InductionofgutregulatoryCD39 Tcells by teriflunomide

Javier Ochoa-RepaacuterazPhD

Sara L Colpitts PhDChristopher Kircher MDEli J KasperKiel M Telesford PhDSakhina Begum-Haque

PhDdagger

Anudeep PantLloyd H Kasper MD

Correspondence toDr Ochoa-Repaacuterazjochoareparazewuedu

Induction of gut regulatory CD391T cellsby teriflunomide protects against EAE

ABSTRACT

Objective To determine whether as an orally delivered treatment teriflunomide an inhibitor of themitochondrial enzyme dihydroorotate dehydrogenase approved to treat relapsing forms of multi-ple sclerosis could affect gut-associated lymphoid tissue (GALT) immune responses functionally

Methods C57BL6 mice were treated orally with teriflunomide and flow cytometric analysis ofimmune GALT cells performed ex vivo and adoptive transfer experiments were used to testthe protective effects of GALT regulatory T (Treg) cells

Results Teriflunomide reduced the percentages of antigen-presenting cells of Peyer patcheswhen compared to controls Conversely a significant increase of the relative frequency ofCD391 Treg cells was observed In vivo the protective effect of GALT-derived teriflunomide-induced CD391 Treg cells was established by adoptive transfer into recipient experimental auto-immune encephalomyelitis mice

Conclusions Our results identify specific GALT-derived CD391 Treg cells as a mechanism ofaction that may contribute to the efficacy of teriflunomide during CNS inflammatory demyelin-ation and as an oral therapeutic in relapsing multiple sclerosis Neurol Neuroimmunol Neuroinflamm

20163e291 doi 101212NXI0000000000000291

GLOSSARYAPC 5 antigen-presenting cell ATP 5 adenosine 59-triphosphate CLN 5 cervical lymph node DHODH 5 dihydroorotatedehydrogenase EAE 5 experimental autoimmune encephalomyelitis GALT 5 gut-associated lymphoid tissue JAK 5 Januskinase MLN 5 mesenteric lymph node MOG 5 myelin oligodendrocyte glycoprotein MS 5 multiple sclerosis PP 5 Peyerpatch PSA 5 polysaccharide A SPL 5 spleen Treg 5 regulatory T

Teriflunomide is an approved oral therapeutic to treat relapsing forms of multiple sclerosis (MS)The mechanism of action is presumed to be the inhibition of de novo pyrimidine synthesis byacting as a reversible noncompetitive inhibitor of mitochondrial dihydroorotate dehydrogenase(DHODH) The importance of teriflunomide as a therapy against MS relies on the antiproli-ferative effects of DHODH inhibition which is preferentially observed in autoreactive T andB cells by blocking cell cycling in the G1 phase1 Because of the effects in autoreactive cellsand proinflammatory pathways associated with MS and other diseases teriflunomide is pro-posed as an immunomodulatory drug23

Although the efficacy of teriflunomide in rat models of experimental autoimmune encepha-lomyelitis (EAE) has been demonstrated4ndash7 its therapeutic protection against EAE in mice is notoptimal Because of its reduced protective effects in mice little is understood regarding anyalternative immunologic mechanisms by which it may regulate CNS demyelinating diseaseIn this study we aimed to determine whether teriflunomide an approved oral therapy againstrelapsing forms of MS targets and modifies the phenotype and function of the gut-associatedlymphoid tissue (GALT)

daggerDeceased

From the Department of Microbiology and Immunology Geisel School of Medicine Dartmouth College Hanover NH JO-R is currently affiliatedwith the Department of Biology College of Science Technology Engineering and Mathematics Eastern Washington University Cheney WA

Funding information and disclosures are provided at the end of the article Go to Neurologyorgnn for full disclosure forms The Article ProcessingCharge was paid by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (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

Neurologyorgnn copy 2016 American Academy of Neurology 1

ordf 2016 American Academy of Neurology Unauthorized reproduction of this article is prohibited

We hypothesized that teriflunomide wouldsignificantly affect the GALT The phenotypicchanges could then influence EAE severitysince the GALT is a known reservoir for proin-flammatory cells with direct function in EAEdevelopment

METHODS Mice and treatments Female 8-week-old

C57BL6 mice were obtained from the Jackson Laboratories

(Bar Harbor ME) All animal care and procedures were in

accordance with Dartmouth College Animal Resources Center

institutional policies for animal health and well-being

Mice were treated with either vehicle or teriflunomide (20

mgkg supplied by Sanofi Genzyme Corporation Cambridge

MA) by daily oral gavage during the duration of each experiment

Dartmouth College Animal Resources Center routinely screens

for a wide range of infectious agents including Helicobacter Mice

were maintained in a restricted access-controlled environment

Cell preparation and flow cytometry Single lymphocyte

preparations from Peyer patches (PPs) mesenteric lymph nodes

(MLNs) spleens (SPLs) or cervical lymph nodes (CLNs) were

stained using conventional methods A livedead fixable

fluorescence-labeled viability dye (Molecular ProbesndashThermo

Fisher Scientific Inc Waltham MA) was used in all staining

protocols for deadlive discrimination and gated only on viable

cells for subsequent analysis Cell subsets were analyzed using

fluorochrome-conjugated monoclonal antibodies against T cell

antigens (CD3 CD4 CD8 CD39) NK and NKT cells

(NK11) and B cells (CD19 B220 CD5 CD1d) (BioLegend

San Diego CA) Intracellular staining for Foxp3 was performed

using fluorochrome-labeled anti-Foxp3 monoclonal antibody

(clone FJK-16s eBioscience San Diego CA) Monocytes

macrophages neutrophils and dendritic cells were analyzed

using CD11b CD11c Gr-1 and CD103 (BioLegend)

