FTY720 (fingolimod) treatment tips the balance towards less immunogenic antigen-presenting cells in...

Multiple Sclerosis Journal

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DOI: 10.1177/ 1352458515574895

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BackgroundFingolimod is a sphingosine-1-phosphate (S1P)-receptor modulator and the first novel oral disease-modifying drug approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). Pharmaceutical trials demonstrated a reduction in relapse rates and magnetic resonance imaging (MRI) measures of inflammation.1 Due to its structural resemblance to S1P, phosphorylated fingolimod can bind and activate four out of five subtypes of S1P receptors (S1P1, S1P3–5),2 which belong to the family

of G protein-coupled receptors. Fingolimod treat-ment downregulates S1P1 leading to entrapment of T cells in the lymph nodes.3

To date the direct effect of fingolimod on antigen-presenting cells (APCs) in multiple sclerosis (MS) is not fully understood. The expression of S1P recep-tors 1–4 has been shown on human monocyte-derived dendritic cells (DCs) and stimulation of human DCs with S1P triggered intracellular Ca2+ transients, actin remodelling and chemotaxis.4 The

FTY720 (fingolimod) treatment tips the balance towards less immunogenic antigen-presenting cells in patients with multiple sclerosis

Felix Luessi, Stefan Kraus, Bettina Trinschek, Steffen Lerch, Robert Ploen, Magdalena Paterka, Torsten Roberg, Laura Poisa-Beiro, Luisa Klotz, Heinz Wiendl, Tobias Bopp, Helmut Jonuleit, Valérie Jolivel, Frauke Zipp and Esther Witsch

AbstractObjective: We aimed to clarify whether fingolimod has direct effects on antigen-presenting cells in mul-tiple sclerosis patients.Methods: Frequency and phenotype of directly ex vivo dendritic cells and monocytes were analyzed in 43 individuals, including fingolimod-treated and untreated multiple sclerosis patients as well as healthy subjects. These cells were further stimulated with lipopolysaccharide to determine functional effects of fingolimod treatment.Results: Absolute numbers of CD1c+ dendritic cells and monocytes were not significantly reduced in fingolimod-treated patients indicating that fingolimod did not block the migration of antigen-presenting cells to peripheral blood. CD86 was upregulated on CD1c+ dendritic cells and thus their activation was not impaired under fingolimod treatment. Quantitative analyses of gene transcription in cells and protein content in supernatants from ex vivo CD1c+ dendritic cells and monocytes, however, showed lower secretion of TNFα, IL1-β and IL-6 upon lipopolysaccharide-stimulation. These results could be matched with CD4+MOG-specific transgenic T cells exhibiting reduced levels of TNFα and IFN-γ but not IL-4 upon stimulation with murine dendritic cells loaded with MOG, when treated with fingolimod.Conclusions: Our data indicate that fingolimod – apart from trapping lymphocytes in lymph nodes – exerts its disease-modulating activity by rebalancing the immune tolerance networks by modulation of antigen-presenting cells.

Keywords: Fingolimod, multiple sclerosis, dendritic cell, monocyte, cytokine, immunotherapy

Date received: 16 September 2014; accepted: 26 January 2015

Correspondence to: Felix Luessi Department of Neurology, Focus Program Translational Neuroscience (FTN), Rhine Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg-University of Mainz, Langenbeckstrasse 1, Building 708, 55131 Mainz, Germany. [email protected]

Felix Luessi Stefan Kraus Steffen Lerch Robert Ploen Magdalena Paterka Torsten Roberg Laura Poisa-Beiro Valérie Jolivel Frauke Zipp Esther Witsch Department of Neurology, University Medical Center of the Johannes Gutenberg-University of Mainz, Germany

Bettina Trinschek Helmut Jonuleit Department of Dermatology, University Medical Center of the Johannes Gutenberg-University of Mainz, Germany

Luisa Klotz Heinz Wiendl Department of Neurology, University of Münster, Germany

Tobias Bopp Institute for Immunology, University Medical Center of the Johannes Gutenberg-University of Mainz, Germany

The first two authors contributed equally to this work.

