RESEARCH ARTICLE

Melanoma Cell Adhesion Molecule– Positive CD8 T Lymphocytes Mediate Central Nervous System Inflammation Catherine Larochelle, MD, PhD,1,2,3 Marc-Andr e L ecuyer, MSc,1 Jorge Ivan Alvarez, PhD,1 Marc Charabati, BSc,1 Olivia Saint-Laurent, MSc,1 Soufiane Ghannam, PhD,1 Hania Kebir, MSc,1 Ken Flanagan, PhD,4 Ted Yednock, PhD,4 Pierre Duquette, MD,1,2,3 Nathalie Arbour, PhD,1,3 and Alexandre Prat, MD, PhD1,2,3 Objective: Although Tc17 lymphocytes are enriched in the central nervous system (CNS) of multiple sclerosis (MS) subjects and of experimental autoimmune encephalomyelitis (EAE) animals, limited information is available about their recruitment into the CNS and their role in neuroinflammation. Identification of adhesion molecules used by autoaggressive CD81 T lymphocytes to enter the CNS would allow further characterization of this pathogenic subset and could provide new therapeutic targets in MS. We propose that melanoma cell adhesion molecule (MCAM) is a surface marker and adhesion molecule used by pathogenic CD81 T lymphocytes to access the CNS. Methods: Frequency, phenotype, and function of MCAM1CD81 T lymphocytes was characterized using a combination of ex vivo, in vitro, in situ, and in vivo approaches in humans and mice, including healthy controls, MS subjects, and EAE animals. Results: Herein, we report that MCAM is expressed by human effector CD81 T lymphocytes and it is strikingly upregulated during MS relapses. We further demonstrate that MCAM1CD81 T lymphocytes express more interleukin 17, interferon c, granulocyte-macrophage colony–stimulating factor, and tumor necrosis factor than MCAM2 lymphocytes, and exhibit an enhanced killing capacity toward oligodendrocytes. MCAM blockade restricts the transmigration of CD81 T lymphocytes across human blood–brain barrier endothelial cells in vitro, and blocking or depleting MCAM in vivo reduces chronic neurological deficits in active, transfer, and spontaneous progressive EAE models. Interpretation: Our data demonstrate that MCAM identifies encephalitogenic CD81 T lymphocytes, suggesting that MCAM could represent a biomarker of MS disease activity and a valid target for the treatment of neuroinflammatory conditions. ANN NEUROL 2015;78:39–53

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ultiple Sclerosis (MS) is an idiopathic inflammatory disease of the central nervous system (CNS) characterized by multifocal infiltration of T lymphocytes across microvascular structures called the blood– brain barrier (BBB). Th1- and Th17-polarized CD41 T lymphocytes have been clearly identified as pathogenic effectors of disease in MS and experimental autoimmune encephalomyelitis (EAE).1–5 Encephalitogenic Th17 lymphocytes preferentially express the che-

mokine receptor CCR66 and melanoma cell adhesion molecule (MCAM/CD146), an adhesion molecule that contributes to their recruitment from the peripheral blood (PB), across the BBB and into the CNS.7–9 We have previously shown that the frequency of MCAM1CD41 T lymphocytes is increased in MS patients as compared to healthy volunteers.9 In the current study, we focus on CD81 T lymphocytes expressing MCAM.

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24415 Received Sep 17, 2014, and in revised form Mar 23, 2015. Accepted for publication Mar 30, 2015. Address correspondence to Dr Prat, Neuroimmunology Research Laboratory (R09–312), CRCHUM, 900 rue Saint-Denis, Tour Viger, Montreal, Quebec, Canada, H2X 0A9. E-mail: [email protected] e de Montr eal (CRCHUM); 2Multiple From the 1Neuroimmunology Research Laboratory, Centre de Recherche du Centre Hospitalier de l’Universit Sclerosis Clinic, Division of Neurology, Centre Hospitalier de l’Universit e de Montr eal (CHUM)–Notre Dame Hospital, and 3Department of Neurosciences, Faculty of Medicine, Universit e de Montreal, Montreal, Quebec, Canada; and 4Prothena Biosciences, South San Francisco, CA, USA

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The exact contribution of CD81 T lymphocytes, including Tc1 (ie, producing interferon c [IFN-c]) and Tc17 (ie, producing interleukin 17 [IL-17]), to the pathogenesis of MS remains controversial. In support of their proinflammatory role in MS, IL-17–producing CD81 T lymphocytes are markedly enriched in a subset of cells expressing CCR6 and CD161.10 Recent studies demonstrate an increased frequency of CD1611CD81 T lymphocytes in the PB and within CNS lesions of MSaffected patients as compared to controls.11,12 Conversely, CD161highCD81 mucosal-associated invariable T lymphocytes are markedly decreased following intensive immunosuppressive therapies in MS.12 Interestingly, IL17–producing CD81 T lymphocytes are enriched in the cerebrospinal fluid (CSF)13 and in active lesions14 of MS subjects, as well as in the CNS of animals with EAE.13,15 Moreover, IL-17 production by CD81 T lymphocytes was recently reported to play an important role in the development of Th17-mediated EAE.13 Overall, these data suggest a potential implication of Tc17 lymphocytes in the pathogenesis of MS. Identification of surface markers and adhesion molecules specifically expressed by Tc17 lymphocytes could provide new therapeutic targets in MS and refine the characterization of this potentially pathogenic subset.16 In the current article, we investigate the expression and function of MCAM in CD81 T lymphocytemediated CNS inflammation and demonstrate that IL17–producing CD81 T lymphocytes with enhanced effector and killing capacities use MCAM to cross the BBB and enter the CNS.