Samples were acquired using a Miltenyi Biotec MACSQuant

(Miltenyi Biotec Bergisch Gladbach Germany) Data were

analyzed with FlowJo software (FlowJo LLC Ashland OR)

EAE induction EAE was induced in C57BL6 mice by subcuta-

neous challenge with 200 mg MOG35ndash55 (Peptides International

Louisville KY) in 200 mL of complete Freundrsquos adjuvant (Sigma-

Aldrich St Louis MO) On days 0 and 2 postchallenge mice

received 400 ng of Bordetella pertussis toxin intraperitoneally (List

Biological Laboratories Campbell CA) Mice were monitored and

scored daily for disease progression as previously shown8

Adoptive transfer of T cells CD391CD41 or CD392CD41

T cell populations were sorted by flow cytometry from pooled

GALT (PPs and MLNs) of teriflunomide- or vehicle-treated

mice CD41 T cells were first enriched with magnetic beads

(Dynal Biotech ASA Oslo Norway) After sorting the cells

were resuspended in sterile phosphate-buffered saline and

injected IV into recipient mice

Statistical analysis Parametric and nonparametric t tests and

2-way analysis of variance followed by Sidaacutek comparison of

multiple groups was applied to show differences in flow cytometric

analysis Two-way analysis of variance followed by Sidaacutek comparison

of multiple groups was applied to show differences in EAE scores

The p values 005 001 and 0001 are indicated

RESULTS Teriflunomide reduces the frequencies of

GALT antigen-presenting cells For our study we treatedC57BL6 mice orally with daily doses of teriflunomide

(20 mgkg) The dosage was selected according to previ-ous studies performed by Sanofi Genzyme CorporationThe proliferation inhibitory capacity of teriflunomide isspecies-specific which results in an increased dosagerequired for protection in mice vs rats9 In mice 20mgkgd is needed to obtain comparable levels ofprotection to those observed in rats treated with 3 and10 mgkgd teriflunomide9 As described later we didnot perform direct protection studies with teriflunomidein mice We compared the frequencies of commonantigen-presenting cell (APC) populations includingdendritic cells monocytesmacrophages and B cells inthe GALT of mice treated orally with teriflunomide orvehicle control Neutrophil frequencies were alsodetermined Daily oral gavages with teriflunomidesignificantly reduced the frequencies and absolutenumbers of several important APC phenotypes presentin the PPs including dendritic cells (CD11c1) and F4802CD11b1 monocytes (figure 1A) A reduced trend inthe frequencies of macrophages (CD11b1F4801) wasalso seen in the PPs of teriflunomide-treated mice whencompared to controls significance that was observedwhen comparing the absolute number of cells (figure1A) No effects on the neutrophil (CD11b1Gr-11)frequencies were observed Although a similar trend inthe reduction of dendritic cells was observed in theMLNs of teriflunomide-treated mice no statisticalsignificant was achieved (not shown) suggestinga tissue differential in response to teriflunomide exposure

Among the GALT dendritic cells that have beenshown to promote immune tolerance by inducingregulatory T (Treg) cell conversion the CD1031 sub-population is perhaps the most studied When com-paring the frequency of the CD103-expressing subsetwithin the CD11c1 cells of the PPs (figure 1B) andMLNs (not shown) following teriflunomide treat-ment we observed no reduction yet a slight enhance-ment was noted (not significant) The total numbersof CD1031 dendritic cells were significantly reducedafter teriflunomide treatment which could corre-spond with the reduced size of the lymph nodesobserved in these mice (not shown)

A significant reduction in the absolute frequenciesof B cells (CD191B2201) was observed in the PPs ofmice treated with teriflunomide (figure 2A) Theabsolute numbers were not significantly reduced

Similarly when B cells were gated on CD5 andCD1d double-positive (CD51CD1d1) cells a signif-icant reduction was observed compared to controls(figure 2B) These B cell subpopulations have beenpreviously documented as regulatory and protectiveduring murine EAE1011 In the MLNs no differenceswere observed in the frequencies of B cells (figure2A) however the absolute numbers were found tobe significantly lower in teriflunomide-treated mice(figure 2A) The discrepancies might reflect the

2 Neurology Neuroimmunology amp Neuroinflammation


reduced size of the lymph nodes observed in micetreated with the drug (not shown) When MLNCD51CD1d1 B cells were compared we observeda reduction in both frequencies and total numbersin teriflunomide-treated mice although no statisticalsignificance was observed (figure 2B)