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Original Research Paper

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increase in intracellular Ca2+ is due to mobilization from intracellular stores by activation of Gi- proteins and phospholipase C.5 In vitro studies have reported that migration of DCs can be mediated by S1P3; an effect that can be antagonized by fingolimod.6 Fingolimod inhibits chemotaxis in human mono-cyte-derived DCs in vitro and leads to reduced T cell stimulatory capacity and changes in cytokine secre-tion profiles in T lymphocytes favoring T helper (Th)2 differentiation in vitro.4,7

Monocytes promote T cell migration through the blood–brain barrier in the central nervous system (CNS) and are a crucial source of inflammatory medi-ators.8 A study demonstrated increased frequencies of monocytes following fingolimod therapy in the cere-brospinal fluid and blood of patients with MS.9 However, in experimental autoimmune encephalomy-elitis (EAE), fingolimod administration led to decreased absolute numbers of circulating monocytes associated with accumulation of monocytes in the spleen and bone marrow.10 In addition, this study pro-vided evidence that in vitro fingolimod acts directly on monocytes to alter surface protein expression. The goal of our study was to clarify the impact of fingoli-mod treatment on circulating DCs and monocytes in MS patients.

Materials and methods

Analysis of DCs and monocytes from MS patientsForty-three individuals were investigated (Table 1). Heparinized peripheral blood was collected with informed written consent and appropriate ethics approval. Some of the healthy donors and untreated RRMS patients had already been investigated as con-trols in a previously published study.11 Conventional CD1c+ DCs and monocytes were magnetically sorted from peripheral blood mononuclear cells (PBMCs) and expression of CD86, HLA-DR and CD1c was assessed. The cytokine and chemokine expression of CD1c+ DCs was analyzed as previously described.11

In vitro generation of DCsHuman monocyte-derived DCs were prepared from blood CD14+ monocytes as described previously.12

C57BL/6 and CD90.2+ 2D2 mice were purchased from our central animal facility. Animal procedures were performed under the supervision of authorized investigators in accordance with the European Union normative. Murine bone marrow-derived DCs were generated as described.12

Flow cytometric analysisFor flow cytometric analysis of human cells, we used the following antibody conjugates: anti-CD11c-APC, anti-CD14-APC, anti-CD40-PE-Cy7, anti-HLA-DR-V450, anti-CD4-V450, anti-CD83-PE, anti-CD86-PE and anti-CD4-PerCP. For analysis of human DC subsets, PBMCs were labelled with anti-CD3-/anti-CD14-/anti-CD19-FITC, anti-HLA-DR-PE-Cy5, anti-CD11c-APC-Cy7, anti-CD1c-Pacific Blue, anti-CD141-APC, anti-CD303-PE and anti-CD16-PE-Cy7.

For murine cells, we used: anti-CD11c-APC, anti-CD11b-PECy7, biotinylated anti-MHC II-IAb, anti-CD80-FITC and anti-CD86-PE. Intracellular stainings for tumour necrosis factor alpha (TNFα), interferon gamma (IFN-γ), interleukin (IL)-10 and IL-4 were performed using the Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany).

RNA isolation and quantitative PCR analysisTotal RNA from cultured human CD1c+ DCs and mono-cytes was isolated using the RNeasy-Mini-Kit (Qiagen, Hilden, Germany). DNase-I treatment was additionally performed to avoid genomic DNA contamination. The total RNA was used to obtain complementary DNA by the superscript-III-first-strand-synthesis-system and random hexamer primers (Invitrogen, Darmstadt, Germany). Real-time polymerase chain reaction (PCR) was performed with amplification primers by using iQ SYBR Green supermix (BioRad, Munich, Germany)

Table 1. Patient characteristics.

Group Women/men Age (years±SD) EDSS (mean) Disease duration (years±SD)

FTY720 treatment duration (years±SD)

Healthy donors (n=17) 11/6 40.7±8.6 (28–54) n.d. n.d. n.d.

