RESEARCH ARTICLE OPEN ACCESS Specific Patterns of Immune Cell Dynamics May Explain the Early Onset and Prolonged Efficacy of Cladribine Tablets A MAGNIFY-MS Substudy Heinz Wiendl, MD, PhD, Klaus Schmierer, MD, PhD, Suzanne Hodgkinson, MBBS, PhD, Tobias Derfuss, MD, Correspondence Dr. Wiendl Andrew Chan, MD, Finn Sellebjerg, PhD, MD, Anat Achiron, MD, PhD, Xavier Montalban, MD, PhD, heinz.wiendl@ukmuenster.de Alexandre Prat, MD, PhD, Nicola De Stefano, MD, PhD, Frederik Barkhof, MD, PhD, Letizia Leocani, MD, Patrick Vermersch, MD, PhD, Anita Chudecka, MSc, Claire Mwape, PhD, Kristina H. Holmberg, PhD,* Ursula Boschert, PhD, Sanjeev Roy, MBBS, MD, on behalf of MAGNIFY-MS Study Group Neurol Neuroimmunol Neuroinflamm 2023;10:e200048. doi:10.1212/NXI.0000000000200048 Abstract Background and Objectives Cladribine tablets cause a reduction in lymphocytes with a predominant effect on B-cell and T-cell counts. The MAGNIFY-MS substudy reports the dynamic changes on multiple peripheral blood mononuclear cell (PBMC) subtypes and immunoglobulin (Ig) levels over 12 months after the first course of cladribine tablets in patients with highly active relapsing multiple sclerosis (MS). Methods Immunophenotyping was performed at baseline (predose) and at the end of months 1, 2, 3, 6, and 12 after initiating treatment with cladribine tablets. Assessments included lymphocyte subtype counts of CD19+ B cells, CD4+ and CD8+ T cells, CD16+ natural killer cells, plasmablasts, and Igs. Immune cell subtypes were analyzed by flow cytometry, and serum IgG and IgM were analyzed by nephelometric assay. Absolute cell counts and percentage change from baseline were assessed. Results The full analysis set included 57 patients. Rapid reductions in median CD19+, CD20+, memory, activated, and naive B-cell counts were detected, reaching nadir by month 2. Thereafter, total CD19+, CD20+, and naive B-cell counts subsequently reconstituted, but memory B cells remained reduced by 93%–87% for the remainder of the study. The decrease in plasmablasts was slower, reaching nadir at month 3. Decrease in T-cell subtypes was also slower and more moderate compared with B-cell subtypes, reaching nadir between months 3 and 6. IgG and IgM levels remained within the normal range over the 12-month study period. Discussion Cladribine tablets induce a specific pattern of early and sustained PBMC subtype dynamics in the absence of relevant Ig changes: While total B cells were reduced dramatically, T cells were *Kristina H. Holmberg was an employee of EMD Serono, Billerica, MA. From the Department of Neurology with Institute of Translational Neurology (H.W.), University of Münster, Germany and Brain and Mind Center, University of Sydney, Australia; The Blizard Institute (K.S.), Centre for Neuroscience, Surgery & Trauma, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, UK; Clinical Board Medicine (Neuroscience) (K.S.), The Royal London Hospital, Barts Health NHS Trust, UK; Ingham Institute for Applied Medical Research (S.H.), University of New South Wales Medicine, Sydney, Australia; Department of Neurology (T.D.), University Hospital Basel, Switzerland; Department of Neurology (Andrew Chan), Inselspital, Bern University Hospital, University of Bern, Switzerland; Danish MS Center (F.S.), Department of Neurology, Copenhagen University Hospital–Rigshospitalet, Glostrup, Denmark; Department of Clinical Medicine (F.S.), University of Copenhagen, Denmark; Multiple Sclerosis Center (A.A.), Sheba Academic Medical Center, Ramat Gan, Israel; Sackler School of Medicine (A.A.), Tel-Aviv University, Israel; Department of Neurology-Neuroimmunology (X.M.), Centre d’Esclerosi Múltiple de Catalunya (Cemcat), Hospital Universitari Vall d’Hebron, Universitat Autonoma de Barcelona, Spain; Department of Neurosciences and CRCHUM (A.P.), Université de Montréal, QC, Canada; Department of Neurological and Behavioural Sciences (N.D.S.), University of Siena, Italy; Department of Radiology (F.B.), VU University Medical Center, Amsterdam, The Netherlands; UCL Institute of Neurology (F.B.), London, UK; Experimental Neurophysiology Unit (L.L.), Vita-Salute San Raffaele University, Milan, Italy; Univ. Lille (P.V.), Inserm U1172 LilNCog, CHU Lille, FHU Precise, France; Cytel Inc (Anita Chudecka), Geneva, Switzerland; InScience Communications (C.M.), Springer Healthcare Ltd, Chester, UK; EMD Serono (K.H.H.), Billerica, MA; and Ares Trading SA (U.B., S.R.), Eysins, Switzerland, an affiliate of Merck KGaA, Darmstadt, Germany. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The article processing charge was funded by the healthcare business of Merck KGaA, Darmstadt, Germany. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary DMTs = disease-modifying therapies; EDSS = Expanded Disability Status Scale; Ig = immunoglobulin; MS = multiple sclerosis; NK = natural killer; PBMC = peripheral blood mononuclear cell; TEMRA = terminally differentiated effector memory RA+; Th = T helper; Treg = T regulatory. affected significantly less. Naive B cells recovered toward baseline, naive CD4 and CD8 T cells did not, and memory B cells remained reduced. The results help to explain the unique immune depletion and repopulation architecture regarding onset of action and durability of effects of cladribine tablets while largely maintaining immune competence. Trial Registration Information ClinicalTrials.gov Identifier: NCT03364036. Date registered: December 06, 2017. Multiple sclerosis (MS) is a chronic inflammatory disease of the relapsing MS. The substudy was part of MAGNIFY-MS CNS for which several disease-modifying therapies (DMTs) are (NCT03364036), which has previously shown significant available to reduce the risk of relapse and worsening disability. reductions in active MRI lesions from month 2 (day 60) Although the pathogenesis of MS is not fully established, nu- onward after treatment initiation with cladribine tablets.19 merous immune cell subtypes are believed to play a role in demyelination and neuroaxonal damage.1,2 Indeed, MS has been historically associated with T cells, specifically T helper cells.3 Methods However, more recently there is increasing evidence of the Study Design and Participants contribution of B cells,4-6 microglial cells,7,8 and potentially A 2-year Prospective Study to Evaluate the Onset of Action of natural killer (NK) cells9-11 in the pathogenesis of MS. Mavenclad in Subjects With Highly Active Relapsing Multiple Sclerosis (MAGNIFY-MS) (NCT03364036) was a 2-year, Current generation DMTs for MS include a range of immune phase IV, open-label, single-arm study in which eligible patients depletion and repopulating agents.12 Such treatments include are scheduled to receive cladribine tablets 3.5 mg/kg cumula- cladribine tablets 10 mg (MAVENCLAD, the healthcare business tive dose over 2 years (eFigure 1, http://links.lww.com/NXI/ of Merck KGaA, Darmstadt, Germany; 3.5 mg/kg cumulative A759). Patients receive 2 weeks of active treatment per course dose over 2 years), a highly efficacious DMT that is approved for (week 1 and week 5 of each year), the start of the first week of the treatment of MS in >80 countries worldwide. Evidence sug- treatment in year 1 being considered baseline. gests that cladribine tablets act as an immune reconstitution therapy, causing a reduction of lymphocytes with predominance Patients aged 18 years or older, with an Expanded Disability Status in B-cell and T-cell counts followed by reconstitution occurring at Scale (EDSS) score ≤5 and a diagnosis of highly active relapsing different times and rates according to cell subtype.13-15 While MS were enrolled between May 28, 2018, and April 23, 2019. In cladribine tablets selectively reduce adaptive immune cell counts, this study, highly active relapsing MS was defined as 1 relapse in the effects on the innate immune system are negligible.16 the previous year and ≥1 T1 gadolinium enhancing (Gd+) lesion or ≥9 T2 lesions while on treatment with another DMT or ≥2 Some long-term evaluations of the effect of treatment with cla- relapses in the previous year whether on DMT or not. dribine tablets on peripheral blood mononuclear cell (PBMC) subtype dynamics have been completed in previous clinical trials Patients were excluded if they had previous exposure to DMT (CLARITY [CLAdRIbine Tablets treating multiple sclerosis (fingolimod, natalizumab, alemtuzumab, mitoxantrone, or orallY], CLARITY Extension, Prospective observational long- ocrelizumab); a lymphocyte count not within normal labo- term safety registry of multiple sclerosis patients who have parti- ratory limits; presence of signs of progressive multifocal leu- cipated in cladribine clinical trials [PREMIERE] and the oral koencephalopathy; tested positive for HIV, hepatitis B or C, cladribine for early multiple sclerosis trial [ORACLE-MS]).17,18 or active/latent tuberculosis; had an active malignancy or had However, the results described only major cell types and effects an allergy or hypersensitivity to gadolinium; and/or any other of cladribine tablets on a range of immune cell subtypes while contraindication to perform MRI. a deeper immune phenotyping was not determined in these studies. Peripheral Blood Sampling This exploratory substudy of MAGNIFY-MS involved a In this study, as part of an exploratory substudy, our research longitudinal evaluation of PBMCs, as summarized in eFig- question concerned PBMC subtype dynamics and immuno- ure1, http://links.lww.com/NXI/A759. Immunophenotyp- globulin (Ig) levels during the first 12 months after patients ing was performed on blood samples collected at baseline had initiated treatment with cladribine tablets for highly active (predose) and at the end of months 3, 6, and 12. Further 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN immunophenotyping was completed at the end of months 1 and 2 for the full B-cell panel, CD4+ and CD8+ T cells, and Table 1 Demographics and Characteristics of Patients CD16+ NK cells. Immune cell subtypes were analyzed by flow Included in This Substudy of MAGNIFY-MS and the cytometry (Becton Dickinson FACSCanto II cell analyzer) and Overall Population From the MAGNIFY-MS Study were detected using surface cell markers; details are provided Total in eAppendix1, http://links.lww.com/NXI/A759. For the MAGNIFY-MS MAGNIFY-MS substudy population purposes of immunophenotyping, CD19+ B cells were ana- population (N = 57) (N = 270) lyzed as part of the TBNK cell panel while B cell subtypes were Female, n (%) 35 (61.4) 180 (66.7) analyzed as part of the full B-cell panel. Age in y, n (%) In parallel, serum levels of IgG and IgM were analyzed by