Abstract

Background: Immune checkpoints (CTLA4 & PD1) are inhibitory pathways that block aberrant immune activity and maintain self-tolerance. Tumors co-opt these checkpoints to avoid immune destruction. Immune Checkpoint Inhibitors (ICIs) activate immune cells and restore their tumoricidal potential, making them highly efficacious cancer therapies. However, immunotolerant organs such as the liver depend on these tolerogenic mechanisms, and their disruption with ICI use can trigger the unintended side effect of hepatotoxicity termed immune mediated liver injury from checkpoint inhibitors (ILICI). Patients with severe ILICI discontinue ICIs and start steroids, to the detriment of their cancer. How liver cells are dying, and which cell death pathways are involved in ILICI is not known. Understanding how to uncouple ILICI from ICI anti-tumor activity is of paramount clinical importance.

Methods: We developed a murine model to recapitulate human ILICI. CTLA4^+/- mice were treated with combined anti-CTLA4 + anti-PDL1 or IgG1 + IgG2 for 14 days. We probed cell death subroutines: apoptosis, necroptosis, and pyroptosis. We tested two forms of antisense oligonucleotides to knockdown (KD) caspase-3 (C3) in a total liver (hepatocytes and NPCs) or in a hepatocyte-specific manner. We employed imaging mass cytometry (IMC), a powerful multiplex platform for immunophenotyping and spatial cell interaction analysis to study immune cross-talk in our model.

Results: ICI treated mice had significant evidence of liver injury. We detected cleaved caspase-3 (cC3), indicating apoptosis was occurring, as well as NLRP3 inflammasome activation, but no necroptosis. Total liver KD of C3 worsened liver injury, increased inflammasome activation, and triggered GSDMD-mediated pyroptosis. In contrast, hepatocyte-specific KD of C3 reduced liver injury and NLRP3 inflammasome activation. IMC-generated single-cell data for 77,692 cells was used to identify 22 unique phenotypic clusters. Spatial analysis revealed cC3+ hepatocytes highly interacted with macrophages and NLRP3^hi myeloid cells. We also observed zones of three-way interaction between cC3+ hepatocytes, CD8⁺ T-cells and macrophages.

Conclusion: Our work is the first to identify hepatocyte apoptosis and NLRP3 inflammasome activation as drivers of ILICI. Our single-cell IMC data demonstrates macrophages and NLRP3^hi myeloid cells are among the nearest neighbors of cC3+ hepatocytes and contribute to injury. We report that the interplay between adaptive and innate immune cells is critical to hepatocyte apoptosis and ILICI.