Two Years of Ofatumumab Reshapes the Immune Landscape in Multiple Sclerosis

Multiple sclerosis has long been framed as a disease of misdirected T cells, but the last decade has belonged to B cells. Anti-CD20 monoclonal antibodies, which strip CD20-expressing B lymphocytes from the circulation, have become among the most effective therapies ever deployed against relapsing forms of the disease. Yet a persistent question has shadowed their clinical success: what else do these drugs do to the immune system over the long term? A new real-world study from a tertiary multiple sclerosis center in Naples, published in the Journal of Neurology, offers one of the most detailed longitudinal answers yet for ofatumumab, a fully human anti-CD20 antibody delivered through monthly subcutaneous injections.

The research team, led by Antonio Esposito and Marcello Moccia of Federico II University of Naples, followed 34 adults with multiple sclerosis who started ofatumumab and remained on treatment for at least two years, with paired immune assessments at baseline and follow-up. Ofatumumab is administered as 20 milligram subcutaneous injections at weeks 0, 1, and 2, and monthly thereafter. Unlike intravenous anti-CD20 agents such as ocrelizumab and rituximab, ofatumumab acts primarily in the peripheral lymphatic compartment, and its distinct epitope binding and complement-dependent cytotoxicity have fueled speculation that its broader immunological footprint might differ from its intravenous cousins.

To map that footprint, the investigators used a standardized whole-blood flow-cytometry workflow. Fifty-microliter blood samples were incubated with fluorochrome-conjugated antibody mixtures, red blood cells were lysed, and the remaining nucleated cells were acquired on a Beckman Coulter Navios cytometer equipped with 405-, 488-, and 638-nanometer lasers. Sequential gating identified the major lymphocyte lineages: CD3-positive T cells and their CD4 and CD8 subsets, CD19- and CD20-positive B cells, CD56-positive natural killer cells, CD27-positive populations, memory B cells carrying both CD27 and CD19, and the small CD3-positive CD20-positive T-cell subset that anti-CD20 therapy is known to eliminate. Absolute counts were obtained using a double-platform approach under routine quality-control standards, including instrument calibration and participation in the UK-NEQAS external quality assessment scheme.

The longitudinal analysis relied on linear mixed-effects regression, with fixed effects for age, sex, follow-up duration, comorbidities, body mass index, and previous disease-modifying therapy, and a random intercept for each participant. The results were striking. Over roughly two years of treatment, total lymphocyte counts rose by an average of about 251 cells per microliter, while CD3-positive T cells increased by roughly 369, CD4-positive T cells by about 240, CD8-positive T cells by about 132, CD27-positive cells by about 275, and CD27-positive CD3-positive T cells by about 452. All of these increases were statistically significant, with confidence intervals that excluded the null. In parallel, the expected depletion was unmistakable: CD19-positive and CD20-positive B cells fell by about 182 cells per microliter, and CD27-positive CD19-positive memory B cells dropped sharply as well.

Not everything moved, however. The ratio of CD4 to CD8 T cells remained stable, as did CD56-positive natural killer cells and the CD3-positive CD20-positive T-cell subset. That stability of the CD4-to-CD8 ratio is important, because it suggests that no major rebalancing occurred between the two broad arms of the T-cell compartment, even as absolute T-cell numbers climbed. The authors are careful to note that the rise in CD27-positive cells cannot be taken as evidence of naive T-cell expansion. CD27 is expressed across both naive and central memory T-cell compartments and also appears on subsets of natural killer and natural killer T cells. Without additional differentiation markers such as CD45RA, CD45RO, and CCR7, the specific CD27-expressing population driving the increase cannot be resolved with the panel used.

What, then, explains the T-cell rise? The authors propose that the pattern may reflect broad reorganization of the T-cell compartment following sustained B-cell depletion, a phenomenon that has been hinted at in other anti-CD20 studies. Previous short-term real-world investigations of ofatumumab generally found CD3, CD4, and CD8 counts to be broadly stable, which makes the two-year increases reported here particularly noteworthy. The discrepancy may be a matter of timing: early immune kinetics differ from the intermediate and long-term reorganization that unfolds as the immune system adapts to life without circulating CD20-positive B cells. Other variables complicate the picture, including prior exposure to therapies such as natalizumab or fingolimod, which reshape peripheral immune pools and trafficking patterns, and differences in measurement approaches, such as whether studies use surface-epitope or intracellular CD20 staining, the latter avoiding steric hindrance by therapeutic antibodies still circulating in the blood.

