T lymphopenia in multiple sclerosis patients treated with ofatumumab: Even with consideration for CD3 ⁺ CD20⁺ cells

Abstract

Background

B cell depleting therapy has become a cornerstone in disease-modifying treatments for relapsing-remitting multiple sclerosis (RRMS). Given that the maximum blood concentration of ofatumumab (OFA) is two orders of magnitude lower than that of ocrelizumab, it was anticipated that OFA would result in fewer adverse events.

Methods

The study included 38 RRMS patients had received at least 5 months of standard OFA administration. CD3 ⁺ CD20⁺ cell counts were assessed prior to OFA administration.

Results

The number of CD3 ⁺ CD20⁺ cells reached up to 75/μL in the treatment groups other than NTZ, and up to 200/μL in the NTZ-treated group, where B cell levels increased. Lymphopenia classifies as Grade 2 (800/μL or less) was observed in 7/33 cases. Beyond the depletion of CD3 ⁺ CD20⁺ cells, a reduction in CD3⁺ cells was noted in 29 of 33 cases (88%), and with seven cases showing progressive T cell decline for up to 5 months after OFA initiation.

Conclusion

In addition to the expected depletion of B cells, there was a greater-than-anticipated reduction in T cells lacking CD20 expression. Long-term continuous BCDT appears to have a profound impact on the immune system. Adjustments to administration intervals should be considered to mitigate the risk of over-treatment.

Graphical abstract.

a. T-cell levels decrease significantly after ofatumumab administration (p < 0.001).

b. In seven cases, T-cell levels progressively decrease for up to 5 months following initiation.

Highlights:

• Analysis of 38 RRMS patients treated with ofatumumab for 5 months.

• CD3 ⁺ CD20⁺ cells reached up to 200/μL in natalizumab-treated patients.

• Grade 2 lymphopenia (≤800/μL) observed in 21%.

• CD3 ⁺  cell reduction occurred in 88%, with 7 cases showing progressive T cell decline over 5 months.

Introduction

Although the exact pathomechanisms of multiple sclerosis (MS) is unknown, genome-wide association studies have shown that genes related to the activation and proliferation of helper T cells are involved in the risk of developing the disease, and it is thought to be caused by T cell-mediated autoimmune mechanisms. ¹ Baker et al. showed that drugs that reduce memory B cells in the peripheral blood were effective in clinical trials in patients with MS. Conversely, clinical trials were stopped halfway through for drugs that increased them, such as atacicept, because MS disease activity increased, suggesting the effectiveness of B-cell depletion therapy (BCDT). ² Furthermore, clinical trials of ocrelizumab (OCR) for Relapsing-remitting MS (RRMS) ³ and Primary progressive MS (PPMS) ⁴ have shown positive results in terms of clinical symptoms and brain MRI findings. It was reported that subcutaneous injections of ofatumumab (OFA), which are easier to administer, significantly reduced disease activity compared to teriflunomide. ⁵

The mechanisms involved include suppressing the activation of helper T cells through antigen presentation, eliminating pathologically pro-inflammatory CD20⁺ T cells, ⁶ and shifting the balance of cytokine secretion from proinflammatory cytokines such as tumor necrosis factor (TNF) α, interleukin-6 (IL-6), and granulocyte macrophage-colony stimulating factor (GM-CSF) to anti-inflammatory cytokines like interleukin-10 (IL-10). Additionally, by inhibiting T cell proliferation in peripheral blood and reducing the production of interleukin-17 (IL-17) and interferon-γ, these mechanisms are thought to suppress the disease activity of MS. ⁷

The efficacy of anti-CD20 monoclonal antibody (mAb) therapy is now recognized as a highly effective for disease-modifying therapy (DMT). ⁸ However, a major challenge with anti-CD20 mAb treatment during the pandemic era is the suppression of neutralizing antibody production following the introduction of the coronavirus disease 2019 (COVID-19) vaccine. While this suppression is less severe compared to the near-complete inhibition observed during fingolimod (FTY) treatment. ⁹ Therefore, starting in November 2023, patients who had completed vaccination with the Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were gradually transitioned from natalizumab (NTZ) to OFA. We examined the changes in T-cell (CD3⁺ cells) and B-cell (CD19⁺ cells) populations in the peripheral blood of RRMS patients after of OFA administration, and analyzed the effects on T cell counts of past FTY or NTZ treatment. We found T cell reduction beyond the extent attributable to CD3⁺CD20⁺ cell depletion following OFA administration.

Materials and methods

RRMS patients received subcutaneous OFA (Kesimpta^Ⓡ) injections for at least 5 months (Figure 1). Only one patient in this cohort developed progression independent of relapse activity (PIRA), which persisted for several months during NTZ treatment.

