Benvenuto, F

Gandoglia, I

Ivaldi, F

Kerlero de Rosbo, N

Laroni, A

Mancardi, G

Solaro, C

Uccelli, A.

Neurology: Neuroimmunology & Neuroinflammation

English

Gandoglia I

Ivaldi F

Laroni A

Benvenuto F

Solaro C

Mancardi G

Kerlero de Rosbo N

Uccelli A.

Archivio istituzionale della ricerca - Università di Genova

Ilaria Gandoglia, MDFederico IvaldiAlice Laroni, MD, PhDFederica Benvenuto, PhDClaudio Solaro, MDGianluigi Mancardi, MDNicole Kerlero de Rosbo,PhD*Antonio Uccelli, MD*Correspondence toDr. Uccelli:auccelli@neurologia.unige.itTeriflunomide treatment reduces B cells inpatients with MSABSTRACTObjective: To study the immunomodulatory effect of teriflunomide on innate and adaptive immunecell populations through a pilot, open-label, observational study in a cohort of patients withrelapsing-remitting MS.Methods: Blood lymphocytes were isolated from 10 patients with MS before and after 3 or 12months of treatment. Adaptive and innate immune cell subsets were analyzed by flow cytometryas follows: B cells (memory, regulatory, and mature subsets), T cells (effector and regulatory sub-sets), and natural killer (NK) cells (CD56dim and CD56bright subsets).Results: Our results show that teriflunomide significantly reduces absolute counts of totalCD191 B cells and mature and regulatory B-cell subsets. T cells were affected to a lesser extent,with a trend in reduction of absolute counts for both T effector CD41 cells (Th1, Th17 and Th1/17) and T regulatory CD81 and CD41 cells. Teriflunomide had no detectable effect on NK-cellnumbers.Conclusions: In our small cohort, teriflunomide treatment affects mainly and significantly on B-cellnumbers, while having a milder effect on T-cell numbers. Larger cohorts are necessary to confirmthese findings and understand the effect of teriflunomide on the functionality of these cells.Neurol Neuroimmunol Neuroinflamm 2017;4:e403; doi: 10.1212/NXI.0000000000000403GLOSSARYEDSS 5 Expanded Disability Status Scale; IFNg 5 interferon gamma; NK 5 natural killer; RR-MS 5 relapsing-remitting MS;Teff 5 effector T cell; Treg 5 regulatory T cell.The main mechanism of action of teriflunomide, a drug approved for treatment of relapsing-remitting MS (RR-MS), relies on the inhibition of dihydroorotate dehydrogenase, which cata-lyzes de novo biosynthesis of pyrimidines. Cells with high proliferative rates, such as activatedlymphocytes, are therefore affected by teriflunomide.1 Accordingly, treatment with terifluno-mide decreases proliferation of T and B lymphocytes.2 Clinical trials (teriflunomide multiplesclerosis oral and teriflunomide oral in people with relapsing multiple sclerosis)3 have shown thatteriflunomide treatment is associated with an early 15% mean decrease in leukocyte counts.However, the effect of teriflunomide on different immune cell subpopulations has not beenproperly addressed so far. Therefore, we have investigated how teriflunomide treatment affectsimmune cell populations in MS.METHODS Flow cytometry analysis. Mononuclear cells were isolated from the peripheral blood by Ficoll gradient centrifugation(Lympholyte-H, Cedarlane), and flow cytometry analysis, using the BD LSRFortessa flow cytometer and FACSDiva 7 Software, wasperformed in 4 tubes using the following conjugated antibodies (Becton Dickinson, Franklin Lakes, NJ): anti-CD3 (V500), anti-CD4(allophycocyanin [APC]-H7), anti-CD8 (PercCP-Cy5.5), anti-CD28 (phycoerythrin [PE], anti-CD25 (PE.Cy7), anti-CD127*These authors contributed equally as last authors to this work.From the Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health Unit (I.G., A.L., F.B., G.M., N.K.d.R., A.U.), and Center of Excellence for Biomedical Research (CEBR) (F.I., A.L., G.M., A.U.), University of Genoa, Italy; Ospedale PoliclinicoSan Martino–IRCCS (A.L., G.M., A.U.), Genoa, Italy; and Rehabilitation Unit (C.S.), Centro di Recupero e Rieducazione Funzionale, M.L.Novarese, Moncrivello, Italy.Funding information and disclosures are provided at the end of the article. Go to Neurology.org/nn for full disclosure forms. The Article ProcessingCharge was funded by the authors.This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CCBY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or usedcommercially without permission from the journal.Neurology.org/nn Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1(BV421), anti-CD45RA (APC), anti-CCR6 (APC), anti-CD161(B421), anti-CCR4 (PE.Cy7), and anti-CXCR3 (PE) for