Abstract