Abstract
OBJECTIVE: We aimed to investigate disease-related and anti-CD20 therapy-related changes in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients compared to healthy controls (HC) using multi-omics single-cell analysis. METHODS: Targeted single-cell sequencing of transcriptomes and epitopes was performed on PBMCs isolated from 64 blood samples collected from MS patients at baseline and at 3 time points following anti-CD20 treatment, alongside HC. Multicolor spectral flow cytometry was performed on 15 of the samples. RESULTS: Cell cluster analysis identified a subpopulation of classical monocytes with significantly high interleukin-1 beta (IL1B) expression and a pro-inflammatory profile compared to other monocyte clusters. This monocyte cluster expressed genes of pro-inflammatory chemokines and cytokines, such as CXCL8, CCL3, CCL4, and TNFα and was a major cell transmitter subset within the intercellular communication network. The IL1B(high) monocyte cluster was prevalent in HC (8.1% of PBMCs), but reduced in untreated MS patients (0.8% of PBMCs). High CXCR4 expression and Gene Ontology-term analysis indicated that IL1B(high) monocytes from MS patients had central nervous system (CNS)-infiltrating abilities in contrast to HC, likely regulated by LEF1. After anti-CD20 treatment, IL1B(high) monocytes showed changes in pro-inflammatory gene expression and MYC-associated regulatory networks, and their abundance increased 6-fold in the blood. INTERPRETATION: Our single-cell analysis identified a unique peripheral IL1B(high) monocyte cluster with a suggested CNS-infiltrating cellular phenotype that may explain its lower abundance in the blood of untreated compared to anti-CD20 treated MS patients. Treatment-associated changes in this population may contribute to the non-B cell related effects of anti-CD20 therapy. ANN NEUROL 2025;98:1283-1298.