Untreated MS patients (n=14) 12/2 36.0±12.1 (24–60) 1.27 (0–3) 3.8±3.59 (0.1–10.2) n.d.FTY720-treated MS patients (n=12)

9/3 40.7±6.2 (32–48) 1.64 (0–3.5) 10.1±7 (2.2–22) 2.2±1.0 (0.3–4.1)

MS: multiple sclerosis; n.d.: not determined or not applicable; EDSS: Expanded Disability Status Scale; FTY720: fingolimod.



F Luessi, S Kraus et al.


and the iCycler iQ (BioRad). Relative changes in gene expression were determined using the ΔΔCt-method with GAPDH, β-actin and EF1α as reference genes.

Cytokine and chemokine assaysSupernatants were collected from sorted human CD1c+ DCs and CD14+ monocytes or murine lipopolysaccharide(LPS)-matured DCs. Cytokine con-centrations were measured with the FlowCytomix (eBioscience) human Th1/Th2/Th9/Th17/Th22 13plex kit or simplex mouse kits for IL-1β, IL-6, IL-10 and TNFα. Chemokine concentrations were measured with the human chemokine 6plex kit.

T cell proliferation assaysMature murine DCs loaded with MOG35–55 peptide were co-cultured with varying ratios of naive 2D2 T cells. Isolated CD4+ T cells were polyclonally stimu-lated with anti-CD3 and anti-CD28. To measure the level of T cell response, cells were cultured for 3 days in 96-well plates followed by an additional 16 h pulse of [3H] thymidine (37 kBq/well; Amersham , Cardiff, UK). Thymidine incorporation was measured in a β-scintillation counter (Microbeta; Wallac , Turku, Finland).

To investigate the phenotype of proliferating T cells we performed proliferation assays using naive 2D2 T cells that were labelled with 2.5 μM carboxyfluorescein succinimidyl ester (CFSE) at 37°C for 10 minutes. After 3 days of co-culture with murine mature DCs loaded with MOG35–55 peptide, the cells were ana-lyzed by flow cytometry.

Phagocytosis measured by FITC-dextran incorporationHuman immature monocyte-derived DCs (1×105) were re-suspended in 100 μl phosphase-buffered saline (PBS) and incubated with FITC-dextran (1 mg/ml; Sigma, Germany) at 37°C and 0°C (negative con-trol) for 30 minutes. The incubations were stopped by adding 2 ml ice-cold PBS containing 1% human serum and 0.02% sodium azide. The cells were washed three times before flow cytometric analysis.

Statistical analysisData were analyzed using PRISM5 (Graphpad). Data are presented as mean±SEM from at least three inde-pendent experiments (in vitro experiments). Statistical analysis of the data was conducted using a non-para-metric test (Mann–Whitney or Kruskal–Wallis tests)

followed by a Dunn’s multiple comparison test and Student’s t test.

For evaluation of the clinical correlation, the annual-ized relapse rate prior to and during treatment with fingolimod was retrospectively assessed. The relative relapse reduction was calculated by dividing the dif-ference of the annualized relapse rates prior to and during fingolimod treatment by the annualized relapse rate prior to fingolimod treatment. The correlation between relative relapse reduction and immune effects of fingolimod was analyzed by the Pearson correlation coefficient r.

Results

Fingolimod treatment increased relative frequencies of DCs and monocytes within PBMCsWe collected blood from MS patients receiving 0.5 mg fingolimod daily, untreated MS patients and healthy donors (Table 1). Age was not considered to be a confounding factor, since the mean age of the different groups was comparable. Fingolimod-treated and untreated MS patients exhibited a similar disabil-ity as assessed by the Expanded Disability Status Scale (EDSS).

Fingolimod is known to trap lymphocytes in the lymph nodes.3 Accordingly, following fingolimod treatment of patients, we found that the amount of PBMCs was significantly decreased (Figure 1(a)). The absolute numbers of isolated conventional CD1c+ DCs and monocytes were not significantly changed in fingoli-mod-treated patients compared to untreated patients (Figure 1(a)). Flow cytometric analyses of PBMCs revealed that fingolimod treatment increased the per-centage of conventional DCs and monocytes com-pared to untreated MS patients. Similarly, the proportion of plasmacytoid DCs (lineage-CD11c-

HLA-DR+CD303+) was significantly elevated (Figure 1(b) and 1(d)). The rather proinflammatory CD1c+ DCs and monocytes were not inhibited in their activa-tion since isolated CD1c+ cells from fingolimod-treated patients, CD86 but not HLA-DR and CD1c were significantly upregulated relative to untreated patients (Figure 1(c) and 1(e)).