Figure 1.

The two patient cohorts. (a). DMT treatment history in patients receiving ofatumumab. For details on the administration methods of FTY and NTZ, refer to the supplement. FTY was reduced over a few years, and the final FTY dosage when switching to NTZ is described in the main text. NTZ and FTY were not started simultaneously. Unlike in Europe and the US, FTY was released before NTZ in Japan. Although both groups consisted mainly of patients with high disease activity, NTZ was selected for patients with family planning. The total treatment duration for each patient is shown in Table 1. During the pandemic era, patients were switched from FTY to NTZ, and after completing the seventh dose of the SARS-CoV-2 vaccine against the omicron strain, they were switched from NTZ to OFA after more than 4-week interval. In this report, we retrospectively analyzed the monitoring results of peripheral blood counts of lymphocytes, T cells, and B cells obtained during routine clinical practice over the first five months of OFA administration. (b). Patients examined for CD3⁺CD20⁺ cell count reference values and relationship between CD3⁺ cells and CD20⁺ cells. Seventy-three of 83 patients were examined during DMT. DMT: disease modifying therapy, FTY: fingolimod, NTZ: natalizumab, DMF: dimethyl fumarate, OFA: ofatumumab, GA: glatiramer acetate, IFNβ: interferon beta, None: before DMT.

OFA was administered at 20 mg as per the prescribed guidelines. In three cases, where OFA was initiated during the pandemic, an unexpectedly significant decrease in lymphocyte count was observed. As a precaution, blood samples were collected immediately before the injections to measure peripheral blood lymphocyte counts and other parameters. The OFA was initiated more than 4 weeks after completion of the Japanese government's SARS-CoV-2 vaccination program, which included vaccines targeting the Omicron strain.

The study involved 38 Japanese patients meeting the 2017 McDonald diagnostic criteria, with a male-to-female ratio of 9/29 (Table 1). All patients tested negative for anti-aquaporin 4 antibodies and anti-myelin oligodendrocyte glycoprotein antibodies in peripheral blood, as determined by the cell-based assay.^10,11 The most common prior treatment was FTY, used in 23 cases.

Table 1.

Patient characteristics.

	Age (yo)	Disease period (M)	EDSS	NTZ (35 cases, period (M)	FTY (23 cases, period, M)	SARS-CoV-2 vaccine	Lym counts at OFA initiation	T cell counts at OFA initiation	BW (kg)	BMI (kg/m2)	BSA (m2)
Range	26–68	130–1680	0–7	25–82	12–135	2–7 times	545–4595	576–2900	41–96	16.6–31.8	1.34–2.10
Average (SD)	47 (11)	519 (342)	2.1 (2.1)	46.6 (14.2)	70.0 (30.9)	4.7 (1.5)	2426 (875)	1441 (602)	58 (12)	21.9 (3.4)	1.59 (0.18)
Median	52	471	1	44.5	70	70	2550	1350	54	21.8	1.55

Note. SARS-CoV-2 vaccine: From spring 2021 to early winter 2023, the Japanese government has been providing free vaccinations to those who wish to receive them, with seven rounds of vaccinations scheduled. ⁵⁹ In the present cohort, all 33 cases with vaccination records in their medical charts had received at least two doses of mRNA vaccine (4.7 ± 1.5 times, median 5 times), with the majority receiving Cominaty^Ⓡ (Pfizer) vaccine, and 13 cases had received six or more doses by November 2023. OFA began administration more than 4 weeks after the final vaccination, mostly starting in January 2024. SD: standard deviation, yo: years old, Period: period of MS, M: months, FTY: fingolimod, Y: years, EDSS: expanded disability status scale, BW: body weight, BMI: body mass index, BSA: body surface area calculated by Du Bois’ formula (Du Bois and Du Bois 1916).

The treatment course prior to OFA administration for 38 patients is shown in Figure 1(A). None received rituximab (RTX) or cytotoxic drugs with bone marrow suppression (e.g., mitoxantrone, azathioprine, cyclophosphamide, and methotrexate). Alemtuzumab and cladribine were also not used, as they are either not commercially available or not approved for MS.

In these patients, the frequency of FTY administration was gradually reduced from daily dosing, aiming to achieve a target peripheral blood CD4 ⁺ CD62L⁺ cell count of 10–80 cells/mm3 ¹² (see Supplementary materials). The final dosing schedules before switching to NTZ were as follows: every 5 days (six patients), twice a week (e.g., Monday and Thursday; nine patients), four times a week (e.g., Monday, Wednesday, Friday, and Saturday; four patients), and daily (four patients, including three who continued daily dosing at their previous clinic). The duration of prior FTY treatment exceeded 3.5 years in more than 80% of the patients, and the overall duration of FTY administration in all patients subsequently treated with ofatumumab (OFA) was 6.0 ± 3.3 years (median, 6.6 years).