Subjects and Methods

processed for flow cytometry analysis as previously published.9 PB was obtained by venous puncture, and immune cells were isolated as previously published.18 Human CD81 T lymphocytes and CD141 monocytes were isolated using MACS isolation columns (positive selection), as previously described.9 CD81 T lymphocytes were cultured in vitro as follows: for nonspecific activation, 0.5 3 106 cells/ml were cultured with plate-bound anti-CD3 (2.5lg/ml incubated 24 hours at 48C; eBioscience, San Diego, CA; clone OKT3) and soluble antiCD28 (BD Pharmingen, San Diego, CA; 2lg/ml) in X-vivo 15 medium (Lonza, Walkersville, MD) without serum and supplemented with 2mM L–glutamine, 100U/ml penicillin, and 100lg/ml streptomycin (Sigma, St Louis, MO). When specified, CD81 T lymphocytes were cultured with autologous CD141 monocytes at a 1:0.6 ratio in the presence of soluble anti-CD3. Cells were cultured for 4 days if not specified otherwise.

Active EAE Disease Induction and Scoring EAE was induced in 6- to 8-week-old female C57BL/6 mice following our previously published protocol,5,9 except for the use of 300ng of pertussis toxin (PTX). All animal procedures were approved by the Centre de Recherche du Centre Hospitalier de l’Universite de Montreal (CRCHUM) Animal Care committee (N11023APs) and followed guidelines of the Canadian Council on Animal Care. When indicated, animals were then injected intraperitoneally (i.p.) with murinized aMCAM monoclonal antibody (mAb; gift from Prothena Biosciences, South San Francisco, CA; clone 158) or mouse immunoglobulin G (IgG) isotype control (Bio X Cell, West Lebanon, NH; clone MOPC-1). The scoring system used was as follows: 0 5 normal, 1 5 limp tail, 2 5 slow righting reflex, 3 5 paralysis of 1 hindlimb, 4 5 paralysis of both hindlimbs, 5 5 moribund. Mice were scored by an investigator blinded to the treatment group.

Transfer EAE

Patients were classified by 2 certified neurologists according to McDonald’s 2010 revised criteria17 as previously described.9 Acute relapse was defined as presence of new gadoliniumenhancing lesion(s) on magnetic resonance imaging (MRI) and/ or acute onset of new neurological deficits lasting >48 hours within 3 weeks before sample was taken. Other noninflammatory CNS disorders (OND) consisted of migraine, leukoaraiosis, hydrocephalus, trigeminal neuralgia, tympanic hydrops, and subjective neurological symptoms with normal neurological examination, MRI, and CSF analysis.

Transfer EAE was performed as previously described.9 Reactivation of cells was performed in the presence of myelin oligodendrocyte glycoprotein (MOG)35–55, recombinant human transforming growth factor b (rhTGFb), recombinant mouse (rm) IL-6, rmIL-23, and anti–murine IFN-c mAb. For lymph nodes (LN), IL-2 and murine IL-15/IL-15 receptor (R) were added on day 0. For splenocytes, only IL-15/IL-15R was added at 48 hours. A total of 5 3 106 CD31CD41 T lymphocytes were injected i.p. to all the recipient female C57BL/6 animals, in combination with 4 3 106 of either total CD31CD81 T lymphocytes or MCAM-depleted CD31CD81 T lymphocytes.

Human Immune Cell Isolation and Culture

TCR1640 Transgenic Model

Informed consent was obtained from every donor prior to sample collection, in accordance with institutional guidelines (Centre Hospitalier de l’Universite de Montreal [CHUM] research ethic committee approval numbers SL05.022 and 023 and BH07.001). CSF samples were obtained from subjects undergoing lumbar puncture for clinical indication. Cells were isolated from supernatant by centrifugation and immediately

TCR1640 single transgenic mice, which express a T-cell receptor (TCR)-specific for MOG92–106, were kindly provided by the group of Dr Wekerle.19 Only male animals depicting chronic progressive disease were used to study the impact of MCAM blockade on chronic neurological disability. A global score was attributed to TCR1640 mice based on functional impact of paralysis and/or ataxia as follows: 1.0 5 do

Patient Classification

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not control tail or mild balance impairment with mild head turn or both (1.5), 2 5 righting reflex impaired either due to paralysis or ataxia or both (2.5), 3 5 reaching a score of 3 on either atypical or typical EAE scoring scale or both (3.5), 4 5 reaching a score of 4 on either atypical or typical EAE scoring scale or both (4.5), 5 5 moribund. When indicated, males were injected i.p. with murinized aMCAM mAb (clone 15) or mouse IgG isotype control (clone MOPC-1).

Intracellular Staining for Flow Cytometry Analysis Extracellular and intracellular stainings were performed as previously described.9 Cells were processed on the same day.

Immunostaining of CNS Material Frozen sections of CNS autopsy specimens obtained from 3 relapsing–remitting MS (RRMS) patients (brain) and of CNS specimens obtained from mice following rapid intracardiac perfusion (brain and spinal cord) were studied as previously described.5,20,21 The following primary antibodies were used: rabbit antimouse and antihuman MCAM (Abcam, Cambridge, MA), mouse antihuman IL-17-A488 (eBioscience), rat antimouse IL-17 (BD Pharmingen), rat antihuman CD8 (Abcam), rat antimouse CD8 (BD Pharmingen), and mouse antihuman CD4 (BD Pharmingen). Fluorescence acquisition was performed using a Leica (Wetzlar, Germany) Confocal Microscope SP5 platform.

