Peripheral HLA-DRhiCD141+ Classical Monocytes Predict Relapse Risk and Worsening in Multiple Sclerosis.

Background and objectives: Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS characterized by a heterogeneous disease trajectory, highlighting the need for biomarkers to predict disease activity. Current disease-monitoring tools primarily reflect existing disease damage rather than impending activity. Peripheral blood mononuclear cells (PBMCs) are an ideal source of potential biomarkers due to their accessibility and their known role in MS pathology. Among PBMCs, myeloid cells are key players in MS pathogenic processes, yet they have not been as extensively studied than lymphocytes. The objective of our study was to identify indicators of MS disease activity through immune profiling.

Methods: We analyzed PBMCs using high-dimensional flow cytometry with a panel focusing on myeloid cells. We performed unsupervised clustering analyses to define a comprehensive immune landscape at a single-cell resolution. Supervised machine learning methods were used to extract immune features indicative of MS activity.

Results: We analyzed PBMCs from 135 individuals with MS with retrospective longitudinal follow-up and 44 healthy controls (HCs). Among the individuals with MS, 53 were untreated and were compared with HCs. Using an elastic-net model, 20 immune features were identified as contributors to the classification of MS and HCs (receiver operating characteristic-AUC 0.8881). To explore associations between immune features and disease activity, we focused on individuals with relapsing-remitting MS (n = 106). We identified a subpopulation of classical monocytes (CMs) with high expression of human leukocyte antigen - DR isotype (HLA-DR) and positive for CD141 (HLA-DR^hiCD141⁺) as a predictor of impending relapses over 2 years (hazard ratio [HR] 2.8, 95% CI 1.6-4.7) and disability worsening in patients with higher relapse activity. HLA-DR^hiCD141⁺ CMs could be retrieved by manual gating using 9 parameters and were similarly indicative of 2-year relapse risk (HR 1.9, 95% CI 1.3-2.8), highlighting its potential as a practical, translational approach. Compared with the widely studied biomarker serum neurofilament light chain reflecting acute activity, HLA-DR^hiCD141⁺ CMs provided a stronger prognostic value for impending relapse risk, suggesting different kinetics related to the underlying pathology.

Discussion: Our findings suggest that the frequency of HLA-DR^hiCD141⁺ CMs could serve as a valuable predictor of disease activity complementary to current clinical tools to guide evidence-based treatment decisions.