Treatment with teriflunomide did not affect the fre-quencies of NK and NKT cells in either PPs or MLNs(not shown) Collectively the flow cytometric analysisof the PPs and MLNs of mice subjected to terifluno-mide suggests that oral treatment with teriflunomidereduces the frequencies and numbers of the major

APC populations of the PPs Similar trends althoughnot significant were also observed in the MLNs

Teriflunomide increases the frequency of a CD391

phenotype in GALT CD41 T cells We next aimed todetermine whether teriflunomide treatment wouldincrease the populations of Treg cells in the GALTThe numbers of CD41 and CD81 T cells isolated fromthe PPs were unaffected by treatment with terifluno-mide (figure 3A) A significant increase in the totalpercentage of CD391Foxp31 Treg cells was observedwhen compared to controls (figure 3A) A similar but

Figure 1 Teriflunomide affects the frequencies and numbers of antigen-presenting cell populations in PPs

C57BL6 mice were treated with teriflunomide (20 mgkg) or vehicle for 14 days After euthanasia PPs of mice were iso-lated and flow cytometric analysis of CD11c1 dendritic cells and CD11b1 monocytes also expressing either F480 or Gr-1was performed (A) (frequencies [Ab] and absolute numbers [Ac] shown) Flow cytometric analysis of CD103 expression wasperformed on the CD11c1 dendritic cells in the PPs (frequencies and absolute numbers shown) (B) Bar graphs show themean 6 standard errors of 1 of 2 repeats (n 5 5 per group) PP 5 Peyer patch

Neurology Neuroimmunology amp Neuroinflammation 3


not significant increase was observed among the totalfrequency of CD391Foxp32 T cells harvested from thePPs of teriflunomide-treated mice (figure 3A) Nodifferences in the frequencies of CD392Foxp31 Tregcells were observed in the PPs of teriflunomide- vsvehicle-treated mice As observed in the analysis ofthe PPs no statistically significant differences in theCD41 and CD81 T cells were seen in the MLNs(figure 3B) although the absolute numbers of bothT cell subpopulations were diminished whencompared with controls A statistically significantincrease in the CD391Foxp31 Treg population wasalso quantified in the MLNs of mice treated withteriflunomide when compared with controls (figure3B) Because the overall numbers of T cells appearedreduced or at least not increased after teriflunomidetreatment the numbers of Treg subsets were mostly

reduced in PPs (figure 3A) and MLNs (figure 3B)although no significance was observed in the analysisof any of the subpopulations compared

Although increases in the frequencies of bothFoxp31 and Foxp32CD391 T cells were observed(figure 3) 14 days of treatment with teriflunomidedid not affect the CD391 percentages within CD41

T cells of PPs and MLNs (not shown) To determinewhether more prolonged treatment would affect thesefrequencies we next performed a kinetic analysis ofthe frequencies of CD39 expression within CD41

T cells The PPs MLNs SPLs and CLNs of micetreated with teriflunomide or vehicle daily for a totalof 28 days were isolated and analyzed using flowcytometry (figure 4) Of interest the increase in therelative frequency of CD391 T cells was not observedin the SPLs and CLNs of mice treated with

Figure 2 Teriflunomide reduces the frequencies of PP B cells and regulatory B cell subsets

Flow cytometric analysis of B2201CD191 B cells was performed in PPs andMLNs as described in figure 1 (Aa) Bar graphs show themean6 standard errorsof 1 of 2 repeats (n5 5 per group) (A) CD51 and CD1d1 double-positive subset of gated B2201CD191 cells were also compared (Ba) For both B cells (Ab)and CD51Cd1d1 B cells (Bb) frequencies on tissue and absolute numbers are shown Analysis as described in figure 1 (n5 5 per group) MLN5mesentericlymph node PP 5 Peyer patch



teriflunomide (figure 4) These results suggest thatthe effects are restricted to the GALT at least innaive mice lacking any overt autoimmune-driveninflammation

Teriflunomide-induced GALT CD391 T cells are

sufficient to reduce EAE severity prophylactically and

therapeutically Our flow cytometric analysis of theGALT compartments of mice treated with terifluno-mide showed an increase in GALT T cells that expressCD39 We next hypothesized that this cellularsubpopulation would be active in vivo and coulddiminish EAE severity Thus CD391CD41 T cellpopulations were sorted by flow cytometry frompooled GALT tissues (PPs and MLNs) of terifluno-mide- or vehicle-treated mice and transferred intountreated recipient mice with EAE We firstevaluated the prophylactic effects of a Treg celltransfer For that 100000 CD391CD41 cellsfrom teriflunomide- or vehicle-treated mice wereadoptively transferred into naive mice 1 day beforethe induction of EAE (figure 5A) An additional

group receiving no cells was used as a positivecontrol for EAE disease As shown in figure 5A theadoptive transfer of CD391CD41 T cells was able tosignificantly reduce the severity of EAE in vivo Nodifferences in the EAE clinical scores were observed inmice receiving CD391CD41 GALT cells fromvehicle- vs teriflunomide-treated donors

We next evaluated the potential therapeutic effectsof CD392CD41 T cells and CD391CD41 T cellsboth isolated from the GALT of teriflunomide-treated mice At the onset of CNS disease (day 17)60000 cells per recipient mouse were transferred(figure 5B) Of note despite the low number of cellstransferred into the recipient mice CD391CD41

