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Spatial transcriptomic profiling of indeterminate antibody-mediated rejection in liver allografts: A pilot study
Nigar Anjuman Khurram1, Karlene Stanton2, Armando P Signore3
1University of Pittsburgh, School of Medicine, Pittsburgh, PA, United States; Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States.
Background:
Antibody-mediated rejection (AMR) of liver allografts remains incompletely defined, particularly in biopsies classified as indeterminate for AMR under the current Banff criteria. Whether this category reflects the biological heterogeneity of antibody-mediated injury or is a distinct alloimmune phenotype remains unclear.
Methods:
We performed Geo Mx spatially resolved transcriptomic profiling of six liver allograft biopsies from four patients representing definite AMR, AMR-indeterminate, a histology-negative, C4d-positive within-patient comparator, T-cell mediated rejection (TCMR), and a stable allograft control. All AMR-spectrum biopsies were from ABO-incompatible grafts. Immunofluorescence-guided segmentation identified 91 regions of interest (ROIs) across the CD68 + macrophage-enriched, hepatocyte, and pan-cytokeratin + epithelial compartments; CD45 + ROIs were recovered only from rejection biopsies (TCMR, n = 5; definite AMR, n = 1). After quality control, 90 ROIs and 12,466 genes were analyzed using compartment-stratified linear models with slide-level adjustments.
Results:
Unsupervised analyses demonstrated that AMR-indeterminate biopsies clustered with definite AMR rather than TCMR, with shared upregulation of innate inflammatory and acute-phase transcripts (PLA2G2A, CRP, and SAA2) most prominent within macrophage-enriched and hepatocyte compartments. A direct comparison of AMR-indeterminate and definite AMR identified subtle distinctions, including IL-32 upregulation and non-significant enrichment of antigen presentation and NK cell-mediated cytotoxicity pathways (HLA-B, KIR3DL1). The TCMR reference biopsy showed upregulation of antigen processing and presentation pathways.
Conclusions:
These pilot findings suggest that, in ABO-incompatible allografts, AMR-indeterminate biopsies may sit within the molecular spectrum of antibody-mediated injury rather than constitute a separate rejection entity. These findings are strictly hypothesis-generating; validation in HLA-DSA-driven, ABO-compatible AMR is a prerequisite for generalization.