Due to risks associated with continuing FTY during the pandemic, we transitioned patients from FTY to NTZ between April and June 2020, in line with observed practices in the US and Europe. Among the patients receiving OFA, 13 had no history of FTY treatment. Since April 2019, none of the patients have experienced relapses or exhibited new brain MRI activity. Furthermore, no intravenous methylprednisolone therapy has been required.

Thirty-five patients, including 23 switched from FTY, received NTZ with body weight-based extended-interval dosing therapy (EID-NTZ; 3–4 mg/kg every 6–7 weeks; supplement).

Reference values for the number of CD3 ⁺ CD20⁺ cells in peripheral blood were obtained from 28 patients immediately before re-injection during NTZ treatment, 13 patients receiving glatiramer acetate (GA), and 3 patients receiving interferon (IFN)β1a (Figure 1(B)). Additionally, the correlation between CD20⁺ cells and CD3 ⁺ CD20⁺ cells was analyzed in 57 patients receiving NTZ and 10 untreated MS patients. Peripheral blood lymphocyte, T cell and B cell counts were analyzed when continuous changes were observed for more than 2 months, except during periods of SARS-CoV-2 vaccination or infectious diseases such as COVID-19. Blood samples were collected for lymphocyte analysis before OFA administration before 9 a.m. and 3 p.m. at the outpatient clinic. Lymphocyte counts were measured in the hospital laboratory. CD3⁺ cells, CD19⁺ cells, or CD3 ⁺ CD20⁺ cells were analyzed using fluorescence-activated cell sorting (FACS) by BML (Tokyo, Japan). Double staining was used to detect the percentages of CD3⁺, CD19⁺, cells and CD3 ⁺ CD20⁺ cells among lymphocytes in a sample of 10,000 mononuclear cells. The absolute number of these cells was calculated based on the lymphocyte counts obtained from blood test. The detection threshold for specific staining in this double-staining system was 5 cells/μL and 0.5% or greater (data not shown). All tests were covered under national medical insurance. It should be noted that FACS analysis, as used here, is part of routine clinical practice and differs from more precise methods that analyze several to ten times more cells.

Statistical analyses

All data analyzed were nonparametric. The Mann‒Whitney test was used to assess significant differences between unpaired groups, while the Wilcoxon signed-rank test was applied for paired groups. The correlation between the presence or absence of a history of FTY treatment and lymphocyte count was evaluated using Pearson's χ² test.

Given reports of increased T-cell counts with NTZ,^13,14 we assessed the risk of T-cell depletion in our cohort using Fisher's exact test, comparing values above and below the lower IQR limit.

Statistical analyses were performed using GraphPad Prism 9J (9.3.1, MDF, Tokyo, Japan). For analyses where paired samples were unavailable (Figures 2 and 3), data from 33 cases were included. The statistical analyses of the correlation between CD20 ⁺ cells and CD3 ⁺ CD20⁺ cells were performed using SPSS 16.0J, IBM Japan, Tokyo.

Figure 2.

CD3 ⁺ CD20 ⁺ cell counts in blood color. (a) CD3 ⁺ CD20 ⁺cell counts in patients treated with natalizumab (n = 28, red circles), glatiramer acetate (n = 6), or interferon β1a (n = 3) were all below 200/μL. (b) Patients treated with glatiramer acetate (13) consistently had CD3 ⁺ CD20⁺ cell counts below 100/μL. (c) CD3 ⁺ CD20⁺ cell counts (Y-axis) were correlated with the CD20⁺ cell counts (X-axis) in MS patients treated with natalizumab (n = 57) or without disease-modifying drugs (n = 10). Spearman's rank correlation: ρ=0.517, p < .001, R² = .7216 (SPSS).

Figure 3.

Changes in lymphocyte counts in peripheral blood after OFA administration. After initiation OFA, the median lymphocyte count dropped by approximately 1300/μL, and stabilized after 2 months. There was a significant reduction between counts at 2 weeks and 5 months (p < .001). Statistical test: Wilcoxon signed-rank test. w: weeks, m: months.