T cells significantly reduced the severity of EAE dis-ease for approximately 1 week past the injectionTogether these results confirm the protective effectsof CD391 T cells isolated from the GALT demon-strating an additional mechanism of action for teri-flunomide during CNS inflammatory demyelinationbased on the induction of protective CD391 T cellsin the GALT

Figure 3 Teriflunomide enhances the frequencies of gut-associated lymphoid tissue CD391 regulatory T cells

Flow cytometric analysis was performed to examine T cells (total T cells CD41 and CD81 T cell numbers are shown) and CD391Foxp32 CD391Foxp31 andCD392Foxp31 subpopulations (after gating on CD41CD31 T cells) in the PPs (A C E) and MLNs (B D F) Bar graphs show the mean 6 standard errors offrequencies and absolute numbers of 1 of 2 repeats (n 5 5 per group) MLN 5 mesenteric lymph node PP 5 Peyer patch



DISCUSSION Teriflunomide inhibits the action ofDHODH in proliferating cells This inhibition re-duces DNA synthesis and affects both B and T cellproliferation which requires de novo synthesis ofpyrimidine as opposed to those resting cells that uti-lize the salvage pathway for pyrimidine requirementsBy blocking DHODH teriflunomide reduces therate of proliferation by reducing the supply of de novosynthesized pyrimidine12 Furthermore other pro-posed mechanisms suggest that teriflunomide inhibitsthe Janus tyrosine kinase (JAK) enzymes JAK1and JAK313 and reduces transforming growth factorandashinduced nuclear factor kB activation14 Teri-flunomide is the active metabolite of leflunomidea drug approved by the Food and Drug Administra-tion for the treatment of rheumatoid arthritis15 Inpatients with rheumatoid arthritis treatment withleflunomide also reduces the transcription of celladhesion molecules and matrix metalloproteinases16

Teriflunomide is partially metabolized by the CYPenzyme family and recycled by ABCG2 an adeno-sine 59-triphosphate (ATP)-binding ldquocassette trans-membrane transporter proteinrdquo present in the liverand gut The half-life of teriflunomide in humansis 2 weeks and it is cleared in the liver Whenthe enterohepatic recirculation is blocked with

cholestyramine the half-life is reduced to 2 days17

Teriflunomide is protective in rat models of EAE4ndash7

However its therapeutic protection against EAE inmice is not optimal As opposed to active EAEinduced in mice using a standard MOG protocolEAE was induced in Dark Agouti rats by injectionwith frozen Dark Agouti rat spinal cord homogenatein combination with complete Freundrsquos adjuvant6

Whether the differences in the experimentalprotocols between mouse and rat EAE modelsaccount for the observed differences in theprotection by teriflunomide remains uncertain79

Nevertheless because of its reduced protectiveeffects in mice little is understood regarding anyalternative immunologic mechanisms by which itmay regulate CNS demyelinating disease

In recent years the importance of gut microbiotaas an immunomodulatory entity has emergedAlthough more and larger studies are needed experi-mental studies suggest the relevance of the microbiotain regulating human MS818ndash27 More directly relevantto MS a recent study has now demonstrated thatapproved therapies and those currently under evalu-ation for the treatment of MS significantly modify thegut microbiome28 Fewer studies have focused on thepotential effects of drugs that target the immune sys-tem on the microbiota or the GALT when drugs areadministered orally Treatment with teriflunomidereduced the overall size of PPs and MLNs (notshown) resulting in reduced numbers of cells Thisreduction affected the main and most common APCs(dendritic cells B cells monocytes and macrophages)(figures 1 and 2) It is remarkable that we did notobserve a reduction in the expression levels of theCD1031 subset in CD11c1 cells in PPs (figure1B) although the total numbers were reduced andMLNs (not shown) CD1031 dendritic cells havea tolerogenic phenotype with a previously describedrole in the induction of Foxp31 Treg cells29 Of inter-est is the significant reduction that treatment withteriflunomide induced in the frequencies and num-bers of B cell populations including theCD51CD1d1 subset of the PPs (figure 2 A andB) In the MLNs no changes in the frequencies ofB cells were observed although the absolute numbersof B cells were reduced possibly because of the overallreduction in the size of lymph nodes (figure 2A) Therole of B cells in MS is increasingly understood pri-marily as an effector cell population B cells also act aspresenting cells and significant reductions in mono-cytes and mainly dendritic cells were also observed inthe PPs when mice were orally treated with terifluno-mide The mechanisms by which GALT APCs appearto be significantly affected by treatment with teriflu-nomide remain unknown The systemic immuno-modulatory effects of teriflunomide might account

Figure 4 CD391 T cells increase following sustained treatment withteriflunomide

Gut-associated lymphoid tissue A flow cytometric-based kinetics analysis of CD41CD391