nephe- ≤40 35 (61.4) 152 (56.3) lometric assay (eAppendix1, http://links.lww.com/NXI/A759). >40 22 (38.6) 118 (43.7) Absolute cell counts and median percentage change from Time since onset of MS in mo, 84.81 ± 93.54 84.9 ± 85.46 mean ± SD baseline were assessed for cell subtypes and Igs. Time since diagnosis in mo, 52.55 ± 67.45 60.86 ± 74.48 Statistical Analysis mean ± SD The analysis of PBMC cell subtypes was an exploratory Time since first relapse in mo, 52.62 ± 80.75 54.4 ± 72.57 substudy of MAGNIFY-MS, and thus, demographical and mean ± SD safety data for the full study cohort were analyzed de- No. of relapses within 12 mo scriptively with no formal statistical testing. All patients who before baseline, n (%) received ≥1 dose of cladribine tablets were included in this 0 2 (3.5) 3 (1.1) analysis. 1 15 (26.3) 103 (38.1) The study protocol and statistical analysis plan for MAGNIFY-MS 2 29 (50.9) 133 (49.3) have been published on ClinicalTrials.gov (NCT03364036).20 >2 11 (19.3) 31 (11.5) Standard Protocol Approvals, Registrations, EDSS score at baseline, n (%) and Patient Consents ≤3 42 (73.7) 204 (75.6) Ethical approval for the MAGNIFY-MS study (NCT03364036) was obtained from independent ethics committees for each trial >3 15 (26.3) 66 (24.4) site, and the study was performed in line with the principles of Median (range) 2.5 (0.0–5.0) 2.0 (0–5) the Declaration of Helsinki. All participants provided written No. of previous DMTs,a n (%) informed consent before participation in the study. 0 30 (52.6) 117 (43.3) Data Availability 1 13 (22.8) 88 (32.6) Any requests for data by qualified scientific and medical re- searchers for legitimate research purposes will be subject to the 2 10 (17.5) 50 (18.5) Data Sharing Policy of the healthcare business of Merck KGaA, >2 4 (7.0) 15 (5.6) Darmstadt, Germany. All requests should be submitted in writing Patients with ≥1 T1 Gd + 24 (42.1) 141 (52.2) to the data sharing portal of the healthcare business of Merck lesion during the baseline KGaA, Darmstadt, Germany, emdgroup.com/en/research/our- period,b n (%) approach-to-research-and-development/healthcare/clinical-trials/ Missing, n (%) 3 (5.3) 12 (4.4) commitment-responsible-data-sharing.html. When the healthcare business of Merck KGaA, Darmstadt, Germany, has a coresearch, Patients with ≥1 active T2 lesion 8 (14.0) 48 (17.8) (without T1 Gd+) during the codevelopment, or comarketing or copromotion agreement, or baseline period,b n (%) when the product has been out-licensed, the responsibility for Missing, n (%) 4 (7.0) 14 (5.2) disclosure might be dependent on the agreement between parties. Under these circumstances, the healthcare business of Merck Abbreviations: DMT = disease-modifying therapy; EDSS = Expanded Dis- KGaA, Darmstadt, Germany, will endeavor to gain agreement to ability Status Scale; Gd+ = gadolinium enhancing; MS = multiple sclerosis. a Prior DMTs include one or more of the following first-line treatments: di- share data in response to requests. methyl fumarate, glatiramer acetate, interferons, and teriflunomide. b The baseline period is defined as the time between the screening visit and baseline visit (median period 36.0 [range 21–91] days). Missing data were due to MRIs not performed at site, not evaluable, or excluded due to steroid treatment. Results Between May 28, 2018, and April 23, 2019, a total of 70 cladribine tablets (13 patients were screening failures). The patients were screened for enrollment to this MAGNIFY-MS treated patient population were mostly aged 40 years or substudy and 57 of these patients initiated treatment with younger (61.4%; median 37.0 [range 20–60] years), were Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 3 Figure 1 Median Percentage Change From Baseline of B-Cell and T-Cell Subtypes CM = central memory; EffM = effector memory; NK = natural killer; TEMRA = terminally differen- tiated effector memory RA+; Th = T helper. predominantly female (61.4%), and had a median EDSS score recovery toward baseline from month 3 onward and largely of 2.5 at baseline. Patient demographics and characteristics shared similar repopulation profiles, with a reduction in median from this MAGNIFY-MS substudy are shown in Table 1 and percentage changes between −34% and −45% at month 12. indicate that the population from this substudy was similar to Naive B cells also reached nadir at month 2 (median percentage the overall population of the MAGNIFY-MS study. change: −90%) but recovered to near baseline levels at month 12 (median percentage change: −5%). Changes in the Adaptive Immune Components While most B-cell subtypes reached nadir by month 2 after B-Cell Subtypes treatment, CD38+ plasmablasts and short-lived plasma cells CD19+, CD20+, memory, naive, and activated (CD69+) B cells reached nadir at month 3 (median percentage change: −71% were reduced early on during treatment, as shown by median and −78%, respectively) with both subtypes reaching a me- percentage changes from baseline to month 1 of −77%, −78%, dian percentage change of −51% at month 12. −74%, −80%, and −64%, respectively (Figures 1–3). Following nadir at month 2, median percentage change of CD19+ After the 3-month time point, the reduction in memory B cells (−90%), CD20+ (−91%), and CD69+ (−81%) B cells showed was sustained to month 12 (median