Real Time Quantitative Polymerase Chain Reaction Activated (day 3–5) CD81 T lymphocytes were magnetically sorted using 2 consecutive positive selections for MCAM (CD146 beads; Miltenyi Biotec, Bergisch Gladbach, Germany). Sorting efficiency was confirmed by quantitative polymerase chain reaction (qPCR) for MCAM, which confirmed MCAM levels around 10 times higher in lymphocytes sorted as MCAM1 as compared to MCAM2. qPCR was performed as previously published.9,22

BBB Adhesion and Transmigration Assay BBB endothelial cells (ECs) were isolated and cultured from nonepileptic surgical human CNS material as previously published.21,23,24 Informed consent was obtained from every donor prior to surgery (CHUM research ethic committee approval number HD04.046). For adhesion assays, human BBB ECs were grown to confluence in gelatin-coated T25 plastic flasks. Gelatin coating was used as control condition. On the day of the experiment, fresh medium was added and ex vivo human CD81 T lymphocytes (2 3 106) were immediately added. Transmigration assays were performed using a modified Boyden chamber as previously published.9,21 Antibodies specific for MCAM (P1H12, 40lg/ml) and/or antibodies specific for very late antigen 4 (VLA4; BBA37, 20lg/ml) or the appropriate isotype control (murine IgG1, 20/40lg/ml) were added 1

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hour prior to immune cells. For inflamed conditions, 1 day prior to the experiments IFN-c and tumor necrosis factor (TNF) 100U/ml were added to the culture media of BBB ECs, and then washed twice before adding lymphocytes.

CD107a Mobilization Assay CD81 T lymphocytes activated in vitro for 4 days were harvested and put in contact for 8 hours with adult human oligodendrocytes, in the presence of CD107a antibody (BD Biosciences, Franklin Lakes, NJ) and of monensin (1lM, Sigma) as previously published.25,26 The oligodendrocytes, isolated from surgical resection and maintained under basal culture conditions in accordance with the guidelines of the Biomedical Ethics Unit of McGill University, were kindly provided by the group of Dr Jack Antel.27–29

Killing Assays To assess the killing capacity of MCAM1 versus MCAM2 cells, activated CD81 T lymphocytes were collected and added to carboxyfluorescein succinimidyl ester (CFSE)-labeled MO3.13 human oligodendrocytic cell line30 to a ratio of 1:1. Cells were cocultured for 18 hours prior to analysis by flow cytometry. Survival of target cells was measured as ratio of CFSE1CD82 cells to CFSE2CD81 cells remaining after coculture, as previously published.31

Statistical Analyses Statistical analyses were performed using Prism (GraphPad Software, San Diego, CA), and results are presented as the mean6 standard error of the mean. Paired or unpaired Student t test followed by Dunnett post-tests were performed when appropriate, and survival curve statistical analysis was performed with Mantel–Cox and Gehan–Breslow–Wilcoxon tests. Only p values < 0.05 were considered statistically significant.

Results MCAM Is Strongly Upregulated on Circulating CD81 T Lymphocytes during Acute MS Relapses and Is Expressed by CNS-Infiltrating CD81 T Lymphocytes The expression of MCAM on CD41 T lymphocytes is associated with IL-17 production in various chronic inflammatory conditions.9,32–34 Given the potential implication of CD81 T lymphocytes in the pathogenesis of MS, we sought to investigate the expression and regulation of MCAM on the surface of PB CD81 T lymphocytes of MS subjects and controls. The proportion of CD81 T lymphocytes expressing MCAM remains comparable in healthy controls (HC) and in most MS patient populations, as well as in OND (Fig 1). However, a significant and striking increase in the proportion of CD81 and of CD81CD45RO1 T lymphocytes expressing MCAM is observed in the PB and in the CSF of MS patients during an acute relapse. Conversely, a reduction in the frequency of MCAM41

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FIGURE 1: Acute relapses of Multiple Sclerosis (MS) are associated with increased frequency of melanoma cell adhesion molecule (MCAM)1 CD81 T lymphocytes ex vivo. (A) Expression of MCAM by CD81 T lymphocytes obtained from peripheral blood (PB) of healthy controls (HC; n 5 46), untreated relapsing–remitting MS (RRMS) patients in remission (n 5 62), acutely relapsing RRMS patients (AR; n 5 17), RRMS patients treated with interferon b or glatiramer acetate (IFNb/GA, n 5 32), with fingolimod (FTY, n 5 5), with dimethyl fumarate (BG12, n 5 9), patients with primary progressive MS (PPMS, n 5 11), patients with secondary progressive MS (SPMS, n 5 6), and patients with other neurological diseases (OND, n 5 21). (B) Expression of MCAM by CD81CD45RO1 T lymphocytes obtained from the PB of controls (HC and OND; n 5 23), RRMS patients untreated and in remission (MS, n 5 48), AR (n 5 10), and RRMS patients under treatment with disease-modifying therapies and in remission (DMT, n 5 26). (C) Expression of MCAM by CD81 T lymphocytes obtained from the PB or cerebrospinal fluid (CSF) of untreated RRMS patients (n 5 22), AR (n 5 5), and OND (n 5 8). (D) Expression of MCAM (red), CD8 (green), and CD4 (blue, only in bottom row) in active MS lesions. Asterisks 5 blood vessels. Arrowhead shows colocalization of MCAM and CD8 staining. Representative of n 5 3 MS patients, total of 15 lesions. Nuclei 5 blue, in upper two rows. Scale bars 5 10lm. *p < 0.05, **p < 0.01. ***p < 0.001.

