Abstract

Background: Pediatric acute liver failure (PALF) remains a poorly-characterized disease entity that affects children of all ages. Emerging data supports an underlying immune-mediated process driving progression and severity. Up to 25% of patients will require liver transplantation (LT), yet there are no reliable indicators of disease trajectory to guide clinicians.

Methods: A single center, retrospective cohort of 58 children was identified: 38 with PALF (25 who recovered (SR) and 13 who underwent LT) and 20 patients who underwent surveillance liver biopsy following LT, whose histologically normal liver tissue is indistinguishable from otherwise healthy liver biopsies. Imaging Mass Cytometry was performed on these specimens using a 25-marker panel. Post-processing using our informatics pipeline generated a spatially resolved, single-cell dataset. Results were stratified by disease, and sub-analyzed by PALF outcome (SR vs LT).

Results: The study population resulted in 337,823 cells including 120,387 immune cells, which were identified based on lineage marker expression, making up 23 immune subpopulations. When comparing normal liver tissue and PALF, PALF patients had more immune cells overall (p<0.01) – specifically macrophages (p<0.01), monocytes (p<0.01) and CD8+ T-cells (p=0.01) (Fig 1A). No difference was observed between overall populations of CD4+ T-cells. When comparing immune populations between PALF and normal liver tissue, PALF demonstrated more proliferating CD8+T-cells (p=0.02) and HLADR+CD4+T-cells (p=0.03) than normal tissue (Fig 1B). In the CD4+ compartment, PD1+HLADR-CD4+Tregs were present in the PALF cohort that were absent in normal tissue. Within PALF, patients with SR demonstrated more CD4+ Tregs compared to LT (p=0.04), specifically, PD1+HLADR-Tregs, as well as PD1+C8+T-cells (Fig 1C/D). When evaluating non-immune populations, PALF tissue demonstrated increased Ki67 and HLADR+ hepatocytes when compared to normal tissue (p<0.01) (Fig 1E).

Conclusion: Our spatially resolved single cell atlas of PALF has determined that PALF is associated with greater proliferative cell populations than normal liver tissue, and PD1+HLADR-CD4+Tregs and are unique to this disease entity. Hepatocyte proliferation as indicated by Ki67 and HLADR predominates PALF. Further, we have determined that favorable clinical outcomes are associated with expansion of regulatory cell populations. Our preliminary analysis has determined that a subset of regulatory and PD1+T cells are characteristic of patients who experience recovery, suggesting an important role for immune exhaustion mediating liver inflammation and injury in PALF. Future studies are needed to evaluate these cell populations as potential biomarkers and therapeutic targets.