BACKGROUND AND OBJECTIVES: The anti-CD20 antibody ofatumumab is an efficacious therapy for multiple sclerosis (MS) through depletion of B cells. The purpose of this study was to examine the derivative effects of B cell depletion on the peripheral immune system and a direct treatment effect on T cells expressing CD20. METHODS: Frequency and absolute numbers of peripheral leukocytes of treatment-naive patients with relapsing-remitting MS (RRMS) and patients treated with ofatumumab for a mean of 482 days were assessed in this observational study by flow cytometry. In addition, effector function and CNS migration of T cells using a human in vitro blood-brain barrier (BBB) assay were analyzed. RESULTS: This study showed that ofatumumab treatment of patients with RRMS increased the control of effector T cells and decreased T cell autoreactivity. It also showed that ofatumumab reduced the level of peripheral CD20+ T cells and that the observed decrease in CNS-migratory capacity of T cells was caused by the depletion of CD20+ T cells. Finally, our study pointed out a bias in the measurement of CD20+ cells due to a steric hindrance between the treatment antibody and the flow cytometry antibody. DISCUSSION: The substantial ofatumumab-induced alteration in the T cell compartment including a severely decreased CNS-migratory capacity of T cells could partly be attributed to the depletion of CD20+ T cells. Therefore, we propose that depletion of CD20+ T cells contributes to the positive treatment effect of ofatumumab and suggests that ofatumumab therapy should be considered a B cell and CD20+ T cell depletion therapy. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that compared with treatment-naive patients, ofatumumab treatment of patients with RRMS decreases peripheral CD20+ T cells, increases effector T cell control, and decreases T cell autoreactivity.