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expressing CD81CD45RO1 T lymphocytes is detected in patients treated with disease-modifying therapies (DMT). Moreover, we found CD81 T lymphocytes expressing MCAM within perivascular infiltrates and in the parenchyma surrounding active lesions in the brain of RRMS patients, as well as on vascular endothelium as previously published.9 We also detected MCAM expression on both CD41 and CD81 T lymphocytes within the same lesion (see Fig 1D, lower panels). Overall, our data demonstrate that MCAM expression on CD81 T lymphocytes is associated with disease activity and suggest a role for MCAM1CD81 T lymphocytes in MS lesion formation. CD81 T Lymphocytes Expressing MCAM Are Proinflammatory Effector Memory To evaluate whether MCAM1CD81 T lymphocytes are regulatory or proinflammatory, we further characterized this subset ex vivo using the PB of MS patients and controls. We found that the majority of MCAM1CD81 T lymphocytes coexpress CD161 and CCR6, and that MCAM expression is associated with CD45RO, CD95, and CD147/extracellular matrix metalloproteinase inducer (EMMPRIN), suggesting a Tc17 memory phenotype (Fig 2). Our characterization further demonstrates that ex vivo CD81 T lymphocytes expressing MCAM are significantly more frequently CD61 (89.4 6 2.3% vs 78.5 6 3.9%, p < 0.001), CCR72CD62L2 (48.5 6 1.8% vs 31.5 6 3.3%, p < 0.001), CD45RA2 (61.7 6 4.0% vs 39.5 6 4.0%, p < 0.001), CD281 (61.6 6 4.4% vs 35.6 6 3.9%, p < 0.001), CD691 (29.6 6 1.9% vs 19.1 6 2.2%, p < 0.01), CD251 (15.5 6 2.0% vs 9.0 6 1.1%, p < 0.001), and CD561 (30.6 6 3.6% vs 13.3 6 1.4%, p < 0.001) than their MCAM2 counterpart, consistent with an effector memory profile. Of note, the proportion of CD31CD81 T lymphocytes that coexpress MCAM and the invariable chain of the TCR is negligible (data not shown). Moreover, the percentage of cells expressing IL17, IFN-c, IL-17/IFN-c, granulocyte-macrophage colony– stimulating factor (GM-CSF), IL-22, and TNF is higher in MCAM1 than in MCAM2 CD81 T lymphocytes, whether they were isolated from the PB of HC, or untreated or DMT-treated RRMS patients. Interestingly, the frequency of MCAM1CD81 T lymphocytes expressing IFN-c alone or coexpressing IL-17 and IFN-c is significantly increased in the PB of untreated RRMS subjects as compared to controls. We also found that nearly half of IL-17/IFN-c double-positive CD81 T lymphocytes are MCAM1, especially in MS patients. Finally, we identified CD81 T lymphocytes coexpressing IL-17 and MCAM within active lesions in postmortem CNS tissue of MS patients. Taken together, our data demonstrate that July 2015

MCAM is expressed by effector memory CD81 T lymphocytes secreting TNF, IL-17, IFN-c, GM-CSF, and IL22, and that MCAM1 Tc17 lymphocytes can be found in active MS lesions. MCAM Is Expressed by Cytotoxic CD81 Tc1 and Tc17 Lymphocytes In Vitro To identify the environmental cues that promote MCAM expression on CD81 T lymphocytes, we performed polyclonal activation of CD81 T lymphocytes in the presence of various cytokines. Anti-CD3/CD28 stimulation by itself elicited a rapid and significant induction of MCAM expression on CD81 T lymphocytes, which paralleled the expression of activation markers CD25, CD69, and HLA-DR (Fig 3). Activated CD81 T lymphocytes were then sorted according to MCAM expression and analyzed by qPCR. MCAM2CD81 T lymphocytes express significantly higher levels of FoxP3, IL4, and IL10 mRNA, whereas MCAM1CD81 T lymphocytes have significantly higher levels of IL17A mRNA. Flow cytometry analyses revealed that in vitro activated MCAM1CD81 T lymphocytes express significantly more IL-17, GM-CSF, TNF, granzyme B (GzB), and perforin than their MCAM2 counterparts. Furthermore, we found the proportion of MCAM1CD81 T lymphocytes to be significantly increased in IL-171 and IL–171/IFN–c1 cells, as compared to IL-41 or IL-172/ IFN-c2 cells. Finally, cytokine-polarizing conditions stimulating MCAM expression on CD81 T lymphocytes were assessed by flow cytometry, and we found the highest increase in MCAM expression under Tc17-polarizing conditions (TGFb 1 IL-6 1 IL-23) and to a lesser extent under Tc1-inducing conditions. CD81 T Lymphocytes Use MCAM to Migrate across CNS ECs We and others have previously demonstrated that human BBB ECs express MCAM, especially under inflammatory conditions.9,35 MCAM interacts with itself36 and with other heterophilic ligands also expressed at the BBB such as laminin 411.8 The presence of MCAM1CD81 T lymphocytes within MS lesions suggests that MCAM may be involved in their transmigration across the BBB. Using an in vitro binding assay, we showed that the absolute number of CD81 T lymphocytes adhering to human BBB ECs was increased under inflammatory conditions, but significantly reduced in the presence of antiMCAM mAb (Fig 4). Moreover, MCAM1CD81 T lymphocytes are significantly enriched among CD81 T lymphocytes adhering to BBB ECs, as compared to nonadherent (floating) cells or to cells adhering to gelatin. Furthermore, because MCAM plays a role during 43