Results

CD3 ⁺ CD20⁺ cells

Before analyzing T cell counts after OFA administration, we first examined the effect on CD3⁺CD20⁺ cells, which are similarly affected as B lymphocytes (CD19⁺CD20⁺) depleted by OFA. This was necessary because this cell population cannot be directly evaluated once OFA has been administered. The CD3 ⁺ CD20⁺ cell count in patients receiving NTZ, GA, or IFNβ treatment was 77 ± 42 (range: 25–198/μL, median: 64/μL). The upper limit of the mean plus 2SD was approximately 160/μL. Since CD3 ⁺ CD20⁺ cells increased in patients who received NTZ immediately prior, it was suggested that OFA's effect on T cell count may result in a decrease of up to 200/μL (Figure 4(A)). This increase is likely related to NTZ's influence on the release of B cells from the bone marrow. ¹⁵ In contrast, the CD3 ⁺ CD20⁺ cell count in the group of 13 patients treated with GA was 40 ± 19/μL (range: 14–65/μL; median: 41/μL). This suggests that when switching patients from treatments other than NTZ to OFA, the reduction in T cell count due to OFA may be up to 100/μL (Figure 4(B)). The correlation between CD20 ⁺ cells and CD3 ⁺ CD20⁺ cells was also analyzed (Figure 4(C)). A significant correlation was observed (Spearman's rank correlation, ρ=0.517, p < .001). However, unlike leukemia, the release of CD20⁺ cells due to NTZ-induced release from bone marrow appears closer to physiological conditions. While the number of CD3 ⁺ CD20⁺ cells in peripheral blood rises as CD20⁺ cells increase, this increase does not seem to be indefinite.

Figure 4.

Changes in T cell counts in peripheral blood after OFA administration. T cell counts decreased significantly 5 months after OFA initiation (p < .001). Of 33 patients, 29 (87.9%) experienced reductions in T cell counts more than 200/μL from baseline. Statistical test: Wilcoxon signed-rank test.

Effect of OFA administration on peripheral blood lymphocyte and B cell counts

Two weeks after the start of OFA administration, B cells were eliminated from the peripheral blood of 30 of 35 cases (85.7%). In the remaining five cases, B cell counts decreased to 6–15/μL (0.8–1.5%), and were completely eliminated by two months after starting OFA. Even at two months, when B cells had almost disappeared from the peripheral blood, the lymphocyte count continued to decline and stabilized at approximately 1300/μL (Figure 2 p < .001). In 14 of the 33 cases observed continuously for up to 5 months, lymphocyte counts dropped to 1000/μL or less, with 7 cases (21%) decreasing to 800/μL or less, classified as Grade 2 according to the CTCAE (CTCAE: Common Terminology Criteria for Adverse Events, version 5.0) (Table 2). Among these seven cases, five had a history of FTY treatment. However, as 62% of the cohort received FTY previously, there was no statistically significant association between FTY treatment history and lymphocyte count reduction (p = 0.6402, Pearson's χ²). Notably, only one of the seven cases maintained a lymphocyte count below 800/μL after 5 months. It was challenging to predict the risk of lymphocyte reduction based on pre-OFA lymphocyte counts. However, no patients with pre-treatment lymphocyte counts of 3000 or more experienced a decrease to 800/μL or less. Compared to pre-OFA levels, lymphocyte counts decreased by more than 30% in 37/39 cases (94.9%) and by more than 50% in 19/39 cases (49%).

Table 2.

Patients with lymphocyte below 800/μL after ofatumumab initiation.

Age at OFA initiation	Disease period (y)	FTY adm period (y)	Switch from NTZ	B cell elimination after 2 w	Lymphocyte count at baseline (/mm3)	T cell count at baseline (/mm3)
52 F	13	13	○	○	1500	770
47 F	13.2	‒	○	○	2700	1900
60 M	18.9	8.9	○	○	2500	1400
45 F	12.4	5.8	○	×	1100	800
52 F	9.1	5.3	○	×	1400	1000
52 F	16	5.8	○	○	1550	1170
56 F	31	‒	‒	○	1100	920

Note. The baseline lymphocyte counts and history of FTY treatment do not predict whether lymphocyte counts will drop below 800/μL. However, no patients with baseline counts exceeding 3000/μL experienced counts below 800/μL. OFA: ofatumumab, FTY: fingolimod, adm: administration, NTZ: natalizumab, after 2w: blood drawn just before third injection of OFA

Unexpected decrease in T lymphocytes due to OFA

Compared to baseline T lymphocyte counts prior to OFA administration, a significant decrease was observed, with a maximum reduction exceeding 200 cells/μL in 33 of 37 cases (89%). After three months of OFA treatment, the average T lymphocyte count had dropped to 60% of the pre-treatment level. In only three cases, the expected decrease in T cells—associated with the presence of CD3 ⁺ CD20⁺ cells—was minimal. In two of these cases (Cases 1 and 3), patients had previously switched from FTY to NTZ in 2020, followed by a transition from NTZ to OFA during the current treatment phase (Table 3). T lymphocyte counts showed a statistically significant reduction (p < .001) five months after OFA initiation. Notably, 29 of 33 patients (87.9%) experienced a decrease exceeding 200 cells/μL from baseline levels (Figure 3). In seven cases, the T lymphocyte count declined progressively up to five months after starting OFA (Figure 5), with reductions ranging from 500 to 1000 cells/μL. However, there was no correlation between the magnitude of the decrease and a prior history of FTY or NTZ treatment in these seven cases.

