Supporting InformationThell et al. 10.1073/pnas.1519960113SI Materials and MethodsPeptide Synthesis.Amino acid were purchased from Iris Biotech orPepChem as follows: Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Boc-Cys(Trt)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH. O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluroniumhexafluorophosphate (HBTU) was from PepChem. N,N-dDimethylformamide (DMF) and dichloromethane (DCM)were from Thermo Fisher Scientific. Diisopropylethylamine(DIPEA), guanidine hydrochloride, reduced and oxidized glu-tathione, piperidine, trifluoroacetic acid (TFA), trichloroaceticacid (TCA), triisopropylsilane (TIPS), 3,6-dioxa-1,8-octanedi-thiol, Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 4-mercaptophenylacetic acid, α-cyano-hydroxy cinnamic acid, and4-nitrophenyl chloroformate were from Sigma-Aldrich. Gel filtra-tion PD-10 Sephadex G-25M columns were from GE Healthcare.Synthesis followed a recent protocol for the generation of thi-

oesterpeptides using Fmoc-SPPS and their use in native chemicalligation, and its adaptation to synthesize cyclotides assisted bymicrowave heating (according to references used in the main text).A rink type 3-(Fmoc-amino)-4-aminobenzoyl AM resin (Dawson’sDBz resin), 100–200 mesh, from Novabiochem (Merck-Millipore)with a substitution value of 0.49 mmol/g was used as starting pointfor the synthesis. Resin was allowed to swell in DMF for 2 h. TheFmoc protecting group was removed using 20% (vol/vol) piperi-dine solution in DMF two times for 5 min. Two equivalents ofamino acid were dissolved in five equivalents of 0.5 M HBTUsolution and activation was achieved by adding 10 equivalents ofDIPEA base. The mixture was rigorously shaken for 1 min beforeadded to the deprotected resin. The manual coupling of the firstamino acid was repeated once. Afterward elongation of all otherresidues was performed on a Liberty1 microwave peptide syn-thesizer (CEM Corp.) applying an optimized microwave assistedFmoc/HBTU SPPS protocol. The Nbz formation was performedusing 4-nitrophenylchloroformate in DCM (16 equivalentsbased on the net weight gain after peptide synthesis). Theacylation reaction was carried out at 23 °C for 1 h. Activationwas achieved using 195 equivalents of DIPEA in DMF for25 min at 23 °C. The Nbz-peptide was cleaved off the resinusing TFA/TIPS/ddH2O 99.5/0.25/0.25% (vol/vol/vol) for 3 h at23 °C. The released Nbz-peptide was precipitated with dieth-ylether. Peptide precipitate was dissolved in 50% CH3CN/0.05% TFA in double-distilled H2O and lyophilized. The thi-oesterification was performed in a ligation buffer containing200 mM mercapto-phenyl acetic acid, 20 mM TCEP, and 6 Mguanidine-HCl in 200 mM phosphate solution adjusted to pH7.0–7.2. Nbz-peptide was dissolved in the ligation buffer in1 mM and the solution was stirred for 24 h. The cyclic peptidewas purified from the ligation reagents using Sephadex G25 gelfiltration columns. Peptide fractions were then lyophilized, be-fore final purification of native cyclotide was achieved usingpreparative HPLC on a diChromKromasil C18 (250 × 20 mm, 10 μm)column (dichrom). Linear gradients from 5 to 80% solvent B[double-distilled H2O/CH3CN/TFA, 10/90/0.1% (vol/vol/vol), sol-vent A 0.1% TFA aqueous] was applied to achieve peptide sepa-ration. Quality control was judged upon A215 and A280 UV tracesfrom an analytical C18 separation using a Phenomenex Kinetex(150 × 3 mm, 2.1 μm) column. Peptide purity ≥90% was acceptedor otherwise submitted to another purification cycle.

Animals and Ethics. Mice used for experiments and ethics per-mission for animal care have been described in the main text.

Genotyping.Mice were earmarked 3 to 4 wk after birth. DNA fromlysed (proteinase K lysis buffer) ear tissue was subjected to directPCR using GoTaq Polymerase (Promega). Using the following2D2 and control primers (Microsynth AG, Balgach, Switzerland)specific PCRs were performed: 2D2 forward: 5′-CCCGGGCA-AGGCTCAGCCATGCTCCTG-3′, 2D2 reverse: 5′-GCGGCC-GCAATTCCCAGAGACATCCCTCC-3′ and internal controlprimer forward: 5′-CTAGGCCACAGAATTGAAAGATCT-3′,reverse: 5′-GTAGGTGGAAATTCTAGCATCATCC-3′.

