*Purpose: Peri-transplant tissue damage driven by innate-adaptive immune cascades resulting from ischemia-reperfusion injury (IRI) is a major concern in vascularized composite allotransplantation (VCA). Here, we targeted the endothelial CD62 – T-cell CD162 signaling pathway with a recombinant soluble form of a tandem-PSGL-1 (TSGL-Ig) to monitor organ preservation-triggered early/late immune responses in VCAs.

*Methods: Orthotopic hindlimb transplantation studies were performed to determine the significance of CD62-CD162 signaling in the context of innate/adaptive immune activation in syngeneic (BL6->BL6) vs. allogeneic (Balb/c->BL6) mouse models with/without TSGL-Ig during 24h cold-storage (UW). To determine whether TSGL-Ig promotes CD4+ T cell exhaustion, allo-VCAs pre-conditioned with TSGL-Ig, were treated with adjunctive anti-TIM3 Ab. Some TSGL-Ig treated allo-VCAs were conditioned with Rapamycin (Rapa), an mTOR inhibitor, to determine whether targeting endothelial CD62 – T-cell CD162 by TSGL-Ig synergizes with immunosuppressive Rapa. Skin and muscle samples were collected and analyzed at 24h post-transplant.

*Results: Treatment with TSGL-Ig exerted anti-inflammatory function in the skin and inhibited thrombosis in the muscle of syn-VCAs. By contrast, TSGL-Ig treated allo-VCAs showed a more remarkable improvement, evidenced by decreased H&E scores (p<0.05), and suppressed T-cell activation markers (e.g. IFNg, T-Bet and CD4/8), as detected by qPCR. TSGL-Ig treatment also led to higher TIM-3 gene expression while mTOR signaling was down-regulated. Anti-TIM3 Ab used as an adjunctive therapy increased tissue damage and T-cell activation, as compared with TSGL-Ig monotherapy, suggesting the beneficial effect of TSGL-Ig may relate to TIM-3 - mTOR axis. Interestingly and by contrast, TSGL-Ig failed to attenuate VCA-induced tissue damage with unchanged TIM-3 levels in allo-VCA groups conditioned with Rapa, suggesting TSGL-Ig improved VCA was mTOR-TIM-3 signaling dependent. Skin samples from Rapa + TSGL-Ig regimen showed increased T-cell activation and inferior long-term VCA survival. Lastly, TSGL-Ig failed to up-regulate TIM-3 in syn-VCA, suggesting TIM-3 signaling is essential in an adaptive immune response in allo-VCA.

*Conclusions: Our results identify the PSGL-1-TIM-3 signaling axis as a novel regulator of adaptive immunity in VCA, with mTOR being the critical checkpoint of this pathway.

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