EXPERIMENTAL ISCHEMIA AND REPERFUSION INJURY

S. Ueki¹, A. Castellaneta¹, O. Yoshida¹, K.S. Ozaki², M. Zhang¹, S. Kimura³, A.W. Thomson⁴, D. Geller¹, N. Murase^5
1Surgery - University Of Pittsburgh, Thomas E Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA, ²Surgery - University Of Pittsburgh, Thomas E. Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA, ³E1505 Biomedical Science Tower, University of Pittsburgh Thomas E Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA, ⁴Surgery, Thomas Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA, ⁵Surgery, University of Pittsburgh Medical Center, Pittsburgh/UNITED STATES OF AMERICA

Body: Background: I/R injury remains as a significant risk factor contributing to high mortality and morbidity after liver transplantation (LTx). The programmed death 1 (PD-1) receptor and its ligand PD-L1 are known to play an important role in regulating local immune responses. We hypothesize that the PD-L1 pathway plays crucial roles during innate immune responses induced by hepatic I/R injury. The hypothesis was directly tested in the mouse LTx model using PD-L1 KO grafts. Methods: PD-L1 KO or WT B6 liver grafts were transplanted into B6 CD45.1 recipients after 24 hrs cold preservation in UW solution. Recipients were sacrificed 1-24 hrs after LTx, and liver grafts were analyzed by immunohistochemistry, PCR, and FACs after collagenase digestion and hepatic NPC isolation. Results: Prolonged cold storage resulted in significant hepatic injury in WT grafts with ALT levels 5759 U/L and numerous necrotic foci at 12 hrs, which associated with prompt PD-L1 induction on hepatocytes, in addition to enhanced constitutive PD-L1 expression on sinusoidal endothelial cells and DC. The injury was significantly augmented in PD-L1 KO grafts with significantly increased T cells in immunohistochemistry, and ICAM and MCP-I mRNA upregulation. FACs of hepatic NPC further revealed significant increases of CD69+ CD8 T cells in KO graft. PD-L1 KO grafts had significantly lower Annexin V+ cells among infiltrating CD8+ T cells, indicating the failure to delete infiltrating CD8+ T cells during I/R injury. Further, to determine if parenchymal or NPC PD-L1 expression regulated innate inflammatory infiltrates, liver grafts lacking PD-L1 on parenchyma or on NPC were created and transplanted into B6 CD45.1 mice. Selective PD-L1 deficiency on patrnchyma or NPC resulted in higher levels of ALT (~6600 U/L) and CD8+ T cell infiltrates compared to WT grafts. However, frequencies of Annexin V+ CD8+ T cells were lower when PD-L1 was deficient in parenchyma than in BM-derived cells (27 vs 46%), suggesting rather significant function of parenchyma PD-L1 in deleting CD8+ T cells, although PD-L1 expression on both parenchyma and NPC plays important roles in I/R injury. Conclusions: The study demonstrates that graft tissue expression of PD-L1 plays a critical role in regulating inflammatory responses during LTx-induced hepatic I/R injury, and suggests that negative regulatory costimulatory signals may have an important function in innate immune responses.

Disclosure: All authors have declared no conflicts of interest.