Treatment with fingolimod in vivo reduced proinflammatory cytokine and altered chemokine gene expression of isolated DCsWe found that LPS-activated induction of TNFα, IL-1β, MIP-1α and MIP-1β gene expression was





reduced in CD1c+ DCs from fingolimod-treated patients (Figure 2(a)).

Analyses of protein content in ex vivo CD1c+ DC supernatants upon LPS stimulation corroborated altered expression of proinflammatory cytokines: IL-1β and IL-6 were significantly reduced compared to untreated patients (Figure 2(b)). TNFα, MIP-1α and

MIP-1β concentrations did not exhibit significant dif-ferences in CD1c+ DCs. Thus CD1c+ DCs from fingoli-mod-treated patients demonstrated a significantly reduced immunogenic responsiveness to LPS stimula-tion without influencing chemokines.

Genes regulating TNFα, IL-1β, MIP-1α and MIP-1β were assessed by quantitative PCR (supplemental

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Figure 1. Fingolimod increases dendritic cell (DC) and monocyte frequencies in multiple sclerosis (MS) patients. Comparison was made of absolute numbers of peripheral blood mononuclear cells (PBMCs), CD1c+ DCs and CD14+ monocytes per millilitre of whole blood in untreated and fingolimod-treated MS patients as well as in healthy donors (a). Comparison was made of peripheral blood conventional DCs (CD1c+ DC, CD16+ DC and CD141+ DC), CD303+ plasmacytoid DCs (b) and monocytes (d) as the percentage of PBMCs. Fingolimod increases significantly CD86 surface expression assessed by flow cytometry on isolated CD1c+ cells, whereas expression of HLA-DR and CD1c remained unchanged (c). Surface expression of CD86 and HLA-DR was also assessed on monocytes (e). Some of the samples from healthy donors and untreated relapsing remitting MS patients were used as controls in a previously published study.10 Data represent results from at least ten donors per group. Error bars represent SEM, *P<0.05, **P<0.01, ***P<0.001.





Figure 1(c)). The reduction of IL-1β by CD1c+ DCs under treatment cannot be attributed to a caspase-1 defi-ciency cleaving the precursor of IL-1β into its active form because caspase-1 was significantly increased in fingolimod-treated patients. The expression of IL-18,

another caspase-1 substrate, was not influenced by fin-golimod. Receptor engagement through CCR7, IL-1R1 and TLR4 leads to activation of the nuclear factor kappa B (NF-κB) pathway and IL-1β has been reported to be positively autoregulated by this pathway.13 We analyzed

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Figure 2. Fingolimod reduces immunogenic function of CD1c+ dendritic cells (DCs) in multiple sclerosis (MS) patients. Gene expression levels of cytokines and chemokines were determined in isolated conventional CD1c+ DCs following stimulation with lipopolysaccharide (LPS) for 4 hours. The experimental results represent the fold induction of the gene expression after stimulation with LPS normalized to GAPDH expression (a). Supernatants of CD1c+ DCs cultured with or without LPS for 24 hours were collected and concentrations of cytokines (TNFα, IL-1β,ΙL-6, IL-10) and chemokines (MIP-1α, MIP-1β) were determined by means of the cytometric bead array system (b). The experimental results represent the fold induction of protein secretion upon stimulation with LPS. Data represent mean results from at least ten independent donors per group. Some of the samples from healthy donors and untreated relapsing remitting MS patients were used as controls in a previously published study.10 Error bars represent SEM, *P<0.05, **P<0.01.





the expression of IL-1R1 and IL-1R2 and found no reg-ulation of IL-1R genes under treatment. In addition, the analysis of ICAM-1, MyD88 and TLR4 proteins impli-cated in the NF-κB signalling did not show regulation of this pathway by fingolimod (supplemental Figure 1(c)).