Table 3.

Patients with minimal T-lymphocyte changes.

	FTY	NTZ	Baseline (/mm3)	2 w (/mm3)	2 m (/mm3)	3 m (/mm3)	5 m (/mm3)
Case 1	○	○	746	610	620	680	680
45 F			3000	1800	1200	1000	1200
Case 2 50 F	×	×	625	675	‒	‒	750
			915	920	740	780	980
Case 3 37 F	○	○	1320	2050	‒	‒	1240
			2400	3320	‒	1990	1590

Overall lymphocyte counts decreased in cases 1 and 3, but T cell counts remained stable. Upper row: T lymphocyte counts. Lower row: Lymphocyte counts.

Figure 5.

Progressive reduction in T lymphocytes color. (a) T lymphocyte counts progressively decreased (500–1000/μL) in seven patients. (b) There was no correlation between this progressive reduction and a history of FTY or NTZ treatment (○: with treatment, ×: without treatment).

The representative case of a woman in her 30s is shown in Table 4. She developed relapsing-remitting MS with pontine lesions and myelitis in January 2013, and subsequently developed contrast-enhancing cerebral lesions. After 4 years of FTY treatment, she was switched to EID-NTZ (3–4 mg/kg; see supplement). Following two doses of OFA, B cells were completely depleted, and T cell counts decreased by half after the ninth administration. As a result, the tenth dose was delayed to a 9-week interval, yet B cells had not recovered. B cells reappeared only after 16 weeks, and the next administration is scheduled for 17 weeks.

Table 4.

Changes in lymphocyte, T cell, and B cell counts after the initiation of OFA treatment in the representative case.

date	DMT	Lym	CD3+	CD3 + CD20+	CD19+
−21 Ma	NTZ 18th (7 W) 130 mgb	1839/μL	1011/μL	55/μL	‒
−16	NTZ 21th (7 W) 180 mg	1978	1068	‒	613/μL
−2	NTZ 32th (7 W) 180 mg	2459	1399	‒	804
0	OFA 2Wc	1392	1140	‒	0
5	OFA 9th (4 W)	690	611	‒	0
7	OFA 10th (9 W)d	754	635	‒	0
14	OFA 13th (12 W)	823	651	‒	0
18	OFA 14th (16 W)	903	688	‒	11

Note. One week after two OFA injections, B cells were eliminated, and T cells were significantly reduced just before the ninth injection seven months after the start of OFA. Therefore, the interval between injections was gradually extended from the tenth injection, and B cells became detectable only after extending the interval to 16 weeks at the 14th injection. No FTY treatment history.

^a The number of months before and after the start of OFA is indicated. −21 M means 21 months ago.

^b NTZ was administered at 3 or 4 mg/kg at 7-week intervals.

^c Blood drawing 2 weeks after the OFA initiation, just before the third administration.

^d Nine weeks after the previous OFA, just before the tenth.

There was no correlation between the proportion of T cells remaining 5 months after OFA initiation (relative to baseline) and the age at OFA initiation (Figure S1A). Similarly, in patients with prior FTY treatment, no correlation was observed between T cell survival rate at five months and the duration of preceding FTY therapy (Figure S1B).

Is T cell depletion associated with a history of NTZ treatment?

Referring to values reported in the Italian cohort, ¹³ which showed an increase in T cells in patients receiving NTZ, we divided patients with a history of NTZ treatment into two groups based on their T cell counts prior to OFA administration. We then examined whether there was a significant difference in the risk of T cell counts falling below 800/μL at five months after OFA initiation (Table 5). Using the lower IQR limit, 1637/μL, at baseline reported in the Italian cohort switched from NTZ to OFA, we compared the proportion of patients in our cohort who had T cell counts of ≤800/μL at five months after OFA initiation. Patients were stratified into those with baseline T cell counts ≥1637/μL and those <1637/μL. In the higher-count group (≥1637/μL), four of 14 patients had T cell counts ≤800/μL, whereas in the lower-count group (<1637/μL) 14 of 19 patients did (two-tailed Fisher's exact test, p = .0152).

Table 5.

Comparison of lymphocyte and T cell counts in our cohort versus Italians who switched from NTZ to OFA.