T cells was performed in Peyer patches (A) mesenteric lymph nodes (B) spleens (C) andcervical lymph nodes (D) of mice treated with teriflunomide (20mgkg) or vehicle for 28 daysTissues were harvested from mice weekly (days 0 7 14 and 28) and stained for flowcytometry (n 5 3 per group) A 2-way analysis of variance followed by Sidaacutek multiple com-parison test was performed p 005 p 001



for such reductions however our results show that inMLNs teriflunomide effects in APC populations arealready diminished (not shown) We speculate andare currently evaluating whether teriflunomide mayhave a direct effect on the microbiota composition ofthe gut We will determine whether potential changesin the gut microbial populations of mice treated withteriflunomide influence the percentages and moreimportantly the functions of these APC populationsof the PPs necessary for the appropriate sampling ofthe gut content

Teriflunomide induced a significant increase in thefrequencies of CD391 cells specifically within theCD41 subset in murine GALT (PPs and MLNs)CD39 (ENTPD1) is an ectoenzyme that mediatesimmune suppression of Treg cells by hydrolysis ofATP and ADP (adenosine 59-diphosphate) into 59AMP(adenosine monophosphate)30 CD391 Treg cells cansuppress the proliferation of autoreactive Th17 cellsand their production of interleukin 1731 The oral immu-nization of mice with an attenuated strain of Salmonella

enterica serovar Typhimurium that expresses on its sur-face the colonization factor antigen 1 fimbriae of theenterotoxigenic Escherichia coli the causative agent ofthe travelerrsquos diarrhea promotes CD391 T cells thatmediate protection against the collagen-induced experi-mental model of arthritis32ndash34 Furthermore the suppres-sive function of CD391 Treg cells from patients withMS is reduced when compared to those obtained fromhealthy individuals35We showed that the immunomod-ulatory effect of polysaccharide A (PSA) produced by thegut commensal Bacteroides fragilis is dependent on inter-leukin 10ndashproducing CD391 T cells2627 In the contextof EAE exposure to PSA promoted the accumulation ofCD391 cells in the CLNs with a regulatory anda migratory phenotype More recently we reported thatthe oral treatment of EAE with the murine-specific anti-CD52 antibody increases in CD391 regulatory T cells36

Treatment of patients with relapsing MS with thehumanized anti-CD52 also promoted an enrichmentof the CD391 subset in circulating Treg cells37 Fingo-limod a sphingosine-1-phosphate receptor 1 (S1P1) an-tagonizer approved for the treatment of MStherapeutically enhances the levels of CD39 messengerRNA and CD391 Treg cell frequencies in the circulatingblood of patients treated for MS38 Based on these find-ings and our previous reports with PSA we propose thatthe induction of CD391 Treg cells is not drug-specificAdditional studies are needed to test this hypothesis

An increase in the relative frequency of CD391

T cells was not observed in the SPLs and CLNs ofmice treated with teriflunomide (figure 4) These re-sults suggest that the effects seen are restricted to theGALT It is important to note however that thesemice were not subjected to EAE Our previous workusing PSA which is also administered orally and in-duces a CD391 Treg phenotype in the gut showedthat EAE induction is required to observe these tol-erogenic changes beyond the gut In naive mice PSAdid not increase the frequencies of CD391 Treg cellsexcept in the GALT27 By contrast in mice withEAE Treg cells and CD391 Treg cells were enhancedin relative frequencies (of total CD41 T cells) andabsolute frequencies in CLNs27 and even the CNSof diseased mice26

Despite the modest although significant increasein the frequencies of GALT CD391 T cells after 3 to4 weeks of treatment these cells were protective inlow numbers Furthermore we observed that CD391

T cells isolated from teriflunomide-treated mice wereprotective when adoptively transferred 1 day beforeEAE onset (figure 5A) and also at clinical onset(figure 5B) Of interest is the observation that GALTCD391 T cells isolated from vehicle-treated micewere also protective (figure 5A) Our results suggestan immunomodulatory role for CD391 T cells in theprotection conferred by teriflunomide We propose

Figure 5 Teriflunomide-induced CD391CD41 T cells are protective against EAE

Peyer patches and mesenteric lymph nodes were isolated from mice treated with terifluno-mide or vehicle for 2 weeks pooled and CD391 and CD392CD41 T cells were sorted by flowcytometry One day before EAE induction 50000 to 100000 CD391CD41 T cells fromeither teriflunomide- or vehicle-treated mice were transferred to recipient mice (A) SimilarlyCD391 or CD392CD41 T cells from teriflunomide-treated mice were next adoptively trans-ferred at the onset of disease (B) (depicted is 1 of 2 experiments performed for n 5 5 pergroup and n5 6 per group respectively) Two-way analysis of variance followed by multiplecomparison tests p 005 p 001 and p 0001 EAE 5 experimental autoim-mune encephalomyelitis



that the immunoregulatory effects provide an addi-tional mechanism to the already established anti-proliferative effects of teriflunomide The adoptivetransfer of CD391CD41 T cells independent ofFoxp3 expression was previously shown to be pro-tective against murine rheumatoid arthritis after oraltreatment with gut living vaccine vectors SalmonellaTyphimurium33 and with Lactococcus lactis39 Basedon our results and although the effect of terifluno-mide on the proliferation of lymphocytes in MS iswell established we postulate that in addition teri-flunomide as an orally delivered treatment alters theGALT immune response due to changes in the colo-nization of gut microbial populations The increase inthe frequencies of inducible Treg cells in patients withrelapsing-remitting MS treated with teriflunomidehas been recently reported as part of the TERI-DYNAMIC study that focused on the immunomod-ulatory effects of the drug40 Investigators showed thatafter treatment with teriflunomide the frequencies ofB and T cells in circulating blood are reduced com-pared to control individuals but inducible Treg cellsare increased Our findings show that in non-EAEmice teriflunomide increases a CD391 Treg popula-tion in the gut Studies to evaluate the effect of teri-flunomide on the gut microbiota are being explored