EAE. C57BL/6 mice of both sexes were used due to the fact thatno sex-specific differences were registered. Mice were immu-nized at day 0 with 75 μL of equal amounts of MOG (MOG35–55,1 mg/mL; Charite Berlin) and incomplete Freud’s adjuvants(Sigma-Aldrich) supplemented with 10 mg/mL Mycobacteriumtuberculosis H37Ra (Difco) s.c. into the left and right flank.Additionally mice received i.p. 200 ng pertussis toxin (Milli-pore,) solubilized in 100 μL PBS at day 0 and day 2. Mice wereobserved daily for clinical signs as described in the main text.C57BL/6 mice were immunized at day 0 according to the pro-tocol described recently in Sahin et al. (44). Progression ofEAE was divided into five clinical stages: score 0, no signs;score 1, complete tail paralysis; score 2, partial paraparesis;score 3, severe paraparesis; score 4, tetraparesis; and score 5,moribund condition. When a mouse meets exclusion criteria(score >4, loss of weight >20%, no water and food uptake, nogrooming) then it is considered moribund. Mice were eutha-nized by deeply anesthetizing them with ketamine reaching ascore of 3–4 due to ethical guidelines. Records were kept ofanimal numbers and treatment details; while scoring the op-erator was blinded to the treatment records. Two independentassistant operators performed random spot checks in a blindedmanner to confirm the assessment of the main operator. Thisresulted in a blinded scoring procedure. For optimized effectsin T-cell detection, histological assessments, and cytokineanalysis following ex vivo restimulation it is critical to finalize theexperiment at the disease peak. Survival analysis, includingmoribund mice, was performed using a Kaplan–Meier plot.

Histology. Euthanized mice were perfused intracardiacally withPBS. Spinal cords were then isolated, fixed in 4% (vol/vol)buffered formalin, and processed for histological evaluation.Sections were stained with H&E and LFB using standard proto-cols. Furthermore sections were analyzed for CD3 surface ex-pression using immunohistochemistry with rat-anti-CD3 (AbDSerotech) and goat-anti-rat (Vector Laboratories) antibodies. Aminimum of three cross-sections of each animal were evaluatedhistologically. Inflammatory index was calculated as follows: Thenumber of perivascular infiltrates in spinal cord cross-sectionswas divided by the number of used cross-sections for each ani-mal. Therefore, a higher inflammatory index indicates more in-flammatory infiltrates. To evaluate the extent of demyelinatedarea, total and demyelinated area of each cross section wasmeasured in the KLB staining. The demyelinated area was thencalculated and plotted as percent of total cross-section. Image J(NIH) was used for all histological evaluations. Stomach and in-testine of euthanized mice were perfused with 10 mL PBS beforefixation in 4% (vol/vol) buffered formalin. Gastrointestinal sec-tions were stained with H&E and evaluated as described above.

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In Vivo Imaging.EAE-induced [T20K]kB1-treated (10 mg/kg i.p. onday −7) and untreated control EAE mice received RediJectD-Luciferin bioluminescent substrate (PerkinElmer) i.v. on day 12after MOG immunization according to the manufacturers’ pro-tocol. Monitoring with the IVIS (PerkinElmer) was performed onday 13 by measuring chemiluminescence signal, induced by theRediJect substrate. Higher chemiluminescence levels representenhanced inflammation in the appropriate regions. Quantificationwas performed with Living Image software (PerkinElmer). VivoTag680 XL (PerkinElmer) peptide was dissolved in 0.1 M NH4HCO3buffer, pH 8.5. A 20-fold molar excess of labeling reagent wasprepared in anhydrous DMSO and the reaction was allowed toproceed at 23 °C for 4 h. Reaction was stopped with 0.1% TFA.Purification of labeled peptide from excess of reagent was achievedby semipreparative HPLC using a diChrom Kromasil C18 column(250 × 10 mm, 5 μm) and linear gradients as indicated above.HPLC fractions were analyzed via MALDI-TOF mass spectrom-etry in the negative reflector mode. Purity of peptide samples wasdetermined to be ≥95% based on analytical HPLC and detectionof VivoTag label in the A280 UV trace. Labeled [T20K]kB1 wasinjected intravenously, intraperitoneally, and per oral gavage intonaïve mice. Fluorescence signal (excitation: 665 ± 5 nm, emission:688 ± 5 nm) was monitored after 5 min, 20 min, 40 min, and 1, 2,4, 8, 24, 32, 48, 56, and 72 h. Organs (spleen, liver, kidney,stomach, and intestine) of euthanized mice were screened for thefluorescence and quantified by using IVIS Living Image software.

Serum Analysis. Blood sampling was performed for indicated ex-periments. Sera were analyzed using the Reflotron Plus System(Roche Diagnostics) according to the manufacturer’s instructions.