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FIGURE 2: Expression of melanoma cell adhesion molecule (MCAM) on human circulating ex vivo CD81 T lymphocytes is associated with an effector memory Tc17/Tc1 phenotype. (A) Representative dot plots of CCR6 and CD161 expression and (B) frequency of CCR61CD1611, CD45RO1, CD1471, and CD951 in MCAM1 (red) versus MCAM2 (blue) CD81 T lymphocytes, as assessed by flow cytometry analysis of freshly isolated ex vivo CD81 T lymphocytes from untreated relapsing–remitting multiple sclerosis (RRMS) patients. n  4 donors. (C) Expression of interleukin 17 (IL-17), interferon c (IFN-c), IL-17/IFN-c, granulocyte-macrophage colony–stimulating factor (GM-CSF), IL-22, tumor necrosis factor (TNF), perforin, IL-4, and granzyme B (GzB) by ex vivo CD81 T lymphocytes from healthy controls (HC), untreated RRMS patients (MS), and RRMS patients treated with interferon b or glatiramer acetate (DMT). Cytokine expression by MCAM1 (1) versus MCAM2 (2) CD81 T lymphocytes was assessed by flow cytometry. n  5 donors per group. (D) Representative dot plot showing the expression of IL-17 and IFNc by ex vivo MCAM1 versus MCAM2 CD81 T lymphocytes freshly isolated from the peripheral blood of untreated RRMS patients. n  9 donors. (E) Representative dot plot showing the expression of MCAM by IL-17/IFN-c double-positive CD81 T lymphocytes of untreated RRMS patients. n  9 donors. (F) Expression of MCAM (red), IL-17 (green), and CD8 (blue) in active MS lesions. Representative of n 5 3 MS patients, 3 lesions each. Asterisk 5 blood vessel. Scale bar 5 10lm. ns 5 not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

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FIGURE 3: Expression of melanoma cell adhesion molecule (MCAM) by in vitro activated human cytotoxic CD81 T lymphocytes. (A) Time course expression of MCAM assessed by flow cytometry on CD81 T lymphocytes following nonspecific activation with aCD3 and autologous CD141 monocytes or with plate-bound aCD3 and soluble aCD28. n  4 donors. (B) Time course expression of CD69, CD25, HLA-DR, and MCAM assessed by flow cytometry on CD81 T lymphocytes following activation with aCD3/ aCD28. n 5 3 donors. (C) mRNA expression of key transcription factors and cytokines by CD81 T lymphocytes following in vitro stimulation with aCD3/aCD28. After 3 to 5 days of activation, cells were sorted according to MCAM expression and were analyzed by quantitative polymerase chain reaction; n  6 donors. Results are presented as ratios (MCAM1/MCAM2) of mRNA transcripts expression, relative to 18S. (D) Percentage of CD81 T lymphocytes expressing interleukin 17 (IL-17), interferon c (IFN-c), granulocyte-macrophage colony–stimulating factor (GM-CSF), granzyme B (GzB), tumor necrosis factor (TNF), and perforin. CD81 T lymphocytes from healthy controls were activated in vitro with aCD3/aCD28 for 4 days before cytokine expression was assessed by flow cytometry, gating on MCAM1 (1) versus MCAM2 (2) lymphocytes. n  11 donors. (E) Percentage of MCAM1 lymphocytes within specific subpopulations of CD81 T lymphocytes. n  5 donors. (F) Expression of MCAM by CD81 T lymphocytes following in vitro activation for 4 days with aCD3/aCD28 (Ctrl) in the presence of indicated cytokines. n  4 donors, as assessed by flow cytometry. Statistical analysis was performed on raw data. For visual display, data are expressed as the percentage of control. *p < 0.05, **p < 0.01, ***p < 0.001.

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FIGURE 4: CD81 T lymphocytes use melanoma cell adhesion molecule (MCAM) to migrate across the blood–brain barrier (BBB) and have the capacity to kill oligodendrocytes. (A) Absolute number of CD81 T lymphocytes adhering to a confluent monolayer of resting human BBB endothelial cells (ECs), to inflamed BBB ECs (tumor necrosis factor [TNF] 1 interferon c [IFN-c]), or to gelatin. CD81 T lymphocytes were purified by positive selection. (B) Expression of MCAM on CD81 T lymphocytes following contact with resting human BBB ECs, with inflamed BBB ECs (TNF 1 IFN-c), or with gelatin. For both A and B, n 5 4 donors on 2 different preparations of human BBB ECs. Dead cells and doublets were excluded from analysis. (C, D) Migration of ex vivo and in vitro activated human CD81 T lymphocytes across resting (C) and inflamed (D) BBB ECs in a modified Boyden chamber assay. A total of 1 3 106 CD81 T lymphocytes were allowed to migrate for 18 hours in the presence of either anti-MCAM monoclonal antibody or isotype control (immunoglobulin G1 [IgG1]). n 5 3–4 experiments using 2 to 3 different preparations of human BBB ECs. Migration is expressed as absolute number of cells in the lower chamber. One pair of dots 5 1 donor. (E) Migration of ex vivo and (F) in vitro activated human CD81 T lymphocytes across BBB ECs following treatment with blocking antibodies against MCAM and/or very late antigen 4 (VLA4), or isotype control. For both E and F, n 5 3 donors on 2 different preparations of human BBB ECs. (G) Expression of the degranulation marker CD107a on MCAM1 and MCAM2 CD81 T lymphocytes activated in vitro and cultured for 8 hours with primary cultures of human oligodendrocytes. Analysis was performed by flow cytometry. Representative of n 5 4 donors, n 5 2 different preparations of human oligodendrocytes. (H) In vitro activated MCAM1 and MCAM2 CD81 T lymphocytes were cocultured for 18 hours with carboxyfluorescein succinimidyl ester–labeled human oligodendrocytic cell line MO3.13. Cell death was assessed using a LIVE/DEAD fixable dead cell kit. n 5 4 donors. ns 5 not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