percentage change: 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN The greatest reduction in cell count for CD4+ T helper (Th)1 + + + Figure 2 Median Values of CD19 and CD20 B Cell, CD4 and CD4+ Th17 cells occurred at month 3, with median per- and CD8+ T Cell, and CD16+/CD56+ Natural Killer centage changes from baseline of −51% and −35%, respectively. (NK) Cell Counts Where Additional Immunophe- notyping Was Performed at Months 1 and 2, in Patients Treated With Cladribine Tablets While we have seen naive B cells recover toward baseline, naive CD4 and CD8 T cells did not. Naive CD4+ and CD8+ T cells showed the greatest median percentage change from baseline at month 6 (−64% and −70%, respectively), followed by some recovery toward baseline at month 12. CD4+ T regulatory (Treg) cells showed a slower and less profound decrease from baseline compared with other T-cell subtypes, reaching the lowest level of median percentage change of −38% at month 6. Thereafter, Treg counts showed a slight recovery toward baseline at month 12. CD8+ terminally differentiated effector memory RA+ (TEMRA) T cells showed the greatest median percentage change from baseline at month 3 (−26%), followed by a recovery of +11% from month 3 to 6. This was followed by a slight decline of −4% The first treatment course of cladribine tablets was administered at baseline and month 1. Data for CD19+ and CD20+ B cells have been slightly offset at at month 12. each time point to improve readability as data points are overlapping. BL = baseline; Q = quartile. Changes in NK Cells Changes in CD16+/CD56+ NK cell counts showed a median percentage decrease at month 1 of −34% with nadir reached at −87%). Reductions in B regulatory (Breg) and B transitional month 2, as represented by a median percentage change from (Btrans) cells were relatively rapid but less profound at month 1 baseline of −40% (Figures 1, 3, and 4). Recovery of these NK (median percentage change: −45% and −61%, respectively), cells toward baseline occurred between months 3 and 12. with Breg beginning to repopulate from month 2 (median CD16bright CD56dim NK cell counts were decreased at month 3 percentage change: −16%). By month 3, cell counts for both (median percentage change: −40%) and remained below base- Breg and Btrans cell subtypes had recovered and were increased line level at month 12 (median percentage change: −12%), while over baseline levels (median percentage change: Bregs +176%; no effect on CD16low CD56bright NK cell counts was noted. Btrans +28%). Breg and Btrans cell numbers continued to show a Impact on Igs sustained increase over baseline levels up to month 12, po- Throughout the study period, serum IgG and IgM levels tentially increasing the ratio of regulatory vs effector B-cell remained within the normal ranges of 5.65–17.65 g/L and subtypes from month 2 and 3 onward. 0.40–2.30 g/L, respectively (eFigure 2, http://links.lww. T-Cell Subtypes com/NXI/A759); however, it should be noted that significant While total B cells were reduced dramatically, T cells were less differences were not expected during the short 12-month affected. CD4+ and CD8+ T cells showed a slower and more observation period. moderate reduction in cell counts compared with B-cell sub- types, as represented by a smaller median percentage change from baseline to month 1 (−22% and −18%, respectively) while Discussion nadir was reached at month 3 followed by a slight recovery The primary analysis of the MAGNIFY-MS study, reported toward baseline (Figures 1–3). elsewhere, was conducted to determine the onset of action of cladribine tablets by observing changes in MRI lesion counts.19 For CD4+ and CD8+ central memory and effector memory In this study, we studied dynamic changes of various cellular T cells, the greatest median percentage change from baseline and noncellular immune components. Cladribine induces a was reached between months 3 and 6. CD4+ central and ef- specific pattern of early and sustained PBMC subtype dynamics fector memory cells showed the greatest median percentage in the absence of relevant Ig changes: While total B cells were change from baseline of −56% and −35% at month 3, re- reduced dramatically, T cells were affected significantly less. spectively, with nadir for effector memory cells sustained into While naive B cells recovered toward baseline, naive CD4 and month 6. CD8+ central and effector memory cells showed the CD8 T cells did not and memory B cells remained reduced greatest median percentage change from baseline of −46% and until month 12. The results from this substudy help to explain −22% at month 6, respectively. All CD4+ and CD8+ central and the unique immune depletion and repopulation architecture effector memory T-cell subtypes showed recovery toward regarding the onset of action and durability of effects of cla- baseline at month 12. dribine tablets while largely maintaining immune competence. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 5 Figure 3 Median Values of B-Cell and T-Cell Counts in Patients Treated With Cladribine Tablets The first treatment course of cladribine tablets was administered at baseline and month 1. BL = baseline; Q = quartile; TEMRA = terminally differentiated effector memory RA+; Th = T helper. The immunologic durability of cladribine tablets beyond this studies that have reported on dynamics of major cell types first year is still to be assessed as part of the full 2-year study during treatment with cladribine tablets 3.5 mg/kg cumulative findings of MAGNIFY-MS. Such results will add to previous dose over 2 years.17,18 Figure 4 Median Values of Natural Killer (NK) Cell Counts in Patients Treated With Cladribine Tablets The first treatment course of cladribine tablets was admin- istered at baseline and month 1. BL = baseline; Q = quartile. 6 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN While we observed a fast and profound reduction in B-cell Ig reduction at 4 weeks.32 Consequently, it is likely that long- subtype counts with cladribine tablets (months 1 and 2), these lived CD138+ plasma cells would not be affected by treatment cell counts recovered well in the following months. By con- with cladribine tablets given the unchanged profiles of total trast, memory B cells remained close to their lower nadir level serum IgG and IgM up to 12 months, although this subtype to month 12. This confirms the findings from a small obser- was not evaluated in our substudy. vational study of Italian patients treated with cladribine tab- lets21 and those from a cross-sectional study of people with Breg and Btrans cell subtypes contribute to the maintenance of MS treated with a different preparation of cladribine.22 The immune tolerance and the modulation of immune strong association between the time course of B-cell subtype responses.33,34 A variety of B-cell subtypes have been docu- depletion and the apparent inhibition of new lesion evolution mented as Breg cells, with CD19+CD24hiCD38hi B cells being on MRI demonstrated in MAGNIFY-MS, as well as the sus- identified as an interleukin (IL)-10–expressing subtype that tained and significant reduction in memory B cells over at may participate in suppressing the increased autoimmune re- least 12 months, suggest an important role of this specific sponses observed in MS. Breg cells have not yet been studied for B-cell subset for sustained disease control in people with all MS DMTs; however, some recent data have shown that after MS.5,23 Monoclonal B-cell depleting antibodies such as alemtuzumab treatment levels of CD19+CD24hiCD38hi, Breg alemtuzumab,24 ocrelizumab,25 or rituximab26 (although the cells are reduced after severe relapses and subsequently repo- latter is not licensed, it is commonly used off-label in people pulate during recovery.35,36 In this substudy, Btrans cells showed with MS)27 lead to similar patterns of rapid and general B-cell a similar repopulation profile to Breg cells. These Btrans cells depletion followed by reconstitution of B-cell subsets, except represent one of the Breg cell subpopulations in healthy indi- for memory B cells. Even with alemtuzumab, although tar- viduals, but the frequency of such cells in circulation may be geting a broad range of T and B lymphocyte subsets, a distinct altered in individuals with MS.34 Btrans cells can also produce pattern of B-cell lysis followed by reconstitution (again, with IL-10, as well as transforming growth factor-β, and can regulate the exception of memory B cells) can be observed.24 CD4+ T-cell proliferation and differentiation toward Th ef- fector cells. Given the sustained decrease of mature B-cell The PBMC subtype dynamics identified in the MAGNIFY- subtypes, especially memory B cells, along with an increase in MS study may not only explain how cladribine tablets effec- Breg and Btrans cells in the first year after treatment with cla- tively reduce MS disease activity and disability accrual but also dribine tablets, it is believed that an increased Breg/B cell indicate why patients treated with cladribine tablets largely (Btrans/B cell) or Breg/B memory (Btrans/B memory) ratio retain immune competence and thus may be able to mount a might contribute to the prolonged clinical efficacy of cladribine relevant vaccine response, such as that seen in response to tablets. Future analyses may determine the intracellular cyto- COVID-19 mRNA vaccines.28 While there was a decrease in kine expression and the Breg vs B effector subtype ratios and B-cell subtypes as early as month 1 after initiating treatment their correlation with clinical efficacy end points. with cladribine tablets, naive B cells started to recover from month 2 and reached near baseline levels at month 12. It is At month 12, the total B-cell count after treatment with these naive B cells that are associated with early immune cladribine tablets was generally more than 30% below responses and the generation of antibodies in response to new baseline levels. At this same time point, an overshoot of the immune challenges (e.g., infections, vaccination). Indeed, total B-cell count of about 30% above baseline has been another substudy of the MAGNIFY-MS population has reported in the pivotal studies of alemtuzumab.24,37 How- reported a favorable humoral response to varicella zoster virus ever, our analysis of the MAGNIFY-MS data reported here and influenza vaccines, including patients vaccinated against revealed that while B memory cells remain depleted, Breg and influenza shortly after initiating treatment with cladribine Btrans cells exceed their baseline values by month 3 and re- tablets.29 main elevated above