effector(Teff) and regulatory T (Treg) cells; anti-CD19 (PE.Cy7), anti-CD38 (APC), and anti-CD24 (PE) for B cells; and anti-CD3(V500), anti-CD16 (PerCp-Cy5.5), and anti-CD56 (BV421)for natural killer (NK) cells.Flow-Count Fluorospheres (Beckman Coulter, Galway,Ireland) were used to obtain absolute cell counts.MS patient cohort. The longitudinal, open-label, observationalstudy was performed on a cohort of 10 patients with RR-MS; ofthese, 3 were analyzed before treatment (T0) and after 3 months(T3), and 7 at T0 and after 12 months (T12) of treatment. The 3patients followed for 3 months (2 men and 1 woman) had a meanage of 43.6 6 1.5 years, a mean Expanded Disability Status Scale(EDSS) score of 3.0 6 1.5 throughout treatment, and a meandisease duration of 15.3 6 10.5 years. One patient had beentreated with mitoxantrone for 6 years previously. The 7 patientsfollowed for 12 months were all women with a mean age of 54.868.9 years; the mean EDSS score was 3.0 6 1.5 and 3.1 6 1.7before and after 12 months of treatment, respectively, and a meandisease duration of 13.46 4.8 years. One patient had been treatedwith cyclophosphamide andmitoxantrone 7 years previously. Otherprevious treatments included interferonb-1a, interferonb-1b,natalizumab, and dimethyl fumarate. Patients did not experiencerelapses during treatment.Patients were enrolled according to the following criteria:age 18 years and older, with a diagnosis of RR-MS (Poser orMcDonald criteria) and an EDSS score of 0–6.5, and able toprovide written informed consent.Ethics approval. This study was approved by the Ethics Com-mittee of Ospedale Policlinico San Martino—IRCCS, Genoa,Italy, No. 190/12.Figure 1 B cells decrease significantly in patients with MS during teriflunomide treatmentB cells are assessed as CD32CD142CD191 of which subpopulations are identified as B memory (B mem CD241CD382), Bmature (B mat CD241CD38low), and B regulatory (B reg CD241CD38high). (A) A clear trend in reduction of B-cell number andpercentage was observed in 3 patients treated for 3 months. (B) B-cell percentage (upper panel) and numbers (lower panel)were reduced after 1 year of treatment with teriflunomide. Data are presented as mean 6 SEM for 7 treated patients withMS. *p, 0.05. (C) While the proportions of the different B-cell subpopulations did not change after 12months of treatment(upper panel), numbers of Bmat and B reg cells were decreased (*p, 0.05), and Bmem cell numbers showed a clear trend todecrease (lower panel). Data are presented as mean 6 SEM for 7 treated patients with MS.2 Neurology: Neuroimmunology & NeuroinflammationStatistical analyses. Statistical analyses were performedusing Graph-Pad Prism v.6.00 (GraphPad Software, SanDiego, CA). Statistical significance was evaluated usingthe paired Student t test (Wilcoxon matched-pairs signed-rank test). p Values #0.05 were considered statisticallysignificant.RESULTS A preliminary assessment by flow cytom-etry of the effect of teriflunomide on broad immunecell subsets (CD31T cells, CD191B cells, andCD561NK cells) in 3 patients after a three-monthtreatment indicated that CD191B-cell percentageand numbers decreased in all 3 patients (figure 1A),while no apparent effect was observed in the other cellpopulations (data not shown). To further define theeffect of teriflunomide, we evaluated its effect on cellnumber and proportion of relevant immune cellsubsets after a 12-month treatment in 7 differentpatients. As expected, the overall lymphocyte countsin all treated patients were slightly decreased (byaround 10%; data not shown).B cells decrease in patients with MS during teriflunomidetreatment. A significant reduction in cell numberand percentage of B cells overall was confirmed inpatients treated for 12 months (figure 1B). Whilethe proportions of the different B-cell subpopula-tions remained stable (figure 1C upper panel), Bmature and B regulatory cell numbers were signif-icantly decreased, and B memory cells alsoFigure 2 T effector and regulatory cell numbers show a slight trend in reduction after 12 months of teriflunomide treatmentEffector T-cell populations were assessed as Th1 (CD31CD41CCR62CD1612), Th17 (CD31CD41CCR61CD1611), and Th1/17(CD31CD41CCR61CD1611CXCR3highCCR4low). Regulatory T-cell subpopulations were assessed as CD31CD41 (including Treg CD251CD1272 and Treg-naive CD45RA1CD25low) and CD31CD81 (including Treg CD282 and Treg–naive CD282CD45RA1). (A) T effector cell numbers show