AUTHOR CONTRIBUTIONSJavier Ochoa-Repaacuteraz conception and design of the study acquisition

and analysis of data and drafting of the manuscript and figures Sara L

Colpitts acquisition and analysis of data Christopher Kircher acquisi-

tion and analysis of data Eli J Kasper acquisition and analysis of data

Kiel M Telesford acquisition and analysis of data Sakhina Begum-

Haque acquisition and analysis of data Anudeep Pant acquisition and

analysis of data Lloyd H Kasper conception and design of the study and

drafting of the manuscript

ACKNOWLEDGMENTThe authors thank the DartLab for their flow cytometry support In the

memory of Dr Sakhina Begum-Haque (December 12 2015)

STUDY FUNDINGThis work was supported by Sanofi Genzyme Corporation (Genzyme

IST Concept Award GF-2014-11153)

DISCLOSUREJ Ochoa-Repaacuteraz received travel funding andor honoraria from EMD

Serono holds a patent for Nutraceutical Composition and Methods for

Prevention or Treating Multiple Sclerosis consulted for Symbiotix Bio-

pharmaceuticals received research support from Sanofi Genzyme Eastern

Washington University National Multiple Sclerosis SL Colpitts

C Kircher EJ Kasper KM Telesford S Begum-Haque and A Pant

report no disclosures LH Kasper received honoraria from a speakers

bureau Go to Neurologyorgnn for full disclosure forms

Received March 14 2016 Accepted in final form September 6 2016

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3 Huang O Zhang W Zhi Q et al Teriflunomide an immu-

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4 Ringheim GE Teriflunomide attenuates immunopatho-

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5 Warnke C Stuumlve O Kieseier BC Teriflunomide for the

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9 Merrill JE In vitro and in vivo pharmacological models to

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LH Induction of a regulatory B cell population in exper-

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commensal microflora Gut Microbes 20101103ndash108

12 Miller AE Teriflunomide a once-daily oral medication for

the treatment of relapsing forms of multiple sclerosis Clin

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13 Xu X Williams JW Bremer EG Finnegan A Chong AS

Inhibition of protein tyrosine phosphorylation in T cells

by a novel immunosuppressive agent leflunomide J Biol

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14 Manna SK Aggarwal BB Immunosuppressive lefluno-

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nuclear factor-kappa B activation and gene expression