Reduced secretion of proinflammatory cytokines in monocytes of fingolimod-treated patientsWhereas quantitative PCR showed only a slight non-significant reduction of IL-1β gene expression in monocytes isolated from fingolimod-treated MS patients (Figure 3(a)), analyses of protein content in supernatants of cultivated monocytes demonstrated altered secretion of proinflammatory cytokines: the response of IL-1β and TNFα upon LPS stimulation was significantly reduced in fingolimod-treated patients (Figure 3(b)). The reductions in chemokine secretion did not reach significance. We observed no difference in the expression of proteins involved in IL-1β signalling (caspase-1, IL-1R1, IL-1R2) and proteins of the NF-κB pathway (MyD88, TLR4, ICAM-1) (supplemental Figure 1(d)). Taken together, monocytes isolated from fingolimod-treated patients demonstrated a significantly reduced responsiveness to LPS stimulation.

Fingolimod impaired the ability of DCs to polarize and stimulate T cellsWe assessed functional relevance of reduced cytokine production by mature DCs on T cells by co-culturing murine bone marrow-derived DCs generated with or without fingolimod and loaded with the MOG35–55 peptide with naive MOG-antigen-specific 2D2 T cell receptor transgenic T cells (2D2 T cells). Intracellular cytokine staining of T cells from this co-culture revealed that the majority of 2D2 T cells produced TNFα, whereas only few cells produced IFN-γ (1.08%), IL-10 (2.01%) or IL-4 (1.34%) when co-cultured with untreated mature bone marrow-derived DCs for 5 days. Pretreatment of bone marrow-derived DCs with fingolimod resulted in significantly less TNFα, IFN-γ and IL-10 production by 2D2 T cells (Figure 4(a)). In addition, fingolimod pretreatment of DCs demonstrated a reduced ability of DCs to induce proliferation in T cells (Figure 4(b)).

Proliferation assays using CFSE-labelled naive T cells showed that cytokines were derived from prolif-erating T cells in response to MOG35–55 peptide pres-entation (Figure 4(c)). Mimicking the conditions in patients, supernatants of fingolimod-treated mature murine DCs displayed reduced pro-inflammatory

cytokines TNFα, IL-1β and IL-6 compared to control cultures (Figure 4(d)).

Activation markers, viability and endocytosis of DCs were not influenced by fingolimodTo address whether fingolimod treatment impairs the differentiation of in vitro generated murine bone mar-row-derived DCs and human monocyte-derived DCs, we investigated the expression of CD11c as a differ-entiation marker and found that it was not affected by fingolimod treatment. Significant differences in either surface expression of the co-stimulatory molecules CD86, CD80, CD40 and MHC II/ HLA-DR in murine and human DCs or in the maturation marker for human DC CD83 were not found. No toxic effect of fingolimod on murine and human DCs was found at 0–100 ng/ml. Fingolimod did not affect the cell num-ber and viability of DCs in vitro. Endocytosis of solu-ble antigens by immature DCs was measured using FITC-dextran uptake and in neither murine DCs nor human DCs fingolimod was FITC-dextran incorpora-tion decreased (supplemental Figure 1(a) and 1(b)).

Clinical and MRI correlations of the fingolimod effect on immune cellsA sufficient patient history to calculate clinical and MRI correlations was available from 10 fingolimod-treated patients in this study. The majority of the par-ticipants were women (75%). Disease duration before fingolimod treatment varied between 2.2 and 22 years (mean 10.1 years). Eleven patients were treated previously (three patients IFN-β, three patients glati-rameracetate, four patients natalizumab, one patient cyclophosphamide).

The mean follow-up duration under fingolimod treat-ment was between 0.3 and 4.1 years (mean 2.24 years) (supplemental Figure 2(a)).

There was a mild correlation between the mean lym-phocyte and PBMC count, and a relative reduction of the annualized relapse rate in individual patients (sup-plemental Figure 2(b) and 2(c)), indicating that lower lymphocyte or PBMC counts might be associated with a more pronounced relative risk reduction.

