Longitudinal analysis of peripheral immune cells in patients with multiple sclerosis treated with anti-CD20 therapy

Objective: Anti-CD20 therapy is a highly effective treatment for multiple scle-rosis (MS). In this study, we investigated MS-related changes in peripheralblood mononuclear cell (PBMC) subsets compared to healthy controls and lon-gitudinal changes related to the treatment.Methods: Multicolor spectral flowcytometry analysis was performed on 78 samples to characterize disease- andtreatment-related PBMC clusters. Blood samples from MS patients were col-lected at baseline and up to 8 months post-treatment, with three collectionpoints after treatment initiation. Unsupervised clustering tools and manual gat-ing were applied to identify subclusters of interest and quantify changes. Results: B cells were depleted from the periphery after anti-CD20 treatment asexpected, and we observed an isolated acute, transitory drop in the proportionof natural killer (NK) and NKT cells among the main populations of PBMC(P=0.03,P=0.004). Major affected PBMC subpopulations were cytotoxicimmune cells (NK, NKT, and CD8(+)T cells), and we observed a higher propor-tion of cytotoxic cells with reduced brain-homing ability and a higher regula-tory function as a long-term anti-CD20-related effect. Additionally, anti-CD20therapy altered distributions of memory CD8(+)T cells and reduced exhaustionmarkers in both CD4(+)and CD8(+)T cells.Interpretation: The findings of thisstudy elucidate phenotypic clusters of NK and CD8(+)T cells, which have previ-ously been underexplored in the context of anti-CD20 therapy. Phenotypicmodifications towards a more regulatory and controlled phenotype suggest thatthese subpopulations may play a critical and previously unrecognized role inmediating the therapeutic efficacy of anti-CD20 treatments.