Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor β (TGFβ) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGFβ (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGFβ treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGFβ treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGFβ.

Tissue‐resident dendritic cells (DCs) are essential for immunological homeostasis and hold promise for a variety of therapeutic interventions. The rare nature of tissue‐resident DCs and their suboptimal description in the lab rat model has limited their characterization. To address this limitation, FMS‐like tyrosine kinase 3 ligand (FLT3L) has been utilized to expand these population in vitro and in vivo for investigative or therapeutic purposes. However, conflicting reports have suggested that FLT3L can either promote immune tolerance or enhance immunogenicity, necessitating clarification of the effects of FLT3L on DC phenotype and functionality. We first paired single‐cell RNA sequencing with multicolour spectral flow cytometry to provide an updated strategy for the identification of tissue‐resident classical and plasmacytoid DCs in the rat model. We then administered FLT3L to Lewis rats in vivo to investigate its effect on tissue‐resident DC enumeration and phenotype in the liver, spleen, and mesenteric lymph nodes. We found that FLT3L expands classical DCs (cDCs) 1 and 2 in a dose‐dependent manner and that cDC1 and cDC2 in secondary lymphoid organs had altered MHC I, MHC II, CD40, CD80, CD86, and PD‐L1 cell‐surface expression levels following FLT3L administration. These changes were accompanied by an increase in gene expression levels of toll‐like receptors 2, 4, 7, and 9 as well as inflammatory cytokines IL‐6 and TNF‐α. In conclusion, FLT3L administration in vivo increases cDC enumeration in the liver, spleen, and mesenteric lymph nodes accompanied by a tissue‐restricted alteration in expression of antigen presentation machinery and inflammatory mediators.

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Purpose of reviewThis review aims to highlight current advances in gene therapy methods, describing advances in CRISPR–Cas9 gene editing and RNA interference in relevance to liver transplantation, and machine perfusion.Recent findingsIn order to minimize rejection, increase the donor pool of available organs, and minimize the effects of ischemia–reperfusion injury, gene therapy and gene modification strategies are, thus, required in the context of liver transplantation.SummaryGene therapy has been used successfully in a diverse array of diseases, and, more recently, this technique has gained interest in the field of organ transplantation. Biological and logistical challenges reduce the rate of successful procedures, increasing the waiting list even more. We explore the exciting future implications of customized gene therapy in livers using machine perfusion, including its potential to create a future in which organs destined for transplant are individualized to maximize both graft and recipient longevity.

Background: Characterization of tissue-resident dendritic cell (DC) subsets, including classical DCs (cDCs) and plasmacytoid DCs (pDCs), is complicated experimentally by their rare nature. Many investigators have artificially increased DC yields through administration of the cytokine FMS-like tyrosine kinase 3 ligand (FLT3L). Despite its previous use experimentally, no study has yet described the effects of in vivo FLT3L administration on rat tissue-resident DCs. This study provides an advanced characterization of rat tissue-resident cDCs and pDCs following FLT3L injection. Methods: Lewis rats were injected once daily for 10 days with PBS vehicle or with 10, 50, or 100μg of FLT3L. Animals were sacrificed on the 11th day and dissected to obtain liver, spleen, and mesenteric lymph nodes. Cells were stained for 13-color flow cytometry and data was acquired on the Cytek Aurora spectral cytometer. Results: cDC and pDC enumeration increased in a dose-dependent manner following FLT3L administration. Expression levels of MHC I, MHC II, CD40, CD80, CD86, and PD-L1 were evaluated on liver, spleen, and mesenteric lymph node-resident cells. MHC II, CD80, and CD86 levels were significantly decreased on liver-resident cDCs but remained unchanged or increased on spleen and lymph node cDCs. Changes in MHC I/II and costimulatory molecule expression levels on pDCs varied by tissue and marker. Conclusions: FLT3L administration in vivo increased cDC and pDC enumeration, generated immature cDCs in the liver, and increased cDC maturation in the spleen and mesenteric lymph nodes. This comprehensive characterization of rat liver, spleen, and lymph node-resident DCs is key to understanding the effects of FLT3L on cDC and pDC phenotype.