migration of Th17 cells through BBB ECs,9 we elected to study the impact of MCAM neutralization using an in vitro human BBB model. MCAM blockade significantly decreased transmigration of both ex vivo and in vitro activated CD81 T lymphocytes in resting as well as in inflammatory conditions. Recent studies have reported 46

that a4 integrin (VLA4) is involved in CD81 T lymphocyte migration to the CNS15,37 and that MCAM can act concomitantly with VLA4 to promote adhesion of human Th17 lymphocytes to ECs.38 Our data now demonstrate that MCAM and VLA4 act synergistically to promote the migration of ex vivo CD81 T lymphocytes Volume 78, No. 1

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across BBB ECs and that MCAM blockade was as effective as VLA4 blockade in preventing the migration of activated CD81 T lymphocytes. These data demonstrate

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that CD81 T lymphocytes interact with human CNS ECs through MCAM in resting and in inflammatory conditions.

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MCAM1CD81 T Lymphocytes Kill Human Oligodendrocytes To evaluate the potential of MCAM1CD81 T lymphocytes as active contributors to the development of demyelinating lesions in the CNS, we cocultured activated CD81 T lymphocytes with human adult oligodendrocytes grown in primary culture. We observed a significant upregulation of MCAM on CD81 T lymphocytes that were put in contact with oligodendrocytes, and measured an increased expression of the degranulation marker CD107a, especially on the surface of MCAM1CD81 T lymphocytes (see Fig 4G). Furthermore, the survival of the oligodendrocytic cell line MO3.13, as assessed by flow cytometry (see Fig 4H) and LIVE/DEAD assay (data not shown), was significantly lower following coculture with activated MCAM1CD81 T lymphocytes, as compared to activated CD81 T lymphocytes depleted for MCAM (MCAM2). Our data demonstrate the lytic capacity of MCAM-expressing CD81 T lymphocytes and emphasize the encephalitogenic nature of these cells. MCAM Is Expressed by Encephalitogenic CD81 T Lymphocytes in EAE To confirm that MCAM1CD81 T lymphocytes play a role in the formation of CNS inflammatory lesions, and to determine whether MCAM could serve as a valuable target to limit neuroinflammation, we assessed the expression of MCAM on CD81 T lymphocytes in MOG35–55-induced EAE mice at different time points. Consistent with our human data, we detected the presence of MCAM1CD81 T lymphocytes in situ within EAE perivascular infiltrates (Fig 5). A time course analysis showed an increased proportion and absolute number of CD81 T lymphocytes expressing MCAM in the LN

(peaking at day 4–8 postimmunization), which then accumulated in the spleen and in the CNS (from day 8 postimmunization) during EAE. The proportion of MCAM1CD81 T lymphocytes from the LN and spleen of EAE animals goes back to preimmunization levels after the early acute phase of the disease (day 12), but the enrichment of MCAM1CD81 T lymphocytes within the CNS is more persistent over time (until day 21). Following reactivation with MOG35–55, the expression of MCAM on murine CD81 T lymphocytes was consistent with an effector memory phenotype (CD44high), similar to our observations in humans. We also found that the expression of MCAM on mouse CD81 T lymphocytes is significantly increased in vitro in the presence of Tc17polarizing conditions and is further enhanced by IL-2/IL15 stimulation. Similar to our human data, MCAM1CD81 T lymphocytes express significantly more IL-17, IFN-c, and IL-17/IFN-c as compared to the MCAM2CD81 T lymphocyte population. To evaluate the pathogenic role of MCAM1CD81 T lymphocytes in vivo, we then transferred to recipient mice either MOG35–55-reactivated CD81 T lymphocytes (13–15% MCAM1; hereon referred to as MCAM1CD81) or MCAM-depleted MOG35–55-reactivated CD81 T lymreferred to as phocytes ( 2.0, whereas 4 of 7 animals injected with the isotype control did. These data are strongly supportive of the role of MCAM in the development of EAE, and of the efficacy of MCAM blockade to control the severity of EAE.