baseline at month 12 at +50% and +36%, respectively. Hence, rebound and overshoot of B-cell The results also show that cladribine is able to directly affect subsets alone may not sufficiently explain why secondary and reduce plasmablasts, which may be associated with dis- B-cell autoimmunities do not seem to occur with cladribine ease pathology, by means of autoantibody production and tablets, yet are so common after treatment with alemtuzu- direct cellular toxicity within the CSF.30 Most current DMTs mab. The reported secondary autoimmunities reported for the treatment of MS do not target the plasmablast and for alemtuzumab include glomerulonephritis, immune plasma cell subtypes directly (including CD20 B cell depleting thrombocytopenia, sarcoidosis, thyroid autoimmunity, and agents),25 a cell subset potentially indicative of more severe vitiligo.38 MS disease.31 As previously suggested, the less pronounced effects of cla- Despite the sustained decrease of memory B cells and a de- dribine on T cells, particularly during the first few months crease in CD38+ plasmablasts in the first 12 months of after treatment, may be key, alongside the described immune MAGNIFY-MS, serum IgG and IgM levels remained within cell dynamics of specific B-cell subsets, for the protection from the normal range over this period. Atacicept, which was secondary autoimmunity with cladribine tablets.24 A recent shown to target long-lived plasma cells, shows a fast onset of in-depth immune phenotyping study38 provided further Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 7 evidence of alemtuzumab-induced changes to lymphocytes most closely associated with the effects of cladribine detected and the underlying immunologic processes involved in sec- on MRI and clinical outcomes.13,17 The contribution of ondary autoimmunity. Autoimmunity after treatment with moderate reductions across T-cell subtypes, as well as the alemtuzumab is associated with homeostatic proliferation of sustained increase of regulatory and transitional B cells from T cells, and it has been suggested that this occurs in the month 3 onward, are less clear. While they might contribute context of a defective thymus.39 However, antibody-mediated to the long-term therapeutic effect of cladribine tablets, the T-cell depletion typically leads to expansion of memory limited reduction of T-cell subsets is likely advantageous for cells and is not associated with the development of B-cell safety. In the context of safety, it should also be noted that, autoimmunities.24,40,41 It is therefore hypothesized that in the despite the sustained decrease of memory B cells and a de- context of B-cell depletion and recovery with alemtuzumab, the crease in CD38+ plasma cells, no relevant changes in serum associated depletion of Treg cells, normally silencing autoreactive IgG and IgM concentrations were observed over the 12- immature B cells exiting the bone marrow as they mature into month study period. naive B cells,42,43 facilitates secondary autoimmunity.24,44,45 Be- cause autoantibody production is likely to be CD4+ T-cell– Acknowledgment dependent, autoimmunity will require T-cell support, and thus, The authors thank patients and their families, investigators, autoimmunity is unlikely to occur until CD4+ T-cell numbers co-investigators, and study teams at each of the participating recover, explaining the delay between the time of B-cell hyper- centers. reactivity and the development of secondary autoimmunity.24 Study Funding The reduction and repopulation profile for CD8+ T cells was This work was supported by the healthcare business of Merck comparable with CD4+ T cells for both alemtuzumab and cla- KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/ dribine, respectively. A further, more recent study has also shown 100009945). alemtuzumab to decrease CD4+ and CD8+ T-cell counts fol- lowed by a period of repopulation, with CD8+ T cells increasing Disclosure above the lower limit of normal at 12 months after treatment.36 H. Wiendl is member of scientific advisory boards/steering For CD8+ T cells, there was a small percentage decrease from committees for Bayer, Biogen, the healthcare business of baseline to week 2, which was then maintained throughout the Merck KGaA (Darmstadt, Germany), Novartis, Roche, study period. In addition to the greater reduction of T cells with Sanofi, and Teva. He received speaker honoraria and travel alemtuzumab, CD4+ effector memory and CD8+ TEMRA support from Bayer, Biogen, CSL Behring, EMD Serono, T cells have been shown to be substantially expanded during the Fresenius Medical Care, Genzyme, the healthcare business of first 6 months after treatment.39 Such clonal expansion has not Merck KGaA (Darmstadt, Germany), Omniamed, Novartis, been observed with cladribine tablets. Sanofi, and Teva. He received compensation as a consultant from Biogen, the healthcare business of Merck KGaA Initial analysis of the full 2-year safety findings from the present (Darmstadt, Germany), Novartis, Omniamed, Roche, and MAGNIFY-MS study have not raised such concerns for cla- Sanofi. He has received research support from Bayer, Biogen, dribine tablets; indeed, no treatment-related serious AEs were the healthcare business of Merck KGaA (Darmstadt, Ger- reported (data on file; the healthcare