Objective:

To determine the global and distinct transcriptomic changes in expression across immune cell subsets, in pathways related to metabolism, inflammation, and apoptosis, in a longitudinal study of MS patients under ozanimod treatment.

Background:

MS is a chronic inflammatory autoimmune disorder characterized by cycles of neurological relapse and remission, as well as ongoing progression. Ozanimod targets Sphingosine-1-phosphate (S1P) signaling, limiting lymphocyte migration from lymph nodes, and mitigating MS inflammation. However, ozanimod’s effects within specific immune cell types remain undefined.

Design/Methods:

Peripheral blood mononuclear cells (PBMCs) were obtained from relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients at baseline and 3 and 12 months after ozanimod treatment, and healthy controls. Viable PBMCs were labeled with 140 oligonucleotide-conjugated antibodies specific for immune subsets. Single-cell transcriptomic data were analyzed using the 10x Genomics platform with differential gene expression (DGE) and pathway analyses. Data analysis using CITE-seq was performed in R with packages including Seurat, clusterProfiler, enricher, ggplot2, and FindMarkers.

Results:

Ozanimod treatment induced compositional and transcriptional changes across immune subsets. Single-cell compositional analysis revealed reduced proportions of circulating lymphocytes and altered subset balance. RRMS and SPMS patients showed similar directional but variable magnitude changes. Globally, significant downregulation of ribosomal protein gene expression and oxidative phosphorylation pathways, particularly in RRMS patients, shifted to a hypometabolic or quiescent immune state. At the cell-type level, distinct subsets exhibited differential modulation of genes governing inflammatory activation, apoptosis, and metabolic regulation, highlighting cell-intrinsic differences in treatment response. Analyses integrating ADT (antibody-derived tags)-based protein data define post-transcriptional regulation and identify biomarkers of response in immune subsets.

Conclusions:

These findings suggest ozanimod exerts broad immunomodulatory effects beyond lymphocyte sequestration, promoting a cell-intrinsic downregulation of ribosomal and mitochondrial pathways. This transcriptional reprogramming may contribute to immune deactivation and therapeutic efficacy in MS immune and brain cells.