The study also looked for clinical meaning behind these immune shifts. Using multivariable logistic regression, the team tested whether the annualized percentage change in each laboratory measure predicted relapses, magnetic resonance imaging activity, progression on the Expanded Disability Status Scale, improvement on that scale, or the composite outcome of no evidence of disease activity, known as NEDA-3. The answer, in every case, was no. No significant associations emerged between changes in any lymphocyte population and any clinical or radiological outcome. Several models could not be reliably estimated at all because clinical and MRI events were so rare, a consequence of treating patients with a highly effective therapy, and because near-complete B-cell depletion left little variability to model.

The authors are refreshingly candid about what these negative findings do and do not mean. With only 34 participants and a low frequency of disease-activity events, the study was never powered to detect subtle immunological-clinical relationships, and the absence of statistical significance should not be read as evidence that such relationships do not exist. The analyses were explicitly designed to be hypothesis-generating rather than to establish mechanistic pathways or predictive biomarkers. Most participants had prior exposure to other disease-modifying therapies, and although previous treatment was included as a covariate, residual carry-over effects on immune composition cannot be excluded. There was also no parallel comparator group receiving ocrelizumab or rituximab, which limits direct inference about how ofatumumab’s subcutaneous route and pharmacology might differ from intravenous anti-CD20 agents, and no standardized intermediate sampling at six and twelve months to reconstruct the early-to-late kinetics of immune change.

Those limitations point directly toward the next generation of experiments. The immunophenotyping panel used here captured broad lymphocyte populations but could not resolve the smaller, functionally critical subsets that other work has implicated in anti-CD20 responses, including Th17 and Th17.1 cells, T follicular helper cells, and their regulatory counterparts. Cross-sectional research during ofatumumab treatment has documented improved Th17.1-to-regulatory-T-cell and T follicular helper-to-follicular regulatory T-cell ratios, reduced pro-inflammatory cytokine production, and impaired migration of residual CD20-positive T cells across an inflamed model of the blood-brain barrier. A multicenter comparison of ocrelizumab and ofatumumab found more pronounced reductions of B and CD8-positive cells with ocrelizumab, while total lymphocytes, CD4 cells, and natural killer cells remained similar between the two drugs. Functional readouts such as cytokine profiling, migration assays, standardized immunoglobulin tracking, and assessments of vaccine responses and infection risk would round out the safety picture.

For now, the study delivers a clear and reassuring headline finding: two years of ofatumumab produces sustained, near-complete depletion of circulating CD19- and CD20-positive B cells, exactly as its mechanism of action predicts, alongside a measurable and statistically robust expansion of broad T-cell compartments that remains clinically silent, at least as far as conventional outcome measures can detect. Whether that T-cell expansion represents a benign consequence of B-cell depletion, a compensatory homeostatic response, or a subtle repositioning of the immune system toward a more anti-inflammatory profile remains an open question. Answering it will require larger prospective cohorts, repeated longitudinal sampling, deeper immunophenotyping panels, and the functional assays that can translate cell counts into biological meaning. Until then, this two-year window into the immune life of ofatumumab-treated patients stands as a valuable benchmark for what long-term B-cell depletion actually looks like in the blood of people living with multiple sclerosis.

Subject of Research: Longitudinal immune profiling of lymphocyte subsets in multiple sclerosis patients treated with the anti-CD20 antibody ofatumumab

Article Title: Ofatumumab in multiple sclerosis: 2-year immune profiling and clinical correlates

Article References: Esposito, A., Corsaro, L., Cave, I. D., Capasso, F., Corsini, G., Matrone, G., Cerbone, V., Petracca, M., Carotenuto, A., Lanzillo, R., Scalia, G., Castaldo, G., Morra, V. B., & Moccia, M. (2026). Ofatumumab in multiple sclerosis: 2-year immune profiling and clinical correlates. Journal of Neurology, 273(10), Article 579. https://doi.org/10.1007/s00415-026-14088-2

Image Credits: AI Generated

DOI: 10.1007/s00415-026-14088-2

Keywords: multiple sclerosis, ofatumumab, anti-CD20, B-cell depletion, T cells, flow cytometry, immune profiling, lymphocytes, NEDA-3, clinical outcomes, immunotherapy, Journal of Neurology