Dissecting the immune discrepancies in mouse liver allograft tolerance and heart/kidney allograft rejection

Abstract

The liver is the most tolerogenic of transplanted organs. However, the mechanisms underlying liver transplant tolerance are not well understood. The comparison between liver transplantation tolerance and heart/kidney transplantation rejection will deepen our understanding of tolerance and rejection in solid organs. Here, we built a mouse model of liver, heart and kidney allograft and performed single-cell RNA sequencing of 66,393 cells to describe the cell composition and immune cell interactions at the early stage of tolerance or rejection. We also performed bulk RNA-seq of mouse liver allografts from Day 7 to Day 60 post-transplantation to map the dynamic transcriptional variation in spontaneous tolerance. The transcriptome of lymphocytes and myeloid cells were characterized and compared in three types of organ allografts. Cell-cell interaction networks reveal the coordinated function of Kupffer cells, macrophages and their associated metabolic processes, including insulin receptor signalling and oxidative phosphorylation in tolerance induction. Cd11b+ dendritic cells (DCs) in liver allografts were found to inhibit cytotoxic T cells by secreting anti-inflammatory cytokines such as Il10. In summary, we profiled single-cell transcriptome analysis of mouse solid organ allografts. We characterized the immune microenvironment of mouse organ allografts in the acute rejection state (heart, kidney) and tolerance state (liver).

FIGURE 1

Single cell transcriptome profiling of mouse liver, heart and kidney allografts. (A) Schematic view of the workflow. (B) The fractions of different immune cells in three allografted organs. ‘Other’ represents the parenchymal cells. (C) UMAP plot showing the clusters of merged single‐cell datasets. (D) Heatmap showing the gene expression correlations of cell types in (C).

FIGURE 2

Bulk RNA‐seq analysis and T cell trajectory in liver allografts. (A) Venn plot showing the overlapped differentially expressed genes between different time points in liver allografts. (B) Volcano plot showing the differentially expressed genes in liver allografts at different stages (bulk RNA‐seq). Significantly up‐regulated and down‐regulated genes (log2FoldChange > 1.5) are labelled (NS: not significant). (C) RNA velocities of major T cell subsets in allografted liver (single‐cell RNA‐seq data).

FIGURE 3

Transcription factor enrichment and correlation analysis of immune cells in normal and allografted mouse liver. (A) Gene module correlations of immune cells in allografted liver. (B) Gene Ontology enrichment of the green yellow gene module (including Gzma, Iglc3, Foxp3 and Tnfrsf4). (C) Correlation of transcription factor modules in normal (left) and allografted mouse liver (right). (D) Venn plot showing the overlapping of transcription factors between normal and allografted mouse liver. (E, F) Gene Ontology enrichment of transcription factor modules in normal (E) and allografted mouse liver (F). (G) Correlation of gene expressions between PBMCs and tissue‐infiltrating immune cells (LM: clusters in tissue and tissue‐infiltrating lymphocytes, LB: clusters in PBMCs). (H) Heatmap showing the Pearson correlation values between PBMCs and tissue‐infiltrating immune cells in allografted liver.

FIGURE 4

Cross‐tissue comparison between liver, heart and kidney allografts. (A) Venn plot showing the overlapped variable genes allografted liver, heart, and kidney. (B) Histogram showing the frequency comparison of immune cell between allografted liver and the other two organs. (‘***’p value ≤ 0.001, ‘*’p value ≤ 0.05). (C) Volcano plot showing the common differentially expressed genes in major immune cells between the allografted liver and the other two organs. Cell types are colour‐coded. (D) Volcano plot showing the common differentially expressed genes in endothelial cells (red dots) and stromal cells (green dots) between the allografted liver and the other two organs. (E) Heatmap showing the KEGG pathway enrichment of differentially expressed genes in major immune cells between the allografted liver and the other two organs.

FIGURE 5

Ligand–receptor interactions in allografted organs. (A, B) The number of ligands and receptors involved in allografted liver (A) and the other two organs (B). (C) Ligand–receptor analysis of cytokines and immune checkpoint in the allografted liver, heart and kidney. Labels of clusters (C1: cluster1) represent the cell types in Figure 1C (D) Representative confocal immunofluorescence images of Cd11c and Cd8 in liver allograft samples at day 7 post‐transplantation.