	Our cohort	Italian cohort
Lymphocyte counts
IQR (Q1–Q3)	1900–3008	2434–4510
Median	2550	3690
Number of patients	33	31
T cell counts
IQR (Q1–Q3)	990–1885	1637–2952
Median	1350	2359
Number of patients	33	23

Note. In our cohort, T cell increases were mild overall, but 14 patients exceeded the Italian cohort's Q1 threshold (1637/μL), which represents the lower IQR limit of T cells in a published report. ¹³ This indicates that NTZ administration increased not only B cells but also CD3⁺ cells in some Japanese patients. In these patients, T cell reductions after OFA were observed but did not fall below 800/μL during the 5-month follow-up. The milder T cell effects of OFA may be explained by its Cmax being two orders of magnitude lower than that of ocrelizumab (OCR; 212 μg/mL vs. 1.43 μg/mL at steady state).^60,61

This study does not establish whether the risk of T cell decline differs according to prior NTZ treatment or low-dose NTZ. Nevertheless, it suggests that switching from NTZ to OFA may reduce the risk of T cell decline if sufficient T cell levels are present at baseline. Possible differences in T cell dynamics related to NTZ exposure could also vary with body size or ethnicity. Exceptions were noted. Among three patients switched from dimethyl fumarate (DMF) to OFA without prior NTZ or FTY, two women in their 30s and 40s had complete T cell data before and after OFA initiation. In the 30-year-old patient, T-cell counts were 1910/μL at baseline but it decreased to 530/μL 5 months after starting OFA.

The longer the treatment period (5 months), the higher the risk of T cell depletion

One week after two OFA administrations, peripheral blood B cells were eliminated in more than 80% of patients. In contrast, analysis of the timing of peak T cell depletion following standard OFA administration showed that the risk of T cell depletion increased with longer treatment duration (Figure 6). When comparing lymphocyte and T cell counts at baseline versus five months after OFA initiation, the median lymphocyte count decreased by 1360/μL, and the median T cell count by 470/μL. Notably, T cell counts were reduced to levels below 200 CD3⁺CD20⁺ cells across the entire cohort.

Figure 6.

Timing of T cell count decline after OFA initiation. The risk of T cell depletion increased with longer treatment duration. 2W: T cell counts measured immediately before the third OFA dose, 2 weeks after initiation. M: months.

Next, the differences in total lymphocyte and T cell count reductions were analyzed based on FTY treatment history (Figure S2). In both groups, there was no significant difference in baseline T cell count (p = 1.042 by Mann‒Whitney test), and total lymphocyte counts decreased by 30–40% within 2 weeks from baseline. However, in patients without prior FTY treatment (13 cases), the reduction in total lymphocyte count was only 23%, indicating a trend toward a smaller reduction compared to those with prior FTY treatment (23 cases) (p = .0562, Mann‒Whitney test). In both groups, there was no significant difference in T cell counts at baseline (p = 1.042, Mann‒Whitney test), nor was there a significant difference in the rate of T cell count decrease between baseline and 5 months later (p = .1435, Mann‒Whitney test). Nevertheless, in the group without prior FTY treatment, T cell counts increased by 20% and 42% in two cases after 5 months compared to baseline.

Discussion

In MS patients treated with DMTs, lymphopenia occurs with agents such as FTY or alemtuzumab due to their intrinsic mechanisms of action. For DMTs with a lower incidence of lymphopenia, the pharmacologic effects ¹⁶ must be well understood, and regular monitoring of lymphocyte counts and subsets at defined intervals is essential. ¹⁷

In our cohort treated with NTZ during the pandemic and subsequently switched to OFA, a clear tendency toward greater T cell reduction was observed within five months of OFA initiation, even when accounting for the elimination of CD3⁺CD20⁺ cells. Although less pronounced than previously reported, ¹³ NTZ was associated with an increase in T cell counts likely attenuated in our patients due to the relatively low single NTZ dose administered. Importantly, when T cell counts were elevated at the time of switching from NTZ to OFA, the risk of subsequent T cell reduction after OFA initiation was reduced.

In clinical trials of OFA, lymphocyte counts decreased by an average of 200/μL within 4 weeks of starting treatment, stabilizing thereafter. ¹⁸ The reduction was consistent across four body weight-based groups. ¹⁹ The present study analyzed changes in the total number of peripheral blood lymphocytes and T lymphocytes in MS patients following OFA administration. Notably, B lymphocytes disappeared from peripheral blood in 30 out of 35 patients (86%) 2 weeks post-OFA initiation. The total peripheral blood lymphocyte count decreased from an average of 2400/μL to 1300/μL. Grade 2 adverse events, defined as lymphocyte counts dropping to 800/μL or less (per CTCAE v5.0, Grade 2), were observed in seven of the 33 cases (21%) where measurements were continuously taken for up to 5 months.