a trend in decreaseafter 12 months of treatment. (B) The proportion between different regulatory T-cell subsets is not affected by teriflunomide (lower panel). The number ofcells is slightly reduced in all subpopulations after 12 months of treatment (upper panel). (C) The proportion between different T effector cell subsets is notaffected by the treatment. Data are presented as mean 6 SEM for 7 treated patients with MS.Neurology: Neuroimmunology & Neuroinflammation 3decreased, albeit not significantly (figure 1Clower panel).Teriflunomide treatment slightly decreases T-cell numbers,albeit nonsignificantly. A slight trend to reduced num-bers was seen in Teff (figure 2A) and Treg (figure 2Bupper panel) cells after 12 months of treatment,whereas the percentage of Teff and Treg cells did notchange (figure 2C and figure 2B, lower panel).Teriflunomide treatment does not decrease NK-cellnumbers. Figure 3 shows that percentage and num-bers of total NK cells remained stable after 12months of treatment (figure 3, A and C), and therewas no difference in the proportion and absolutecounts of CD56bright and CD56dim NK cells(Figure 3, B and D).DISCUSSION MS is an inflammatory autoimmunedisease involving several types of immune cells witheffector and regulatory functions including T and Blymphocytes. While autoreactive CD41 Teff cellsare believed to drive autoimmune diseases when theyescape central tolerance, several mechanisms haveevolved to regulate their function in the peripheryand prevent autoimmunity. Different Treg-cell sub-sets have been characterized as being able to suppresslymphocyte effector functions either directly or byacting on antigen-presenting cells, therefore pre-venting the development of autoimmunity. In MS,Treg suppression has been reported to be dysfunc-tional. The role of Th1 interferon gamma (IFNg)-secreting cells is well known as a major protagonist indemyelinating damage in MS4; more recently, otherTeff-cell subpopulations have been discovered to havea major role in the inflammatory process of MS:Th17 and Th1/17 lymphocytes secreting IFNg and/or interleukin-17. In particular, Th1/17 cells areincreased in patients with MS compared with healthydonors during active phases of disease.5 Our datashow that the numbers of circulating Teff and Tregcells are slightly, albeit not significantly, decreased byteriflunomide treatment. As teriflunomide affectspredominantly cells that are highly proliferative, thissuggests that, in our cohort of patients who did notsuffer any relapse during the period of observation,the rate of proliferation of these cells is likely low.B lymphocytes are emerging as key player in thepathogenesis of MS. Three B-cell subsets have beendescribed: effector, regulatory, and memory cellscharacterized by different roles depending on theproduction of antibody and of different cytokinetypes. Aberrant profiles of both types of cytokineresponses have been observed in B cells from pa-tients with MS.6 Our study shows that terifluno-mide significantly decreases cell numbers of effectorB-cell subsets, confirming the importance of targetingB cells to obtain clinical and radiologic stabilizationof disease, as shown with B-cell–specific treatmentssuch as rituximab7 and ocrelizumab.8 The effect ofteriflunomide on mature B cells may be of clinicalimportance in view of the observed recirculation ofB cells between peripheral tissues and the CNS,9Figure 3 NK-cell subsets are not influenced by teriflunomide treatmentNK cells are assessed as CD32CD142CD56bright and CD32CD142CD56dim cells. The percentage of the overall population(A) or the CD56bright and CD56dim subsets (B) did not change on treatment, nor did the actual cell number (C and D,respectively). Data are presented as mean 6 SEM for 7 treated patients with MS.4 Neurology: Neuroimmunology & Neuroinflammationwith a possible pathogenetic effect. However, teri-flunomide also decreases the Breg-cell subset, as its effecton lymphocytes is not specific toward a single cell pop-ulation, but rather targets proliferating cells. It is as yetunknown what are the roles of Breg cells in MS and theclinical relevance of changes induced by treatments onthis cell population. However, it should be notedthat efficient specific B-cell–depleting treatments suchas rituximab and ocrelizumab, would also affect theBreg-cell population. In this context, beyond its currentutilization as first-line therapy in MS subjects, terifluno-mide could