*Purpose: Although CD4 T cells are critical in the mechanism of allograft rejection, little is known about their role in the innate immune-driven ischemia-reperfusion injury (IRI) in the allogeneic setting. In this translational study, we utilized clinically relevant allogeneic and syngeneic mouse orthotopic liver transplantation (OLT) models as well as screened human OLT biopsies (Bx) to determine the role of CD4+ T cells in the pathophysiology of hepatic IRI and OLT outcomes.

*Methods: Human liver Bx collected at 2h after reperfusion (prior to abdominal closure) from fifty-five liver transplant patients, recruited under IRB protocol, were analyzed by RT-PCR. In the experimental arm, C57BL/6 recipient mice were transplanted with Balb/c (allogeneic [Allo]-OLT) or C57BL/6 (syngeneic [Syn]-OLT) liver grafts subjected to 18 h of ex-vivo cold storage; liver graft/blood samples were collected at 6h post-reperfusion.

*Results: Post-reperfusion CD4 levels in clinical Bx correlated positively with the expression of CD28 (r=0.942, p<0.001); CD80/CD86 (r=0.893, p<0.001/r=0.849, p<0.001); TLR4 (r=0.885, p<0.001); CD68/Cathepsin G (r=0.780, p<0.001/r=0.783, p<0.001); and CXCL10 (r=0.755, p<0.001). Indeed, patients with high-CD4 expression (n=27) post-OLT were characterized by higher sAST levels (POD1 710±355 vs 372±92 IU/L; POD3 259±174 vs 81±15 IU/L; POD6 39±3 vs 33±4 IU/L, p<0.05) and longer post-transplant hospital stay (48±9 vs 36±5 days) as compared to low-CD4 expression group (n=28). To determine whether the presence of CD4+ T cell dictates IRI severity, donor/recipient mice were pretreated with or without anti-CD4 depleting mAb (GK1.5, 20μg/g i.v.) at 48 hours prior to OLT. Both in Allo-OLT and Syn-OLT groups, CD4 mAb treatment diminished CD4+ cell infiltration (flow cytometry, p<0.05), while mitigating IRI, evidenced by sALT (Allo: 3088±2239 vs 8538±3794, n=9/11, p<0.05; Syn: 4481±921 vs 8341±1256, n=5/6, p<0.05), Suzuki's histological grading (p<0.05) and frequency of TUNEL+ cells (p<0.05). The CD4 depletion, both in Allo- and Syn-OLT groups, depressed mRNA levels coding for CD28 (p<0.05), decreased frequency of CD80/CD86 cells in OLT and peripheral blood (flow cytometry, p<0.05), suppressed neutrophil/macrophage infiltration (immunohistochemistry, p<0.05), and decreased mRNA levels coding for CXCL10/MCP1/IL1β/CXCL1/TNFα (p<0.05).

*Conclusions: Our translational study documents the critical contribution of CD4+ T cells in the exacerbation of innate-immune dominated IRI, indicating that CD4+ T cells in the early post-reperfusion phase may not only serve as potential bio-marker but also therapeutic target to ameliorate hepatic IRI in OLT recipients.

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