OBJECTIVES: In many pediatric acute liver failure (PALF) cases a diagnosis is not identified, and the etiology is indeterminate (IND-PALF). Our pilot study found dense CD8 T-cell infiltrates and increased T-cell clonality in liver specimens from IND-PALF patients. We aimed to validate these findings in a multicenter cohort with investigators blinded to diagnosis. METHODS: PALF Study Group registry subjects with IND-PALF (n=37) and known diagnoses (DX-PALF) (n = 18), age 1-17 years, with archived liver tissue were included. Liver tissue slides were stained for T-cells (CD8 and CD4), B-cells (CD20), macrophages (CD163), perforin, and tissue resident-memory T-cells (Trm, CD103), and scored as minimal, moderate, or dense. Lymphocytes were isolated from frozen liver tissue for T-cell receptor beta (TCRβ) sequencing. RESULTS: Dense hepatic CD8 staining was found in significantly more IND-PALF (n = 29, 78%) compared to DX-PALF subjects (n = 5, 28%) (p = 0.001). IND-PALF subjects were more likely to have dense or moderate perforin (88% vs 50%, p = 0.03) and CD103 (82% vs 40%, p = 0.02) staining compared to DX-PALF subjects. TCRβ sequencing of 15 IND-PALF cases demonstrated increased clonal overlap compared to 6 DX-PALF cases (p = 0.002). CONCLUSIONS: Dense infiltration of effector Trm CD8 T-cells characterizes liver tissue from IND-PALF subjects. Increased clonality suggests the T-cell expansion is antigen(s) driven as opposed to a non-specific inflammatory response. These findings support CD8 staining as a new biomarker of the activated CD8 T-cell PALF phenotype. Future studies are needed to characterize potential antigens, host risk factors, and inflammatory pathways with the goal of developing targeted therapies.