J Immunol 19991622095ndash2102

15 Breedveld FC Dayer JM Leflunomide mode of action in

the treatment of rheumatoid arthritis Ann Rheum Dis

200059841ndash849

16 Kraan MC Reece RJ Barg EC et al Modulation of

inflammation and metalloproteinase expression in synovial

tissue by leflunomide and methotrexate in patients with

active rheumatoid arthritis findings in a prospective ran-

domized double-blind parallel-design clinical trial in

thirty-nine patients at two centers Arthritis Rheum

2000431820ndash1830

17 Warnke C Meyer zu Houmlrste G Hartung HP Stuumlve O

Kieseier BC Review of teriflunomide and its potential in

the treatment of multiple sclerosis Neuropsychiatr Dis

Treat 20095333ndash340

18 Mielcarz DW Kasper LH The gut microbiome in mul-

tiple sclerosis Curr Treat Options Neurol 201517344



19 Lee YK Menezes JS Umesaki Y Mazmanian SK Proin-

flammatory T-cell responses to gut microbiota promote

experimental autoimmune encephalomyelitis Proc Natl

Acad Sci USA 2011108(suppl 1)4615ndash4622

20 Berer K Mues M Koutrolos M et al Commensal micro-

biota and myelin autoantigen cooperate to trigger autoim-

mune demyelination Nature 2011479538ndash541

21 Ochoa-Repaacuteraz J Mielcarz DW Ditrio LE et al Role of

gut commensal microflora in the development of experi-

mental autoimmune encephalomyelitis J Immunol 2009

1836041ndash6050

22 Yokote H Miyake S Croxford JL Oki S Mizusawa H

Yamamura T NKT cell-dependent amelioration of

a mouse model of multiple sclerosis by altering gut flora

Am J Pathol 20081731714ndash1723

23 Lavasani S Dzhambazov B Nouri M et al A novel pro-

biotic mixture exerts a therapeutic effect on experimental

autoimmune encephalomyelitis mediated by IL-10 produc-

ing regulatory T cells PLoS One 20105e9009ndashe9011

24 Ochoa-Repaacuteraz J Mielcarz DW Ditrio LE et al Central

nervous system demyelinating disease protection by the

human commensal Bacteroides fragilis depends on polysac-

charide A expression J Immunol 20101854101ndash4108

25 Nichols FC Housley WJ OrsquoConor CA Manning T Wu S

Clark RB Unique lipids from a common human bacte-

rium represent a new class of Toll-like receptor 2 ligands

capable of enhancing autoimmunity Am J Pathol 2009

1752430ndash2438

26 Wang Y Begum-Haque S Telesford KM et al A commen-

sal bacterial product elicits and modulates migratory capacity

of CD39(1) CD4 T regulatory subsets in the suppression

of neuroinflammation Gut Microbes 20145552ndash561

27 Wang Y Telesford KM Ochoa-Reparaz J et al An intes-

tinal commensal symbiosis factor controls neuroinflamma-

tion via TLR2-mediated CD39 signalling Nat Commun

201454432

28 Cantarel BL Waubant E Chehoud C et al Gut micro-

biota in multiple sclerosis possible influence of immuno-

modulators J Investig Med 201563729ndash734

29 Jaensson E Uronen-Hansson H Pabst O et al Small

intestinal CD1031 dendritic cells display unique func-

tional properties that are conserved between mice and

humans J Exp Med 20082052139ndash2149

30 Antonioli L Pacher P Vizi ES Haskoacute G CD39 and

CD73 in immunity and inflammation Trends Mol Med

201319355ndash367

31 Nanzer AM Chambers ES Ryanna K et al Enhanced

production of IL-17A in patients with severe asthma is

inhibited by 1a25-dihydroxyvitamin D3 in a glucocorti-

coid-independent fashion J Allergy Clin Immunol 2013

132297ndash304e3

32 Kochetkova I Golden S Holderness K Callis G Pascual

DW IL-35 stimulation of CD391 regulatory T cells

confers protection against collagen IIndashinduced arthritis

via the production of IL-10 J Immunol 20101847144ndash

7153

33 Kochetkova I Thornburg T Callis G Holderness K

Maddaloni M Pascual DW Oral Escherichia coli coloni-

zation factor antigen I fimbriae ameliorate arthritis via

IL-35 not IL-27 J Immunol 2014192804ndash816

34 Kochetkova I Thornburg T Callis G Pascual DW

Segregated regulatory CD391CD41 T cell function

TGF-b-producing Foxp32 and IL-10-producing

Foxp31 cells are interdependent for protection against

collagen-induced arthritis J Immunol 20111874654ndash

4666

35 Fletcher JM Lonergan R Costelloe L et al

CD391Foxp31 regulatory T cells suppress pathogenic

Th17 cells and are impaired in multiple sclerosis


36 Pant A Wang Y Mielcarz D et al Anti-mu CD52 treat-

ment induces CD391 regulatory cells in the GALT and

suppresses inflammatory cytokine expression within the

CNS of EAE mice Mult Scler 201521244

37 Mielcarz D Bergeron A DeLong J et al Anti-CD52

therapy induces significant changes in T and B cell

subset frequency and cytokine expression by B cells

in multiple sclerosis patients Mult Scler 20131974ndash

558P542

38 Muls N Dang HA Sindic CJM van Pesch V Fingolimod

increases CD39-expressing regulatory T cells in multiple

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39 Maddaloni M Kochetkova I Jun S Callis G Thornburg

T Pascual DW Milk-based nutraceutical for treating

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TGF-b-producing CD391 regulatory T cells PLoS

ONE 201510e0117825

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DYNAMIC exploring the impact of teriflunomide on

immune cell population size receptor repertoire and

function in patients with RRMS Neurology 201685

(16 suppl)P5282



DOI 101212NXI000000000000029120163 Neurol Neuroimmunol Neuroinflamm

Javier Ochoa-Repaacuteraz Sara L Colpitts Christopher Kircher et al T cells by teriflunomide protects against EAE+Induction of gut regulatory CD39

This information is current as of October 12 2016

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This article cites 40 articles 15 of which you can access for free at

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2016 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

We hypothesized that teriflunomide wouldsignificantly affect the GALT The phenotypicchanges could then influence EAE severitysince the GALT is a known reservoir for proin-flammatory cells with direct function in EAEdevelopment

METHODS Mice and treatments Female 8-week-old

C57BL6 mice were obtained from the Jackson Laboratories

(Bar Harbor ME) All animal care and procedures were in

accordance with Dartmouth College Animal Resources Center

institutional policies for animal health and well-being

Mice were treated with either vehicle or teriflunomide (20

mgkg supplied by Sanofi Genzyme Corporation Cambridge

MA) by daily oral gavage during the duration of each experiment

Dartmouth College Animal Resources Center routinely screens

for a wide range of infectious agents including Helicobacter Mice

were maintained in a restricted access-controlled environment

Cell preparation and flow cytometry Single lymphocyte

preparations from Peyer patches (PPs) mesenteric lymph nodes

(MLNs) spleens (SPLs) or cervical lymph nodes (CLNs) were

stained using conventional methods A livedead fixable

fluorescence-labeled viability dye (Molecular ProbesndashThermo

Fisher Scientific Inc Waltham MA) was used in all staining

protocols for deadlive discrimination and gated only on viable

cells for subsequent analysis Cell subsets were analyzed using

fluorochrome-conjugated monoclonal antibodies against T cell

antigens (CD3 CD4 CD8 CD39) NK and NKT cells

(NK11) and B cells (CD19 B220 CD5 CD1d) (BioLegend

San Diego CA) Intracellular staining for Foxp3 was performed

using fluorochrome-labeled anti-Foxp3 monoclonal antibody

(clone FJK-16s eBioscience San Diego CA) Monocytes

macrophages neutrophils and dendritic cells were analyzed

using CD11b CD11c Gr-1 and CD103 (BioLegend)

