Single cell RNA-sequencing identifies the effect of Normothermic ex vivo liver perfusion on liver-resident T cells

Abstract

Background: Normothermic ex vivo liver perfusion (NEVLP) is an exciting strategy to preserve livers prior to transplant, however, the effects of NEVLP on the phenotype of tissue-resident immune cells is largely unknown. The presence of tissue-resident memory T cells (TRM) in the liver may protect against acute rejection and decrease allograft dysfunction. Therefore, we investigated the effects of NEVLP on liver TRMs and assessed the ability of anti-inflammatory cytokines to reduce TRM activation during NEVLP.

Methods: Rat livers underwent NEVLP with or without the addition of IL-10 and TGF-β. Naïve and cold storage livers served as controls. Following preservation, TRM T cell gene expression profiles were assessed through single cell RNA sequencing (scRNA-seq). Differential gene expression analysis was performed with Wilcoxon rank sum test to identify differentially expressed genes (DEGs) associated with a specific treatment group. Using the online Database for Annotation, Visualization and Integrated Discovery (DAVID), gene set enrichment was then conducted with Fisher's exact test on DEGs to highlight differentially regulated pathways and functional terms associated with treatment groups.

Results: Through scRNA-seq analysis, an atlas of liver-resident memory T cell subsets was created for all livers. TRM T cells could be identified in all livers, and through scRNA-seq, DEG was identified with Wilcoxon rank sum test at FDR < 0.05. Based on the gene set enrichment analysis of DEGs using Fisher's exact test, NEVLP is associated with downregulation of multiple gene enrichment pathways associated with surface proteins. Furthermore, NEVLP with anti-inflammatory cytokines was associated with down regulation of 52 genes in TRM T cells when compared to NEVLP alone (FDR <0.05), most of which are pro-inflammatory.

Conclusion: This is the first study to create an atlas of liver TRM T cells in the rat liver undergoing NEVLP and demonstrate the effects of NEVLP on liver TRM T cells at the single cell gene expression level.

Keywords: Liver transplantation; Liver-resident cells; Memory T cell; Normothermic perfusion.

Fig. 1.. Identification of rat liver-resident T cells using scRNA-seq.

After liver digestion and sorting on live cells from a naïve rat liver, scRNA-seq was performed to identify specific cells. A. Counts of memory T cell subsets within the liver. B. t-SNE plot of all cells present in the naïve liver. C. TRM T cells were identified based on canonical gene expression: CD4 TRM (CD4+, CD69+), CD8 TRM (CD8+, CD69+), CD8 TEM (CD8+, CD62L−, CD69−), CD4 TEM (CD4+, CD62L−, CD69−), CD8 TCM (CD8+, CD62L+,), CD4 TCM (CD4+, CD62L+). D. Heatmap and cluster analysis of important genes among memory T cell subtypes within the liver.

Fig. 2.. Effect of SCS on rat liver-resident memory T cells.

A. Counts of memory T cell subsets within the liver after four hours of SCS. “Other” T cells were not included in differential gene expression analysis. B. DAVID enrichment analysis of downregulated pathways between Naïve (reference group) and SCS.

Fig. 3.. Liver function during NEVLP.

A. Lactate clearance during NEVLP shows livers are functioning and all livers (including the ones undergoing scRNAseq have lactate returning to baseline by the end of perfusion. B. Histological analysis of livers from the naïve, SCS NEVLP and NEVLP-cyt groups showed low levels of liver damage. The livers that ultimately underwent scRNAseq from the NEVLP and NEVLP+cyt groups (triangle inserts) were similar to other livers from these groups.

Fig. 4.. Effect of NEVLP on rat liver-resident memory T cells.

A. Counts of memory T cell subsets within the liver after NEVLP. “Other” T cells were not included in differential gene expression analysis. B. DAVID enrichment analysis of downregulated genes between Naive (reference group) and NEVLP. C. DAVID enrichment analysis of downregulated genes between SCS (reference group) and NEVLP.

Fig. 5.. Effect of NEVLP on rat liver-resident memory T cells.

A. Counts of memory T cell subsets within the liver after NEVLP with the addition of TGF-β and IL-10. B. DAVID enrichment analysis of downregulated genes between NEVLP (reference group) and NEVLP-cyt.