Abstract
Recent advancements in genetically engineered pigs have spurred research into pig-to-human xenotransplantation, particularly involving heart, kidney and liver. Here we characterize immune cell populations cells in both peripheral blood and transplanted liver in a human decedent who received a pig liver xenograft and who was monitored over the course of 10 days. Using single-cell RNA sequencing and spatial RNA sequencing, we found that T cells were progressively activated in the peripheral blood, whereas γδT cells and exhausted T cells infiltrated the pig liver extensively, indicating impaired adaptive immunity. Additionally, we identified two distinct monocyte clusters that may influence the coagulation and immune response after xenotransplantation. First, at an early phase after transplantation, THBS1+ monocytes had the potential to regulate coagulation through interaction with platelets via the THBS1–CD36 signaling pathway. Second, at a later phase, C1QC+ monocytes infiltrated the pig liver, potentially promoting T cell exhaustion through induction of CD274 (PD-L1) expression. In summary, our study highlights how innate immune cells may affect thrombotic and immune pathways after liver xenotransplantation and should spur further research to clarify the roles of THBS1+ and C1QC+ monocytes.
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Data availability
The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genom. Prot. Bioinform., 2021) in the National Genomics Data Center (Nucleic Acids Res., 2024), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA011658) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/s/0k9JnPMz. scRNA-seq data for heathy liver tissue were obtained from the DISCO database (https://disco.bii.a-star.edu.sg; these data are freely downloadable from the website). Published scRNA-seq data from liver transplantation patients and healthy liver specimens were acquired from the National Genomics Data Center (https://ngdc.cncb.ac.cn/, HRA007802; these data and raw data of the present study are freely available; researchers should submit applications to the database to obtain data) and the Gene Expression Omnibus database (GSE237622), respectively. Source data are provided with this paper.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (82325007 and 92468202, both funding to L.W.); the National Key R&D Program of China (2021YFA1100502, funding to L.W.); the National Natural Science Foundation of China (82371793, funding to K.-F.D.); the National Natural Science Foundation of China (U24A20656, funding to K.-S.T.); and the National Key R&D Program of China (2024YFC3406800, funding to K.-S.T.).
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K.-S.T. and Z.-X.Y. majorly conducted the surgery. Y.-W.L. and X.Z. prepared the donor pig and collected the experimental data. H.-T.Z. monitored the patient throughout the investigation. S.-Q.Y., Y.-L.Y., W.-J.S., D.-S.W., Z.-C.L., H.-M.L., Y.C. and R.D. participated in the surgery. L.L. and M.L. provided important assistance to the recipient management. R.Z. provided professional advice in immunology. Z.-B.L., H.X. and D.W. prepared the draft of the manuscript. H.Z. and J.-L.D. performed histological observation. E.-W.L. and L.Z. provided the experimental pigs. D.-K.P. established the gene-edited donor pig. K.-F.D. and L.W. designed and supervised the whole study.
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Nature Medicine thanks Leo Buhler, Peter Cowan and Joshua Weiner for their contribution to the peer review of this work. Primary Handling Editor: Michael Basson, in collaboration with the Nature Medicine team.
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Extended data
Extended Data Fig. 1 Flow cytometry for Xenotransplantation.
a. Gating strategy for detecting binding of serum IgG and IgM to porcine PBMC b. Analysis of porcine cells (p-CD45+ cells/h-HLA- Cells) in positive control gene-modified pig sample. c. Analysis of porcine cells in negative control healthy human sample. d. Analysis of porcine cells in liver xenotransplantation patient’s sample. All the sample size was one and performed 3 technical replicates.
Extended Data Fig. 2 Identification of monocyte clusters in PBMCs.
a. Unsupervised clustering of the monocytes in PBMC samples. b. Density plots for specific markers for each monocyte cluster. c. Dot plot for the expression of CD14 and CD16 to identify the classical and non-classical monocytes.
Extended Data Fig. 3 Identification of infiltrated human cells in porcine liver.
a. Identification of the major human cell components in porcine liver. b. Dot plot of specific markers for each cell type.
Extended Data Fig. 4 scRNA-Seq data of Rat allotransplantation model.
a. Unsupervised clustering of the single cells in transplanted liver samples and identification of the major cell components in transplanted liver. b. Dot plot of specific markers for each monocyte cell type. c. UMAP Plots show the distribution of monocytes subtypes in normal liver, rejected liver and non-rejected liver group. d. Bar charts for absolute cell counts (left) and percentage (right) changes of Thbs1+ and Pdl1+ monocytes calculated by scRNA-Seq data (the cell number of different groups were shown in the figure).
Extended Data Fig. 5 scRNA-Seq data of liver allotransplantation patients.
a. Identification of the major cell components in transplanted liver. b. Dot plot of specific markers for each cell type. c. Density plot for THBS1 expression of monocyte clusters. d. UMAP Plots show the distribution of monocytes subtypes in normal liver, rejected liver and non-rejected liver group.
Extended Data Fig. 6 T cells clusters in xenotransplantation patient’s PBMC.
a. Unsupervised clustering of the T cells in PBMC samples. b. Violin plots illustrated the CD4 and CD8 expression of each T cell cluster. c. Heatmap for functional marker expression of each T cell cluster. d. UMAP plots showed the subtypes of T cells. e. Cytotrace analysis evaluated the differentiation status of T cell clusters (Sample size=1, the cell number of different cell type were shown in the figure). Data are presented as median and the upper and lower quartiles, the bounds of box represent the upper and lower quartiles. f. Cell trajectory analysis for T cell clusters by Monocle2.
Extended Data Fig. 7 T cells infiltrated in porcine liver.
a. Unsupervised clustering of the infiltrated T cells in integrated porcine liver scRNA-Seq samples. b. Violin plots illustrated the CD4 and CD8 expression of each T cell cluster. c-d. Heatmap for the functional marker expression of each T cell cluster.
Extended Data Fig. 8 γδT and exhausted T cells in normal liver and allotransplant liver.
a. UMAP plots showed the major cell components of cells in normal liver in DISCO database. b. UMAP plots showed the subtypes of T cells in normal liver in DISCO database. c. UMAP Plots show the distribution of T cell subtypes in normal liver, rejected liver and non-rejected liver group. d. Bar charts for absolute cell counts and percentage changes of γδT and exhausted T cells calculated by scRNA-Seq data (the cell number of different groups were shown in the figure).
Extended Data Fig. 9 Graphical summary.
a. Graphical summary of immune cells dynamics in the periphery and xenograft.
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Source Data Figs. 1–6 and Extended Data Figs. 4, 6 and 8 (download XLSX )
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Tao, KS., Ling, YW., Zhang, X. et al. Immune cell landscape in a human decedent receiving a pig liver xenograft. Nat Med 31, 2611–2621 (2025). https://doi.org/10.1038/s41591-025-03860-y
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DOI: https://doi.org/10.1038/s41591-025-03860-y