Samples were acquired using a Miltenyi Biotec MACSQuant

(Miltenyi Biotec Bergisch Gladbach Germany) Data were

analyzed with FlowJo software (FlowJo LLC Ashland OR)

EAE induction EAE was induced in C57BL6 mice by subcuta-

neous challenge with 200 mg MOG35ndash55 (Peptides International

Louisville KY) in 200 mL of complete Freundrsquos adjuvant (Sigma-

Aldrich St Louis MO) On days 0 and 2 postchallenge mice

received 400 ng of Bordetella pertussis toxin intraperitoneally (List

Biological Laboratories Campbell CA) Mice were monitored and

scored daily for disease progression as previously shown8

Adoptive transfer of T cells CD391CD41 or CD392CD41

T cell populations were sorted by flow cytometry from pooled

GALT (PPs and MLNs) of teriflunomide- or vehicle-treated

mice CD41 T cells were first enriched with magnetic beads

(Dynal Biotech ASA Oslo Norway) After sorting the cells

were resuspended in sterile phosphate-buffered saline and

injected IV into recipient mice

Statistical analysis Parametric and nonparametric t tests and

2-way analysis of variance followed by Sidaacutek comparison of

multiple groups was applied to show differences in flow cytometric

analysis Two-way analysis of variance followed by Sidaacutek comparison

of multiple groups was applied to show differences in EAE scores

The p values 005 001 and 0001 are indicated

RESULTS Teriflunomide reduces the frequencies of

GALT antigen-presenting cells For our study we treatedC57BL6 mice orally with daily doses of teriflunomide

(20 mgkg) The dosage was selected according to previ-ous studies performed by Sanofi Genzyme CorporationThe proliferation inhibitory capacity of teriflunomide isspecies-specific which results in an increased dosagerequired for protection in mice vs rats9 In mice 20mgkgd is needed to obtain comparable levels ofprotection to those observed in rats treated with 3 and10 mgkgd teriflunomide9 As described later we didnot perform direct protection studies with teriflunomidein mice We compared the frequencies of commonantigen-presenting cell (APC) populations includingdendritic cells monocytesmacrophages and B cells inthe GALT of mice treated orally with teriflunomide orvehicle control Neutrophil frequencies were alsodetermined Daily oral gavages with teriflunomidesignificantly reduced the frequencies and absolutenumbers of several important APC phenotypes presentin the PPs including dendritic cells (CD11c1) and F4802CD11b1 monocytes (figure 1A) A reduced trend inthe frequencies of macrophages (CD11b1F4801) wasalso seen in the PPs of teriflunomide-treated mice whencompared to controls significance that was observedwhen comparing the absolute number of cells (figure1A) No effects on the neutrophil (CD11b1Gr-11)frequencies were observed Although a similar trend inthe reduction of dendritic cells was observed in theMLNs of teriflunomide-treated mice no statisticalsignificant was achieved (not shown) suggestinga tissue differential in response to teriflunomide exposure

Among the GALT dendritic cells that have beenshown to promote immune tolerance by inducingregulatory T (Treg) cell conversion the CD1031 sub-population is perhaps the most studied When com-paring the frequency of the CD103-expressing subsetwithin the CD11c1 cells of the PPs (figure 1B) andMLNs (not shown) following teriflunomide treat-ment we observed no reduction yet a slight enhance-ment was noted (not significant) The total numbersof CD1031 dendritic cells were significantly reducedafter teriflunomide treatment which could corre-spond with the reduced size of the lymph nodesobserved in these mice (not shown)

A significant reduction in the absolute frequenciesof B cells (CD191B2201) was observed in the PPs ofmice treated with teriflunomide (figure 2A) Theabsolute numbers were not significantly reduced

Similarly when B cells were gated on CD5 andCD1d double-positive (CD51CD1d1) cells a signif-icant reduction was observed compared to controls(figure 2B) These B cell subpopulations have beenpreviously documented as regulatory and protectiveduring murine EAE1011 In the MLNs no differenceswere observed in the frequencies of B cells (figure2A) however the absolute numbers were found tobe significantly lower in teriflunomide-treated mice(figure 2A) The discrepancies might reflect the



























































































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints




























































































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints



















































































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints










































































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints































































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints


















































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints






































20134169



2013115S90ndashS94






347203ndash211






















Ther 2015372366ndash2380




Chem 199527012398ndash12403







200059841ndash849







2000431820ndash1830



















1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints














1836041ndash6050

















1752430ndash2438








201454432










201319355ndash367





132297ndash304e3





7153










4666













558P542








ONE 201510e0117825





(16 suppl)P5282














Reprints















Reprints




Documents

InductionofgutregulatoryCD39 Tcells by teriflunomide