Multiplex Immunofluorescence Assay with Opal Reagents for Identifying Mononuclear Cell Subsets in Kidney Allograft Rejection Types

Antibody-mediated rejection (ABMR) remains a leading cause of kidney allograft failure, yet the mechanistic roles of innate immune cell subsets such as monocytes and natural killer (NK) cells remain incompletely understood. In this retrospective cohort study, we applied OPAL-based multiplex immunofluorescence (mIF) to human kidney allograft biopsies from 38 recipients with biopsy-proven ABMR (n = 19), T-cell-mediated rejection (TCMR, n = 12), or no rejection (NR, n = 7), enabling spatially resolved quantification of immune subsets in situ. Fluorescence thresholds were pathologist-validated, and co-expression phenotypes were defined using standardized segmentation and spectral unmixing. We observed a significantly higher density of CD1^4+CD11c⁺ monocyte-derived cells in ABMR versus TCMR (<i>p</i> = 0.011), and of cytotoxic CD3^-PAX8^-CD16⁺CD57⁺ NK cells in ABMR versus TCMR (<i>p</i> = 0.008), implicating both subsets in ABMR pathogenesis. Spatial clustering of these populations was evident in ABMR biopsies, suggesting organized immune infiltration. A logistic regression model combining both subsets yielded an area under the ROC curve of 0.79 (95% CI: 0.65-0.93), indicating moderate discriminatory power for ABMR. While Cox regression did not reveal statistically significant associations with graft survival, CD3^-PAX8^-CD16⁺CD57⁺ cells showed a trend toward increased risk (HR = 2.73, <i>p</i> = 0.09). These findings support a mechanistic role for monocyte and NK cell subsets in ABMR and demonstrate the utility of OPAL mIF for high-resolution immune profiling in human allografts. Our study advances understanding of cellular immune contributors to ABMR and highlights the potential diagnostic value of intragraft mononuclear cell phenotyping.