CD20, the antigen targeted by OFA, is also expressed on T cells. CD3 ⁺ CD20⁺ T cells originate via trogocytosis, where CD20 molecules are transferred from B cells to T cells during T/B cell interactions. ²⁰ Furthermore, CD8 ⁺ CD20⁺ T cells, a subset of CD3 ⁺ CD20⁺ T cells, are myelin-specific ²¹ and elevated in the cerebrospinal fluid of patients with PPMS. ²² Assessing lymphocyte and T lymphocyte counts post-OFA requires understanding the normal levels of these cells in peripheral blood. CD3 ⁺ CD20⁺ T cells typically account for fewer than 100/μL, even in NTZ-treated patients experiencing B cell release from bone marrow, where CD3 ⁺ CD20⁺ T cell counts remain below 200/μL as reported in a previous result. ²³ Studies measuring CD3 ⁺ CD20⁺ T cells in MS patients during OCR or OFA treatment face challenges due to the presence of OCR or OFA in the blood, precluding evaluation. Consequently, anti-CD20 monoclonal antibody trials typically measure B cells using CD19 rather than CD20. Post-OFA, mean T lymphocyte counts decreased from 1440/μL to 900–950/μL over 3–5 months. If the expected maximum decline in T lymphocyte counts was within 200/μL, unexpectedly higher reductions were observed in 33/37 cases (89%). A decline in T cells beyond the levels accounted for by CD3 ⁺ CD20⁺ T cells suggests that OFA targets lymphocytes other than T and B cells carrying CD20 molecules.

Safety concerns regarding vaccines and T cell functions have been noted with BCDT. In MS patients treated with OCR, the predecessor to OFA, only a subset produced neutralizing antibodies in response to the SARS-CoV-2 vaccine, suggesting that might suppress vaccine-induced immune responses. However, even in FTY-treated patients, who experience stronger suppression of antibody production, antibodies can still be produced after repeated vaccinations, indicating that vaccines are not entirely ineffective. ²⁴ OCR has also been linked to a higher risk of breast cancer, which is thought to be related to reduced immune functions. ²⁵ The peak blood concentrations (Cmax) of OCR are two orders of magnitude higher than the subcutaneously administered OFA, and the risk of immune dysfunction associated with OCR may not be directly applicable to OFA.

Strategies to mitigate risks associated with long-term use of anti-CD20 Abs have been proposed. For example, EID-NTZ is being explored to minimize overdosage. Such approaches could also inform safer use of OCR and OFA in clinical practice. ²⁶

The Du Bois formula for BSA is still widely used worldwide. There remains no clear consensus on whether BSA or body mass index (BMI) is a better indicator of circulating plasma volume in relation to drug concentration. BMI holds a unique role in the epidemiology of MS; not only is a high BMI in late adolescence a known risk factor for developing MS, ²⁷ but it also influences the progression of physical severity. ²⁸

In patients with a high BMI, faster recovery of B lymphocytes under OCR treatment has been observed, ²⁹ suggesting lower blood drug concentrations and reduced efficacy in such patients. In OCR clinical trials, the drug effectively suppressed relapses regardless of BMI, but patients with a BMI of 25 kg/m² or less showed significantly greater efficacy in slowing disease progression (p < .001) compared to those with a BMI of over 25 kg/m². ³⁰

Although BMI is generally used as an indicator of obesity, it also serves as a rough measure of body size. However, BMI may not correlate well with circulating plasma volume in individuals who are short and extremely obese or tall and very thin. The reported BMI of MS patients in clinical trials from Europe and the United States is typically 26.0 ± 6.0 kg/m². ³¹ In contrast, Japanese MS patients enrolled in clinical trials exhibit lower BMIs: 21.8 ± 3.3 kg/m² for FTY ³² and 22.1 ± 3.5 kg/m² for dimethyl fumarate. ³³ In the present study, the BMI of Japanese patients was 21.9 ± 3.4 kg/m², consistent with previously reported data. Their BSA was 1.59 ± 0.18 m², with a median of 1.55 m².

Using the same Du Bois formula, German MS or neuromyelitis optica spectrum disorder patients administered RTX had a first-quartile mean BSA of 1.65 m², slightly larger than the BSA of Japanese patients in the current study. In the German cohort, delayed B-cell recovery was significantly observed in the first-quartile group compared to the fourth-quartile group (BSA 2.33 m²). ³⁴ Although the sample size was small and statistical significance was not assessed, Japanese participants demonstrated higher blood concentrations than Russian patients, particularly at weeks 1 and 12 (mean values, 0.84 vs. 0.48 and 0.66 vs. 0.38, respectively). This difference appeared to be consistent with the lower mean body weight of Japanese patients (54.1 kg) compared to Russian patients (62.4 kg). ³⁵

