Spatial Transcriptomics Differentiates Donor-Specific Antibody-Positive and -Negative Microvascular Inflammation

Background: In kidney allografts, microvascular inflammation (MVI) is a hallmark of antibody-mediated rejection and can be seen in patients with and without donor-specific antibodies (DSA). The pathophysiology of MVI in DSA-negative patients is speculated to be mediated by non-HLA antibodies or cellular immune responses. Methods: Kidney allograft biopsies from 2018-2022 that had donor-derived cell-free DNA (dd-cfDNA) and DSA testing were reviewed for allograft pathology. Clinical and pathologic data were collected. Biopsy material from representative DSA+ (n= 12) and DSA- (n= 16) cases with and without MVI were analyzed using digital spatial profiling (GeoMx, Nanostring) and compared to control biopsies. Biopsies were hybridized with the Whole Transcriptome Atlas and bound probes were collected, sequenced and quantified from glomerular and tubulointerstitial regions of interest (ROI). Results: We analyzed 237 ROIs from glomeruli (113) and tubulointerstitium (124) from the allograft biopsies. There was strong concordance in differentially expressed genes (DEG) between MVI+ and cfDNA+ biopsies (~80% overlap). In contrast, DSA+ and DSA- biopsies with MVI showed distinct but overlapping patterns of DEG compared to controls (~30% overlap)(Fig. 1). Pathway analysis of the differentially expressed glomerular genes revealed DSA- biopsies with MVI were enriched in interferon-gamma and T cell receptor signaling compared to DSA+ biopsies with MVI. Conclusion: DSP reveals molecular differences between DSA+ and DSA- biopsies with MVI, supporting distinct pathophysiological mechanisms for MVI-induced renal allograft pathology in the presence and absence of DSA. These results provide additional insight into the molecular pathophysiology of DSA- MVI that may help provide insight into the superior outcome and prognosis reported for these patients.Figure 1. DSP ditinguishes DSA+ and DSA- MVI. A) Glomerulus with DSA+ MVI, and B) histologically indistinguishable glomerulus with DSA- MVI. C) Venn diagram of DEG from DSA+ and DSA- glomeruli with MVI relative to controls.