Characterizing Immune Cell Subsets and Interferon in the Kidneys of Lupus Nephritis Patients

Objectives Up to 65% of patients with systemic lupus erythematosus (SLE) develop lupus nephritis (LN), a major cause of renal failure. Approximately 30% of LN patients fail to respond to standard immunosuppressive therapy, emphasizing the need for biomarkers that predict therapeutic response at flare onset. Interferon-induced gene (IFI-G) expression is a promising candidate biomarker, as elevated IFI-G levels in blood and renal tissue have been linked to more severe disease. We have previously shown that IFI-Ps (ISG15, MX1, IFNAR1) serve as reliable surrogates for IFI-G expression. This study aimed to develop and validate an imaging mass cytometry (IMC) panel to spatially characterize IFI-Ps and immune cell subsets in LN kidney biopsies, and to explore whether IFI-P expression correlates with treatment response. Methods We optimized an IMC panel to simultaneously evaluate IFI-P expression and characterize immune cell subsets directly within kidney tissue. Paraffin-embedded biopsies from LN patients in the Lupus Nephritis New Emerging Team and University of Toronto Lupus Clinic cohorts were analyzed. A pseudo-tissue composed of IFN-stimulated and unstimulated peripheral blood mononuclear cells in a clot that was embedded paraffin was used as a positive and negative control for antibody specificity. The panel includes 34 metal-conjugated antibodies to detect renal resident cells, infiltrating immune cells (T cells, B cells, monocytes, macrophages, dendritic cells), IFI-Ps, and fibrosis. Results The IMC panel successfully detected renal and immune cell subsets, as well as distinct patterns of IFI-P expression in various renal compartments (Figure 1A). IFI-P expression demonstrated a moderate to strong correlation between the different kidney compartments (Figure 1B). Preliminary analysis of 10 LN biopsies revealed a trend to increased IFI-P staining intensity in all of the renal compartments of nonresponders when compared to responders (Figure 1C). This achieved statistical significance (p < 0.05) for IFNAR1, MX1, and PKR in the glomerulus and PKR in the nonproximal tubules, consistent with the concept that elevated renal levels of IFN are associated with poorer response to treatment. Figure 1. Spatial profiling of interferon-induced proteins in LN kidney biopsies by imaging mass cytometry (IMC). (A) Representative IMC images of kidney biopsies from a treatment responder and non-responder, showing expression of ISG15 (yellow). MX1 (cyan). PKR (green), and IFNAR1 (red) overlaid with DNA (blue). Non-responders exhibited stronger and more diffuse IFI-P staining across renal compartments. Scale bar = 100 μm. (B) Correlation of MX1 expression between kidney compartments demonstrates coordinated interferon activity within tissue. MX1 is shown as a representative IFI-P, with similar correlations observed for the other IFI-P markers. Strong correlation was observed between proximal and non-proximal tubules (R = 0.84. p = 0.0045) and a moderate correlation between proximal tubules and glomeruli (R = 0.64. p = 0.054). (C) Quantitative comparison of mean IFI-P intensity across kidney regions in responders (n = 6) and non-responders (n = 4). Non-responders showed higher expression of IFNAR1 (p= 0.00952), MX1 (p= 0.0381), and PKR (0.0190) in the glomerulus, and increased PKR expression in the non-proximal tubules (p= 0.0381). Asterisks (*) denote statistically significant differences between treatment groups. Conclusion This study establishes a validated IMC-based approach for high-resolution spatial profiling of interferon signatures and immune cell subsets in LN kidney biopsies. Preliminary data suggest that elevated IFI-P expression is associated with nonresponse to therapy, supporting further investigation of IFI-Ps as predictive biomarkers. Application of this panel to larger LN cohorts may enable early patient stratification and guide precision treatment strategies to improve renal outcomes.