Single-cell Unperturbed Activation Profiling by mass cytometry captures

Introduction: Systemic lupus erythematosus (SLE) and other chronic inflammatory diseases are complex, multifactorial conditions where patient heterogeneity poses a major challenge to treatment. Despite the availability of numerous omics technologies, reliable biomarkers for precision medicine remain scarce, and many cell-based approaches are difficult to integrate into clinical settings. In this proof-of-concept study, we hypothesized that interrogating the cells' imprinting by the inflammatory environment through Unperturbed Activation Profiling (UACT) without prior restimulation would allow for the assessment of individual immune status in chronic rheumatic diseases. Methods: We developed a highly standardized mass cytometry-based workflow to assess signaling protein phosphorylation in peripheral blood leukocytes from active SLE patients (n=20) and healthy controls (n=20) at the single-cell level. Importantly, the entire workflow from blood drawing to stabilization and preparation of barcoded samples was optimized to ensure necessary robustness of the procedure for clinical studies. Results: We identified leukocyte-specific in vivo-like signaling patterns in line with type I interferon-driven signatures, such as elevated pSTAT1 in naïve T cells and pSTAT3 in classical monocytes and naïve CD8 T cells. These patterns captured patient variability and immune activation, although they did not correlate with SLEDAI-2K disease activity scores. We further explored the utility of UACT for treatment monitoring in four SLE patients treated with the JAK inhibitor baricitinib under compassionate use, where baseline pSTAT3 levels across multiple cell subsets seemed to be associated with treatment response. Discussion: Our results thus demonstrate that single-cell UACT without restimulation of cells can detect immune activation in chronic inflammatory diseases, supporting both biomarker discovery and the identification of novel therapeutic targets.