business of Merck KGaA, many), Novartis, Sanofi, and Teva, as well as German Ministry Darmstadt, Germany). Regarding lymphopenia, very few pa- for Education and Research (BMBF), German Research tients experienced grade 4 events (0.7%), with 24.4% and Foundation (DFG), Else Kröner Fresenius Foundation, 54.4% of patients, respectively, experiencing grade 3 and grade Fresenius Foundation, Hertie Foundation, NRW Ministry of 1 or 2 lymphopenia (data on file; the healthcare business of Education and Research, Interdisciplinary Center for Clinical Merck KGaA, Darmstadt, Germany). Studies (IZKF) Muenster, and RE Children’s Foundation; K. Schmierer has received research support from Biogen, the While PBMC counts in this study were measured in peripheral healthcare business of Merck KGaA (Darmstadt, Germany), blood samples, it should be remembered that cladribine pen- and Novartis; speaking honoraria from, and/or served in an etrates into other tissues including the CNS shortly after ad- advisory role for, Amgen, Biogen, EMD Serono, the health- ministration,46 raising the possibility of direct effects on CNS care business of Merck KGaA (Darmstadt, Germany), resident lymphocyte subtypes. Further studies to explore these Novartis, Roche, Sanofi, and Teva; and remuneration for potential effects are under way.47 teaching activities from AcadeMe, Medscape, and the Neu- rology Academy; S. Hodgkinson serves on advisory boards for One shortcoming of this study is that we did not directly Bayer, Biogen, the healthcare business of Merck KGaA correlate the MRI findings with the PBMC subtype dynamics (Darmstadt, Germany), Novartis, Roche, and Sanofi; she has reported here. Our data, however, suggest an early and sub- received money for travel and speaker honoraria from Bayer, sequent sustained effect of cladribine tablets in a specific Biogen, the healthcare business of Merck KGaA (Darmstadt, pattern of depletion and reconstitution of B-cell and T-cell Germany), Novartis, Roche, and Sanofi; T. Derfuss serves on subtypes. The pronounced reduction of B-cell subsets from scientific advisory boards for Bayer, Biogen, GeNeuro, Med- month 2 and the sustained depletion of memory B cells seem Day, the healthcare business of Merck KGaA (Darmstadt, 8 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN Germany), Mitsubishi Pharma, Novartis Pharmaceuticals, support from Biogen, the healthcare business of Merck KGaA Roche, and Sanofi; has received funding for travel and/or (Darmstadt, Germany), and Novartis; P. Vermersch has re- speaker honoraria from Bayer, Biogen, the healthcare business ceived honoraria or consulting fees from AB Science, Biogen, of Merck KGaA (Darmstadt, Germany), Novartis Pharma- Celgene (BMS), Imcyse, the healthcare business of Merck ceuticals, Roche, and Sanofi; and receives research support KGaA (Darmstadt, Germany), Novartis, Roche, Sanofi, and from Biogen, the European Union, Novartis Pharma, the Teva and research support from Novartis, Roche, and Sanofi; Swiss MS Society, and the Swiss National Foundation; A. A. Chudecka is an employee of Cytel Inc., Geneva Branch, Chan has received speakers’/board honoraria from Actelion Switzerland, and was funded by the healthcare business of (Janssen/J&J), Almirall, Bayer, Biogen, Celgene (BMS), the Merck KGaA (Darmstadt, Germany) to perform statistical healthcare business of Merck KGaA (Darmstadt, Germany), analyses for this study; K.H. Holmberg was an employee of Novartis, Roche, Sanofi, and Teva, all for hospital research EMD Serono, Billerica, MA, at the time the study was con- funds and research support from Biogen, Sanofi, and UCB; F. ducted and is currently an employee of Biogen, Cambridge, Sellebjerg has served on scientific advisory boards, been on MA; C. Mwape is an employee of inScience Communications, the steering committees of clinical trials, served as a consul- Springer Healthcare Ltd., Chester, UK. inScience Commu- tant, received support for congress participation, received nications received funding from the healthcare business of speaker honoraria, or received research support for his labo- Merck KGaA Darmstadt, Germany, for medical writing as- ratory from Biogen, EMD Serono, the healthcare business of sistance for this article; U. Boschert and S. Roy are employees Merck KGaA (Darmstadt, Germany), Novartis, Roche, of Ares Trading SA, Eysins, Switzerland, an affiliate of Merck Sanofi, and Teva; A. Achiron has received over the last 5 years’ KGaA, Darmstadt, Germany. Go to Neurology.org/NN for honoraria or consulting fees for participating in advisory full disclosure. boards related to clinical trials design, trial steering commit- tees, and data and safety monitoring committees from Biogen, Publication History BMS, Novartis, the healthcare business of Merck KGaA Received by Neurology: Neuroimmunology & Neuroinflammation (Darmstadt, Germany), Roche, and Sanofi and research January 28, 2022. Accepted in final form September 6, 2022. Submitted support for investigator-initiated trials and MS patients’ and externally peer reviewed. The handling editor was Friedemann Paul, benefits activities from Biogen, BMS, the healthcare business MD. of Merck KGaA (Darmstadt, Germany), Novartis, Roche, and Sanofi; X. Montalban has received speaking honoraria and travel expenses for participation in