Discussion Although the contribution of major histocompatibility complex class I allele HLA-A to MS susceptibility is established39 and the presence of CD81 T lymphocytes in MS lesions is undisputable,40–44 the exact contribution of CD81 T lymphocytes to MS pathogenesis is not yet fully understood.16,45 Their contribution to EAE also remains controversial in part because the classical model July 2015

of EAE relies primarily on CD41 T lymphocyte activation, in contrast to virus-driven demyelination models.16 However, much of the controversy regarding the role of CD81 T lymphocytes in MS and EAE arises from CD8 subsets not being well characterized. Some studies have demonstrated a proinflammatory function for CD81 T lymphocytes in MS or EAE,11,12,46 whereas others have suggested a suppressive or regulatory function.47–49 Nevertheless, to date there are no reliable phenotypic or functional markers that allow one to distinguish proinflammatory from regulatory CD81 T lymphocytes in MS. The findings presented herein, using blood and CNS samples of humans affected with MS, and supported by data using 3 different EAE models, demonstrate that MCAM is a reliable and stable surface marker for pathogenic CD81 T lymphocytes in MS and EAE. These data also show that MCAM is increased during acute MS relapses, and that it is used by encephalitogenic CD81 T lymphocytes to enter the CNS compartment. We had previously shown that the frequency of MCAM1CD41 T lymphocytes is increased in stable MS patients as compared to HC or OND, and more so during a relapse.9 Our data now demonstrate that the proportion of CD81 T lymphocytes expressing MCAM is strongly and significantly increased in the blood and CSF of MS subjects exclusively during acute relapses. MCAM is rapidly induced upon activation on CD81 T lymphocytes and more so in the presence of IL-23, IL12, IL-6, and IL-15. This increased frequency of MCAM1 CD81 T lymphocytes could be attributed to the induction of MCAM on activated CD81 T lymphocytes, probably resulting from a shift toward a proinflammatory environment during relapses. Although an increased proportion of MCAM1CD81 T lymphocytes has been reported in other inflammatory diseases, such as Sj€ogren syndrome, birdshot chorioretinopathy, and Behcet disease,50,51 the proportion of CD81 T lymphocytes expressing MCAM seems considerably higher in our relapsing MS population. Interestingly, in the PB of relapsing MS subjects, the frequency of MCAM1CD81 T lymphocytes surpasses the frequency of CD41 T lymphocytes expressing MCAM (mean 5 8.2 6 1.3% on CD8, as compared to 7.2 6 0.7% on CD4).9 Most importantly, we report herein that MCAM1IL171CD81 T lymphocytes are present within perivascular MS infiltrates. These data suggest that the expression of MCAM on CD81 T lymphocytes is closely associated with disease activity, and identifies encephalitogenic CD81 T lymphocytes. The presence of MCAM1CD81 T lymphocytes in CNS material from MS patients is in line with previous studies reporting CD1611CD81 and Tc17 in MS lesions.11,12,14 Moreover, MCAM1CD81 T 49

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FIGURE 6: Melanoma cell adhesion molecule (MCAM)1CD81 T lymphocytes contribute to chronic neurological disability in spontaneous progressive experimental autoimmune encephalomyelitis (EAE). (A) Incidence and (B) prevalence of EAE in TCR1640 transgenic male animals, a model of spontaneous progressive EAE. (C) Percentage of CD41 and CD81 T lymphocytes expressing MCAM in presymptomatic TCR1640 males and wild-type littermates. n 5 12 animals. (D) Representative dot plots of MCAM expression by CD81 T lymphocytes isolated from lymph nodes (LN), spleen, and central nervous system (CNS) of TCR1640 mice. Animals (males) were sacrificed in the presymptomatic (35 days postnatal [dpn]) or the progressive (120 dpn) phase of disease, and compared to wild-type littermates. (E, F) TCR1640 transgenic animals (males) were treated thrice weekly (between postnatal days 45 and 80) with anti-MCAM monoclonal antibody (mAb; 200mg intraperitoneally [i.p.]) or isotype control (murine immunoglobulin G; 200mg i.p.). Data shown are representative of 2 independent experiments, with n 5 7–8 animals per group. (E) Clinical disease severity in TCR1640 animals (males) following prophylactic treatment with anti-MCAM mAb or isotype control. (F) Kaplan–Meyer representation of time to reach score of 2.0 (representing clinically significant symptoms) for animals treated with anti-MCAM mAb (gray line) or isotype control (black line). p 5 0.0241. Bar charts and time course shown are mean 6 standard error of the mean. ns 5 not significant, *p < 0.05, **p < 0.01.

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lymphocytes are abundant in immune infiltrates in psoriasis, suggesting a role in extravasation to inflamed tissue.52 Schneider-Hohendorf et al38 have not been able to identify MCAM1CD81 T lymphocytes in the CNS of MS patients. The discrepancy between their findings and the data presented herein is most likely technical; whereas we are using snap-frozen MS brain specimen, they have used formalin-fixed paraffin-embedded sections. Nevertheless, although we were able to identify MCAM1CD81 T lymphocytes in 3 of 3 of our MS CNS collection, we cannot exclude that MCAM1CD81 T lymphocytes can be found only in a subset of MS patients. Dagur et al51 have recently reported that MCAM is associated with a Tc17 phenotype. We further demonstrate that MCAM1CD81 T lymphocytes have an effector memory Tc17/Tc1 profile, express the costimulatory factor CD161,11,12 as well as CCR6 and CD147, which are associated with migration and invasion of the CNS parenchyma,6,53,54 and carry cytolytic function after activation. When compared to the MCAM2 subset, MCAM1CD81 T lymphocytes also express more CD28, and less IL-10, IL-4, and FoxP3, confirming that they are not regulatory lymphocytes.45,47–49,55 A growing body of evidence suggests that Tc17, much like Th17 lymphocytes, are plastic, can proliferate, can coexpress Tbet and ROR-c, produce both IL-17 and IFN-c, and acquire cytotoxic functions upon IL-12 stimulation.11,56,57 Sarin et al have recently shown that polymorphisms in the IL23R gene lead to decreased IL-23 signaling, reduce the number of circulating Tc17 lymphocytes, and confer significant protection against autoimmune diseases.58 Our own findings demonstrate that cytokines of the IL-12 family drive MCAM expression on CD81 T lymphocytes, and more so in combination with IL-23. Most importantly, MCAM1CD81 T lymphocytes display an enhanced killing capacity toward oligodendrocytes likely dependent on IFN-c, TNF, Fas, GzB, and perforin.16 This cytotoxic nature of MCAM1CD81 T lymphocytes toward oligodendrocytes provides the rationale for therapeutic interventions aiming at blocking their entry into the CNS, with the goal of reducing long-term CNS damage. We and others have previously demonstrated a significant contribution of MCAM to adhesion and migration of CD41 Th17 lymphocytes to BBB ECs.7,9,38 In the present study, we demonstrate that neutralizing MCAM significantly reduces both adhesion and transmigration of CD81 T lymphocytes on human BBB ECs, especially under inflammatory conditions. SchneiderHohendorf et al38 have demonstrated that adhesion of July 2015

