Spatial Transcriptomic and Proteomic Landscape of Human Liver Allograft Rejection

Formalin-fixed, paraffin-embedded (FFPE) liver biopsy samples were collected from the Department of Pathology at Beijing Tsinghua Changgung Hospital. The cohort included post-transplant needle biopsies from patients with and without pathologically confirmed T cell–mediated rejection (TCMR). All TCMR samples were obtained prior to anti-rejection therapy. Diagnoses were established according to the Banff criteria and independently reviewed by two senior pathologists. The study was approved by the institutional ethics committee, and informed consent was obtained from all participants. To characterize spatial cellular organization during allograft rejection, Xenium subcellular-resolution spatial transcriptomics was performed using a customized 5,000-plex gene panel on 23 liver biopsies (5 non-rejection [NR] and 18 rejection [R]). In parallel, GeoMx Digital Spatial Profiling (DSP) was used to generate spatial proteomics data from 20 biopsies (10 NR and 10 R). A total of 39 regions of interest (ROIs) were selected based on PanCK, CD45, α-SMA, and DNA staining to identify the portal area (PA), including 9 NR_non-PA, 10 R_non-PA, 10 NR_PA, and 10 R_PA regions. Additionally, 8 PanCK⁺ portal areas of illumination (AOIs), representing cholangiocytes, were profiled for protein-level validation (4 NR and 4 R).