be also considered as maintenance treatmentafter induction therapy.Our data show that teriflunomide treatment influ-ences immune cell subsets involved in the pathogen-esis of MS, more specifically B cells and, to a lowerextent, T cells. However, further studies with largercohorts and targeting cell functionality are necessaryto clarify the basis of its efficacy in MS.AUTHOR CONTRIBUTIONSIlaria Gandoglia: acquisition of data, analysis, and interpretation. Federico Ival-di: acquisition of data. Alice Laroni: study concept and design and critical revi-sion of the manuscript for important intellectual content. Federica Benvenuto:acquisition of data. Claudio Solaro: critical revision of the manuscript forimportant intellectual content. Gianluigi Mancardi: study supervision. NicoleKerlero de Rosbo: study concept and design, critical revision of the manuscriptfor important intellectual content, and study supervision. Antonio Uccelli: crit-ical revision of the manuscript for important intellectual content, studysupervision.ACKNOWLEDGMENTThe authors thank the nursing staff of the Neurological Clinic of Genoa fortheir help with patients, and the patients themselves for their cooperation.STUDY FUNDINGNo targeted funding reported.DISCLOSUREI. Gandoglia has received funding for travel from Biogen, Novartis, andGenzyme and honoraria from Amirall. F. Ivaldi reports no disclosures.A. Laroni has received consulting honoraria and/or speaker fees from No-vartis, Genzyme, Biogen, Sanofi, Merck Serono, and Teva and receivedresearch support from Biogen. F. Benvenuto reports no disclosures.C. Solaro has served on the advisory boards for Biogen, TEVA, Novartis,Genzyme, Merck Serono, and GW Pharma; received speaking honorariafrom Biogen, Merck Serono, Almirall, Teva, Novartis, GW Pharma, andGenzyme; and received research support from the Italian Multiple Scle-rosis Foundation. G. Mancardi has received honoraria and financial sup-port for travel expenses and research from Biogen, Genzyme, Teva,Merck Serono, Bayer Schering, Sanofi-Aventis, and Novartis and receivedresearch support from the Italian Multiple Sclerosis Society. N. Kerlero deRosbo reports no disclosures. A. Uccelli has received consulting honorariaand/or speaker fees from Genentech, Athersys, Sanofi-Aventis, TohokuUniversity Graduate School of Medicine, Roche, Biogen, Genzyme, Teva,Novartis, and Merck Serono; served on the scientific advisory board forGenentech, Roche, and Allergan; and received research support fromBayer Schering, Sanofi-Aventis, the Italian Ministry of Health, RegioneLiguira Progetto LIMONTE, Ricera Sanitaria Regionale, and FondazioneCARIGE. Go to Neurology.org/nn for full disclosure forms.Received April 26, 2017. Accepted in final form August 10, 2017.REFERENCES1. Bar-Or A. Teriflunomide (Aubagio(R)) for the treatment ofmultiple sclerosis. Exp Neurol 2014;262:57–65.2. Li L, Liu J, Delohery T, Zhang D, Arendt C, Jones C. Theeffects of teriflunomide on lymphocyte subpopulations inhuman peripheral blood mononuclear cells in vitro.J Neuroimmunol 2013;265:82–90.3. Confavreux C, O’Connor P, Comi G, et al. Oral terifluno-mide for patients with relapsing multiple sclerosis(TOWER): a randomised, double-blind, placebo-controlled,phase 3 trial. Lancet Neurol 2014;13:247–256.4. Kleinewietfeld M, Hafler DA. Regulatory T cells in auto-immune neuroinflammation. Immunol Rev 2014;259:231–244.5. Jones AP, Kermode AG, Lucas RM, Carroll WM, Nolan D,Hart PH. Circulating immune cells in multiple sclerosis.Clin Exp Immunol 2017;187:193–203.6. Duddy M, Niino M, Adatia F, et al. Distinct effector cyto-kine profiles of memory and naive human B cell subsets andimplication in multiple sclerosis. J Immunol 2007;178:6092–6099.7. Hauser SL, Waubant E, Arnold DL, et al. B-cell depletionwith rituximab in relapsing-remitting multiple sclerosis. NEngl J Med 2008;358:676–688.8. Hauser SL, Bar-Or A, Comi G, et al. Ocrelizumab versusinterferon beta-1a in relapsing multiple sclerosis. N Engl JMed 2017;376:221–234.9. Stern JN, Yaari G, Vander Heiden JA, et al. B cells populatingthe multiple sclerosis brain mature in the draining cervicallymph nodes. Sci Transl Med 2014;6:248ra107.Neurology: Neuroimmunology & Neuroinflammation 5

Teriflunomide treatment reduces B cells in patients with MS.

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Teriflunomide treatment reduces B cells in patients with MS.

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