For IL-1β and IL-6 in the supernatant of conven-tional CD1c+ DCs, there was only minimal correla-tion with the relative reduction of annualized relapse rates (supplemental Figure 2(d)). The correlation between the gene expression of TNFα and MIP-1β and the annual relapse rates was not significant (sup-plement Figure 2(e)).





DiscussionInnate immune cells shaping the adaptive immune response play an important role in MS and other auto-immune diseases. To date, the role of fingolimod on APCs, namely DCs and monocytes, has not been clari-fied in MS. Contradictory results exist on monocytes and no study has investigated the effect of oral

fingolimod treatment on the DC compartment in MS patients. We found that fingolimod treatment leads to significant distinct changes in circulating DCs and monocyte populations in MS patients. CD86 was upregulated in CD1c+ DCs indicating that activation was positively influenced (Figure 1(c)). The expression of ICAM-1 and CCR7 on CD1c+ DCs was unchanged

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Figure 3. Fingolimod affects monocyte function in multiple sclerosis (MS) patients. Gene expression level of cytokines and chemokines were determined in isolated monocytes following stimulation with lipopolysaccharide (LPS) for 4 hours. The experimental results represent the fold induction of the gene expression after stimulation with LPS normalized to GAPDH expression (a). The supernatants of monocytes cultured with or without LPS for 24 hours were collected and concentrations of cytokines (TNFα, IL-1β,ΙL-6, IL-10) and chemokines (MIP-1α, MIP-1β,MCP−1) were determined using the cytometric bead array system. The experimental results represent the fold induction of protein secretion after stimulation with LPS (b). Data represent mean results from at least nine donors per group. Error bars represent SEM, *P<0.05.





(supplemental Figure 1), suggesting that adhesion and extravasation of DCs into inflamed tissue mediated by CCR7 was not impaired in fingolimod-treated MS patients.14 A recent study showed that the absolute number of circulating eosinophils, neutrophils and monocytes was not altered after administration of fin-golimod,15 which is in line with our findings.

CD86 expressed by DCs was suggested to be involved in Th2 responses, whereas CD40 and CD80 were shown to be involved in Th1 responses.16 Upregulation of CD86 on ‘semi-mature’ DCs was associated with tolerance induction.17 In addition,

estriol, a pregnancy-specific estrogen with therapeu-tic efficacy in MS,18 generated tolerogenic conven-tional DCs with upregulation of CD86, leading to an ameliorated disease outcome in EAE.19 Therefore, the observed upregulation of CD86 on CD1c+ DCs in the blood of fingolimod-treated patients suggests that fingolimod induces a rather semi-mature differ-entiation-locked tolerogenic DC population.

There is significant evidence that S1P signalling is involved in lymphocyte trafficking,3 but recent litera-ture has shown that S1P is also implicated in other immunoregulatory functions.6,7,20,21 Fingolimod has

(c) Control FTY-720

TNF-

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N-γ

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Control

FTY-720 20 ng/ml

Figure 4. Fingolimod modulates T cell differentiation and proliferation; 20 ng/ml fingolimod-treated mature bone marrow-derived dendritic cells (DCs) loaded with MOG35–55 peptide were co-cultured with varying ratios of naive anti MOG-specific T cell receptor transgenic 2D2 T cells. Intracellular staining of DC:T cell ratio 1:10 was performed. This resulted in significantly fewer TNFα, IFN-γ and IL-10 producing T cells compared to controls (a). The proliferative response was measured by [3H] thymidine incorporation of the co-culture (b). Plotted is the normalized [3H] thymidine incorporation (n=4). Fingolimod significantly reduced (P=0.028) the T cell proliferative capacity of murine DCs at the ratio of 1:10. Cytokine production in proliferating T cells and proliferation of T cells labelled with carboxyfluorescein succinimidyl ester was assessed after 3 days of co-culture with mature bone marrow-derived DCs loaded with MOG35–55 peptide and treated with 400 ng/ml fingolimod or solvent control (dimethyl sulphoxide) (c). Mature DCs from mouse bone marrow were generated in the presence or absence of 20 ng/ml fingolimod (d). Cell culture supernatants were collected and cytokines and chemokines were measured using a cytometric bead array system. Experimental results are summarized as mean values of relative cytokine and chemokine production. Data represent mean results from three independent experiments. Error bars represent SEM, *P<0.05, **P<0.01.