Th17 lymphocytes to BBB ECs was dependent on MCAM when VLA4 was neutralized. In line with these results, we found an additive effect of neutralizing both MCAM and VLA4 on the migration of ex vivo CD81 T lymphocytes. However, when using activated CD81 T lymphocytes, we found no synergistic impact of simultaneously blocking MCAM and VLA4. Altogether, our data on MCAM-expressing CD41 and CD81 T lymphocytes identify MCAM as an adhesion molecule involved in the adhesion and migration of proinflammatory Tc17 and Th17 cells to the CNS. Although MCAM was recently shown to bind to laminin 411,8 our in vitro human experiments did not allow us to evaluate whether anti-MCAM neutralizing mAbs affected MCAM–laminin 411 or MCAM–MCAM homophilic interactions. We can therefore only speculate on the ligand of MCAM involved during the recruitment of CD81 T lymphocytes to the CNS. In accordance with our previous and current results showing a role of MCAM in the recruitment of both Th17 and Tc17 lymphocytes through the BBB, MCAM blockade in vivo reduced long-term disability in the MOG-induced C57BL/6 EAE model and prevented the development of chronic neurologic deficits in the spontaneous TCR1640 EAE model. Moreover, cotransfer of MCAM-depleted CD81 T lymphocytes, as opposed to MCAM1 CD81 T lymphocytes, resulted in a milder and delayed EAE, consistent with the study of Huber et al,13 in which they demonstrate the pathogenic role of Tc17 lymphocytes in EAE. The EAE data provided herein strongly suggests that MCAM1CD81 T lymphocytes can contribute to CNS damage in MS and EAE, and would potentially constitute a therapeutic target to decrease cumulative neurological deficits. Collectively, our data suggest that MCAM1CD81 T lymphocytes are encephalitogenic Tc17/Tc1 lymphocytes that are associated with MS disease activity and have the capacity to kill oligodendrocytes. Most importantly, because MCAM is expressed on BBB ECs and is also a marker of encephalitogenic Th17 lymphocytes, it could emerge as a valuable therapeutic target to dampen CNS infiltration of proinflammatory CD41 and CD81 T lymphocytes and therefore to impact on long-term disability in MS.

Acknowledgment This study was supported by operating grants from the Canadian Institutes of Health Research (CIHR; MOP89885) and the Multiple Sclerosis Society of Canada (MSSC). C.L., M.C., and O.S.-L. hold scholarships and fellowships from the MSSC. M.-A.L. holds a 51

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scholarship from Fonds de Recherche du Quebec-Sante. J.I.A. holds the David L. Torrey endMS Transitional Career Development Award from the MSSC. N.A. holds a New Investigator Award from the CIHR. A.P. holds a Senior Scholar Award of the Fonds de Recherche du Quebec-Sante and holds a senior Canada Research Chair in Multiple Sclerosis. We thank L. Bourbonnie`re, S. Larouche, S. Terouz, D. Beauseigle, and C. Pittet for their excellent technical assistance.

13.

Huber M, Heink S, Pagenstecher A, et al. IL-17A secretion by CD81 T cells supports Th17-mediated autoimmune encephalomyelitis. J Clin Invest 2013;123:247–260.

14.

Tzartos JS, Friese MA, Craner MJ, et al. Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol 2008; 172:146–155.

15.

Ifergan I, Kebir H, Alvarez JI, et al. Central nervous system recruitment of effector memory CD81 T lymphocytes during neuroinflammation is dependent on alpha4 integrin. Brain 2011;134(pt 12):3560–3577.

16.

Denic A, Wootla B, Rodriguez M. CD8(1) T cells in multiple sclerosis. Expert Opin Ther Targets 2013;17:1053–1066.

Potential Conflicts of Interest

17.

Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011;69:292–302.

18.

Ifergan I, Kebir H, Bernard M, et al. The blood-brain barrier induces differentiation of migrating monocytes into Th17-polarizing dendritic cells. Brain 2008;131(pt 3):785–799.

19.

Pollinger B, Krishnamoorthy G, Berer K, et al. Spontaneous relapsing-remitting EAE in the SJL/J mouse: MOG-reactive transgenic T cells recruit endogenous MOG-specific B cells. J Exp Med 2009;206:1303–1316.

H.K., N.A., A.P.: patent, MCAM modulation and uses thereof. K.F., T.Y.: patent pending, MCAM antagonists and methods of treatment.

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