Even in Western countries, small-sized patients exist, highlighting the need for personalized medicine. Indicators such as the degree of B-cell recovery could guide tailored treatment. It has been reported that many patients require over six months for B-cell recovery, even with OCR and RTX.^36,37 As blood drug concentration varies with body size and impacts treatment outcomes, strategies such as adjusting dosage based on body weight^38–40 or spacing dosing intervals using blood concentration and other markers are increasing being explored for immune-mediated neurological disorders.^12,37

Several studies have recently reported that EID-OCR^41–45 can maintain efficacy while reducing costs ⁴⁶ and mitigating IgM depletion associated with standard dosing. ⁴⁷

The study is limited by its small sample size, single-center design, and a high proportion of cases transitioning from NTZ treatment, which is associated with increased B cells in peripheral blood. Consequently, the observed changes following OFA treatment may appear more pronounced compared to patients switching from other DMTs or treatment-naïve patients. Despite the influence of prior NTZ use, the marked decrease in T cells after OFA administration is noteworthy. Although specific T-cell subsets were not analyzed, the observed changes likely reflect random damage rather than targeting a particular subset. However, the significant reduction in helper T cells-a key subset-raises concerns about potential impacts on immune responses. This phenomenon warrants attention.

Several issues remain to be addressed. First, attention must be paid to viral infections, such as herpes zoster, and to carcinogenic risks, particularly breast cancer. In anti-CD20 monoclonal antibody therapy, the risk of severe infection increases with comorbidities, older age, longer treatment duration, and higher EDSS (Expanded Disability Status Scale) scores, whereas only lymphocyte counts are independently associated with the risk of varicella zoster virus (VZV) infection. ⁴⁸ According to FDA post-marketing reports, among the DMTs used to MS, with the exception of alemtuzumab (which exerts strong immunosuppression akin to autologous stem cell transplantation), the risk of VZV infection is higher with OCR than with FTY, although the risk of disseminated disease and death is lower with OCR. ⁴⁹ For long-term administration beyond 10 years, it is essential to avoid excessive immune suppression beyond B cells and to minimize the risk of cancers such as breast cancer.^25,50,51 Second, EID, as used for NTZ, is also being applied to B cell depleting therapies. We introduced a case of EID-OFA (Table 4). Interval extension is being guided by maintaining CD19⁺ cell counts at 10–30/μL at re-dosing, with most patients achieving this target at intervals of 10–15 weeks (Tanaka M, oral presentation, Japanese Society of Neuroimmunology, August 8–9, 2025, Chiba, Japan). The threshold of 30/μL was derived from clinical trial data, which showed that stable patients with CD19⁺ counts below this level experienced no contrast-enhancing lesions. ⁵² It is necessary to consider whether the B cell recovery count as a biomarker is appropriate for each patient.

Third, due to its significantly lower Cmax (Cmax of OCR and OFA, 212 and 1.43 μg/mL, respectively, FDA) although OFA may not provide the same degree of disability progression suppression as OCR—likely due to its substantially lower Cmax (OCR: 212 μg/mL; OFA: 1.43 μg/mL, FDA), a meta-analysis of progressive MS trials demonstrated that suppression of disability progression is largely attributable to the anti-inflammatory effects of DMTs. ⁵³ This suggests that controlling inflammatory lesions may also help reduce PIRA under even OFA therapy. ⁵⁴ Since PIRA in patients with incomplete B cell depletion under OCR ⁵⁵ may also be relevant to OFA. Since PIRA can emerge immediately after CIS, ⁵⁶ the OFA reduction strategy used in stable patients is inappropriate for cases with early evidence of PIRA, in which full B cell elimination should remain the therapeutic goal. Finally, in addition to B cell counts, serum neurofilament light chain (NfL) levels may serve as a valuable biomarker to prevent both excessive immune suppression, which risks rebound disease activity or PIRA, and inadequate suppression of inflammatory lesions. However, the lack of standardization and biological variability remain challenges.^57,58

Although the T cell reduction observed with OFA may partly result from nonspecific cytotoxicity and contribute to its anti-inflammatory effect, excessive immune cell depletion poses a significant burden on MS patients and must be carefully avoided.

Conclusion

The study reveals that OFA administration not only depletes B cells but also causes a greater-than-expected reduction in T cells in the peripheral blood of MS patients, beyond the disappearance of CD3 ⁺ CD20⁺ T cells. This finding suggests that prolonged and continuous OFA use could impair defenses against infections, particularly viral infections, and tumor immunity. Therefore, excessive administration should be avoided. Since single-dose adjustments are not feasible, dosing intervals are generally gradually extended, guided by peripheral blood CD19⁺ cell counts.