been reported to reduce the capacity of mature DCs to promote a pro-inflammatory T cell response in vitro. This effect was mediated by a decrease in IL-12 and an increase in IL-10 production by DCs leading to a Th2 rather than Th1 response by effector T cells.7 We report here that fingolimod-treated mature murine bone marrow-derived DCs secreted less TNFα, IL-6 and IL-1β in vitro (Figure 4(c)).

In fact, in fingolimod-treated patients both DCs and monocytes were altered in their immunogenicity, highlighting the translational relevance of our in vitro results. Interestingly, the modulation of cytokine expression was also found at the messenger RNA level, suggesting an influence of fingolimod on gene transcription, specifically TNFα, IL-1β, MIP-1α and MIP-1β genes (Figure 2(a)). With regard to cytokine secretion by DCs, IL-1β and IL-6 were significantly reduced under fingolimod (Figure 2(b)). It is likely that the IL-6 production by DCs directly affects the Th17 differentiation since DCs have been shown to be the critical source for IL-6 in EAE to promote CNS-reactive CD4+ T cells in IL-6 deficient mice.22

Critical in vivo evidence for an association between the increase in chemokine expression in the CNS and neurological dysfunction has been published by sev-eral groups.23 MIP-1α and MIP-1β concentrations were reported to be increased in and around MS lesions and, particularly, in perivascular cells.24 As DCs are known to localize around the vessels,25 they may be the relevant source of chemokine production in EAE and MS. A crucial role of MIP-1α in preven-tion of EAE and infiltration of mononuclear cells into the CNS was demonstrated.26 We found no significant differences in the secretion of the chemokines MIP-1α and MIP-1β in the supernatant of DCs (Figure 2(b)).

We show here that fingolimod treatment in vitro had no effect on the viability of DCs and the proportions of all assessed DC subsets among the PBMCs in the periph-eral blood were increased (Figure 1(a)); the increase in frequency is most likely a result of less sequestration of DCs as opposed to lymphocytes in response to fingoli-mod treatment in vivo. We observed a relative increase in conventional DCs (CD1c+, CD16+ or CD141+) as well as blood plasmacytoid DCs in fingolimod-treated patients (Figure 1(b)). Plasmacytoid DCs are consid-ered to play a pivotal role in the immunoregulatory net-work in MS because of their tolerogenic capacity. In EAE, plasmacytoid DCs negatively regulated encepha-litogenic CD4+ T cell responses,27 and there is evidence that plasmacytoid DCs are an important cofactor for the amelioration of EAE with S1P receptor agonists.20 Immunomodulatory therapy with IFN-β in MS has

been shown to act at the level of plasmacytoid DC homeostasis, suggesting a therapeutic relevance of fin-golimod-induced elevation of plasmacytoid DCs in the blood.28 Overall, we conclude that fingolimod treat-ment had no harmful or sequestrating effects on DC subsets in MS and their preservation might be benefi-cial for disease outcome.

In our study, monocytes also exhibited a relative increase in frequency in fingolimod-treated compared to untreated MS patients (Figure 1(c)), whereas the absolute number of monocytes was not significantly altered by fingolimod treatment compared to untreated patients (Figure 1(a)). In animal models, modulation of monocytes through S1P receptor agonists has been described.10

We found that monocytes from fingolimod-treated MS patients showed a reduction in IL-1β secretion upon LPS stimulation (Figure 3(a)), which is in line with previous murine data suggesting a direct effect on monocyte activation.10

Proliferation of murine T cells co-cultured with fin-golimod-treated bone marrow-derived DCs was reduced in vitro (Figure 4(b) and 4(c)), indicating that fingolimod reduced the T cell stimulatory capacity of DCs, which has been shown to be necessary for the survival of encephalitogenic T cells in the effector phase of the immune response.29

