*Purpose: T cell immunoglobulin domain and mucin domain-3 (TIM-3) has been reported to induce T cell exhaustion. By alleviating hepatic ischemia-reperfusion injury, we have explored whether activation of TIM-3 function may be explored as a novel therapeutic target in orthotopic liver transplantation (OLT). Although carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) was recently discovered as a novel cellular TIM-3 ligand, whether and how CEACAM1/TIM-3 signaling axis can affect OLT outcomes remains unknown. We have generated TIM-3 transgenic/CEACAM1 knockout (TIM-3Tg/CC1KO) double-mutant mice to investigate the role of CEACAM1/TIM-3 axis on host CED4+ T cells in OLT.

*Methods: In a clinically-relevant mouse OLT model, wild-type (WT; C57BL/6) livers, subjected to the extended cold storage (4°C/18h) in UW solution, were transplanted to groups of: WT, TIM-3 Tg, TIM-3 Tg/CC1 KO, and CC1 KO mouse recipients. Liver graft and serum samples were collected at 6h post-reperfusion. Mouse splenocytes from these animals groups were probed in standard cell activation culture systems. Human liver graft biopsies (2h post-reperfusion), collected from 55 adult OLT patients, were analyzed for immunological status by Western blots/RT-PCR.

*Results: CEACAM1 signaling, reduced hepatocellular injury in TIM-3 Tg recipient mice, compared to WT hosts, as evidenced by serum ALT/AST levels, and Suzuki’s histological grading of WT-OLT (n=8/group, p<0.01). In contrast, in the absence of recipient CEACAM1, the alleviation of WT-OLT injury in TIM-3 proficient recipients of double-mutants was curtailed (TIM-3 Tg/CC1 KO versus WT: n=7/8, p=0.737; TIM-3 Tg/CC1 KO versus CC1 KO: n=7 per group, p=0.11), suggesting CEACAM1 was essential to elicit TIM-3 inhibitory regulation in OLT. In agreement with our in vivo findings, RT-PCR analysis revealed that splenocytes from TIM-3Tg/CC1KO mice were highly susceptible to CD3 stimulation, while TIM-3 proficient T cells were immune suppressive. In the clinical arm, the expression of TIM-3 gene was significantly and negatively correlated with mRNA levels coding for TLR4, CD68, CD80, CD86, CXCL10, and cathepsin G in human OLT liver samples.

*Conclusions: CEACAM1 serves as a critical ligand for TIM-3 to suppress T cell activation, leading to alleviation of liver innate immune-driven injury in the peri-transplant period.

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