scientific meetings, has been a steering committee member of clinical trials or par- Appendix Authors ticipated in advisory boards of clinical trials in the past years with Abbvie, Actelion (Janssen/J&J), Alexion, Bayer, Biogen, Name Location Contribution Celgene (BMS), EMD Serono, Immunic, Janssen (J&J), Heinz Department of Neurology Drafting/revision of the MedDay, the healthcare business of Merck KGaA (Darm- Wiendl, MD, with Institute of manuscript for content, PhD Translational Neurology, including medical writing for stadt, Germany), Mylan, Nervgen, Novartis, Roche, Sandoz, University of Münster, content; major role in the Sanofi, Teva, TG Therapeutics, Excemed, MSIF, and NMSS; Münster, Germany acquisition of data; study concept or design; analysis A. Prat has received speaking honoraria and travel expenses or interpretation of data for participation in scientific meetings, has been a steering Klaus The Blizard Institute, Centre Drafting/revision of the committee member of clinical trials or participated in advisory Schmierer, for Neuroscience, Surgery & manuscript for content, boards of clinical trials in the past years, and/or received MD, PhD Trauma, Barts and The including medical writing for operating grants from Alexion, Bayer, Biogen, Celgene London School of Medicine & content; study concept or Dentistry, Queen Mary design; analysis or (BMS), EMD Serono, Billerica, MA, Novartis, Roche, Sanofi, University of London, interpretation of data and Teva; N. De Stefano is a consultant for Bayer, Biogen, the London, UK; Clinical Board Medicine (Neuroscience), healthcare business of Merck KGaA (Darmstadt, Germany), The Royal London Hospital, Novartis, Roche, Sanofi, and Teva; has grants or grants Barts Health NHS Trust, London, UK pending from FISM and Novartis; is on the speakers’ bureaus of Biogen, the healthcare business of Merck KGaA (Darm- Suzanne Ingham Institute for Applied Drafting/revision of the Hodgkinson, Medical Research, University manuscript for stadt, Germany), Novartis, Roche, Sanofi, and Teva; and has MBBS, PhD of New South Wales content, including received travel funds from the healthcare business of Merck Medicine, Sydney, NSW, medical writing for Australia content; study concept KGaA (Darmstadt, Germany), Novartis, Roche, Sanofi, and or design; analysis or Teva; F. Barkhof is supported by the NIHR Biomedical Re- interpretation of data search Centre at UCLH and is a consultant to Biogen, Tobias Department of Neurology, Drafting/revision Combinostics, IXICO, the healthcare business of Merck Derfuss, MD University Hospital Basel, of the manuscript for Basel, Switzerland content, including KGaA (Darmstadt, Germany), and Roche; L. Leocani has medical writing for received honoraria for consulting services or speaking activi- content; study concept or ties from Biogen, Novartis, the healthcare business of Merck design; analysis or interpretation of data KGaA (Darmstadt, Germany), and Roche and research Continued Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 9 Appendix (continued) Appendix (continued) Name Location Contribution Name Location Contribution Andrew Department of Neurology, Drafting/revision of the Claire inScience Communications, Drafting/revision of the Chan, MD Inselspital, Bern University manuscript for content, Mwape, PhD Springer Healthcare Ltd., manuscript for content, Hospital, University of Bern, including medical writing for Chester, UK including medical writing for Bern, Switzerland content; study concept or content design; analysis or interpretation of data Kristina H. EMD Serono, Billerica, MA Drafting/revision of the Holmberg, manuscript for content, Finn Danish MS Center, Drafting/revision of the PhD including medical writing for Sellebjerg, Department of Neurology, manuscript for content, content; analysis or PhD, MD Copenhagen University including medical writing for interpretation of data Hospital–Rigshospitalet, content; study concept or Glostrup, Denmark; design; analysis or Ursula Ares Trading SA, Eysins, Drafting/revision of the Department of Clinical interpretation of data Boschert, Switzerland, an affiliate of manuscript for content, Medicine, University of PhD Merck KGaA, Darmstadt, including medical writing for Copenhagen, Copenhagen, Germany content; study concept or Denmark design; analysis or interpretation of data Anat Multiple Sclerosis Center, Drafting/revision of the Achiron, MD, Sheba Academic Medical manuscript for content, Sanjeev Roy, Ares Trading SA, Eysins, Drafting/revision of the PhD Center, Ramat Gan, Israel; including medical writing for MBBS, MD Switzerland, an affiliate of manuscript for content, Sackler School of Medicine, content; major role in the Merck KGaA, Darmstadt, including medical writing for Tel-Aviv University, Israel acquisition of data; study Germany content; major role in the concept or design; analysis acquisition of data; study or interpretation of data concept or design; analysis or interpretation of data Xavier Department of Neurology- Drafting/revision of the Montalban, Neuroimmunology, Centre manuscript for content, MD, PhD d’Esclerosi Múltiple de including medical writing for Catalunya (Cemcat), content; study concept or References Hospital Universitari Vall design; analysis or 1. 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