In vitro fingolimod treatment of murine mature DCs significantly reduced their production of the pro-inflammatory cytokines TNFα, IL-1β and IL-6 (Figure 4(c)), mimicking the result with CD1c+ DCs and monocytes from fingolimod-treated MS patients (Figure 2(b) and Figure 3(b)). These cytokines have been shown to promote CNS tissue damage in EAE.30 In humans, IL-1β is required for Th17 differentia-tion.31 In addition to its potential direct toxicity, TNFα perpetuates the inflammatory process through recruit-ment of cells.32 In this study, we found that fingoli-mod treatment reduced the ability of MOG35–55-loaded murine DCs to prime antigen-specifically naive 2D2 T cells into pro-inflammatory Th1 cells.

Previous studies in EAE reported that fingolimod treatment resulted in reduced numbers of Th1 and Th17 cells in the CNS.33 In accordance with our find-ings, allogeneic naive T cells co-cultured with fingoli-mod-treated human DCs have been reported to differentiate into a markedly lower quantity of IFN- γ-producing T cells.7 Interestingly, fingolimod did not affect Th1 and Th17 differentiation of unspecifically stimulated murine CD4+ T cells, which emphasizes an





APC-mediated effect of fingolimod in the altered Th differentiation.34

There was a mild but not significant correlation between mean lymphocyte and PBMC count, and relative reduc-tion of annualized relapse rate in individual patients of our cohort (supplemental Figure 2(b) and 2(c)). In addi-tion, the TNFα and MIP-1β gene expression by CD1c+ DCs from fingolimod-treated patients showed a trend towards correlation with the annual relapse rate (supplemental Figure 2(e)). This relationship must be confirmed in future studies including more patients.

Despite different mechanisms of action, a similar reduction of the Th1-polarizing capacity of human DCs in MS has been reported recently for fumarate.35 Glatirameracetate has also been shown to modulate the properties of both monocytes and DCs.16 In addi-tion, daclizumab, a humanized monoclonal antibody against CD25, has been reported to inhibit potently activation of antigen-specific T cells by modification of mature DCs.36

ConclusionsTaken together, our data indicate that fingolimod exerts its disease-modulating activity in MS patients by rebal-ancing the immune tolerance networks by modulation of APCs in addition to its effect on lymphocyte traf-ficking. This highlights the relevance of APCs as a therapeutic target for influencing MS pathology.

AcknowledgementsThe authors would like to thank Andreas Zymny, Heike Ehrengard, Christine Oswald, Birgitt Hohmann and Ilona Kirchhoff for technical assistance and Darragh O’Neill for proofreading the manuscript.

Authors’ contributions Dr Luessi: data acquisition, data analysis, drafting of the manuscript, and review of manuscript for impor-tant intellectual content. Mr Kraus: data acquisition, data analysis, and drafting of the manuscript. Dr Trinschek: data acquisition, and data analysis. Mr Lerch: data acquisition, and data analysis. Dr Roberg: data analysis, and review of manuscript for important intellectual content. Dr Poisa-Beiro: data acquisition, and data analysis. Dr Klotz: review of manuscript for important intellectual content. Dr Wiendl: review of manuscript for important intellectual content. Dr Bopp: review of manuscript for important intellectual content. Dr Jonuleit: study design and concept, and review of manuscript for important intellectual con-tent. Dr Jolivel: data acquisition, data analysis, and drafting of the manuscript. Dr Witsch: drafting of the

manuscript, and review of manuscript for important intellectual content. Dr Zipp: study design and con-cept, drafting of the manuscript, and review of manu-script for important intellectual content.

Conflict of interestThe authors declare no financial conflict of interest.

FundingThis study has been supported by the German Research Foundation (DFG, SFB-TR 128/B4 to FZ), the Federal Ministry for Education and Research (BMBF, Competence Network on Multiple Sclerosis (KKNMS)), and the Johannes Gutenberg-University Mainz (JGU) to FL (MAIFOR grant and grant from the Inneruniversitäre Forschungsförderung (Stufe I)).

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