Serum Uptake Analysis of Cyclotides After Oral Administration.C57B1/6mice were treated orally with 20mg/kg [T20K]kB1 peptide and freshblood was used for analysis of peptide in blood after 1 and 2 hpostadministration. The total citrated blood (∼1.5 mL) was sub-mitted to homogenization and cell lysis using the Precellyser 24(Bertin Technologies). Proteins were removed using TCA pre-cipitation with a final concentration of 10% (wt/vol) TCA. To en-hance peptide recovery 45% (vol/vol) CH3CN (final) was added tothe solution. The precipitation was allowed to proceed for 1.5 h at4 °C and afterward proteins were pelleted by centrifugation at12,000 × g for 30 min. The supernatant was lyophilized and thendissolved in 100 μL 0.1% TFA (aqueous) and centrifuged for10 min at 12,000 × g before analysis. The samples were desalted andconcentrated using ZipTips (Millipore). For mass spectrometryanalysis a MALDI-TOF/TOF 4800 Analyzer was used (AB Sciex).The desalted samples (0.5 μL) were mixed with 6 μL of α-cyano-hydroxy cinnamic acid matrix, saturated in double-distilled H2O/CH3CN/TFA 50/50/0.1% (vol/vol/vol) and 0.5 μL were spotted ontoa target plate and allowed to air-dry in the dark. External calibrationwas performed on a daily basis applying calibration mix 1 (Laser-biolabs). Mass spectra were recorded in the range of 2,500–3,500Da with optimized settings for laser intensity number of shots peraverage spectrum and digitizer adjustment to obtain acceptablespectra. As a control measurement, the corresponding amount of1% [T20K]kB1 of the total dose of 20 mg/kg was spiked into freshblood. After 1 h of settling time at 4 °C the samples were processedaccordingly as described above.

Splenocyte Isolation and Restimulation. Spleens of euthanized micewere prepared in RPMI media (Sigma-Aldrich) supplementedwith 10% FCS (Sigma-Aldrich), penicillin (100 U/mL; Sigma-Aldrich), and streptomycin (100 μg/mL; Sigma) on ice. To re-ceive a homogeneous cell suspension, spleens were meshedthrough 40-μm nylon sieves and centrifuged for 5 min at 300 × g.The cell pellet was incubated for 1 min with 1 mL erythrocytelysis buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mMNa2EDTA, pH 7.2–7.4). The reaction was stopped by addition of

10 mL full media and centrifugation for 5 min at 300 × g. Spleniccells (3 × 106/mL) were stimulated ex vivo with or without 30 μg/mL MOG for 48–72 h at 37 °C in humidified atmosphere of 5%CO2. Treatment of splenocytes was performed according to theappropriate experiments, described in figure legends.

CNS T-Cell Isolation. Immune cells were isolated from the CNS bydigesting brain of appropriate animals with a mixture of 5 mL col-lagenase D (0.233 U/mg; Roche) and DNase I (Roche) (0.17 U/mLcollagenase D and 0.01 mg/mL DNase I per organ). Brains wereincubated for 30 min at 37 °C in a shaking incubator. For furtherdisruption of the tissue EDTA (pH 8.0 in PBS) was added for a finalconcentration of 2 mM and suspension was pipetted up and downfor 5 min at 23 °C before filtering through a 70-μm cell strainer. Cellswere washed with PBS at 400 × g for 8 min at 4 °C before re-suspension in RPMI media. Cells were used for FACS analysis orseeded at a concentration of 3 × 106/mL and stimulated ex vivo with30 μg/mL MOG. Supernatants of stimulated cells were used fordetection of cytokine secretion using an ELISA.

T-Cell Proliferation and Flow Cytometry. Isolated T cells were in-cubated with 5 μM carboxyfluorescein diacetate succinimidylester (CFSE; eBioscience) in PBS per 1 × 107 cells for 10 min at37 °C. The reaction was stopped by adding media containing 10%FCS. Cells were washed with media and incubated according to theappropriate protocols. Before analysis, cells were stained with AlexaFluor 647 anti-mouse CD3, clone 17A2 (BioLegendsany) accordingto the manufacturer’s instructions. FACS acquisition was performedon a BD canto flow cytometer (BD Biosciences). Further analysiswas performed using BD FACSDIVA software (BD Biosciences).For quantification of CD3-, CD4-, and CD8-positive cells in theCNS, the following antibodies from eBioscience were used: CD3e(APC-eFluor780, 1465-2C11), CD4 (FITC, GK1.5), and CD8a(AF700, 53-6.7). Cells were incubated for 20 min at 4 °C on a shaker,before adding 150 μL FACS buffer to spin down at 500 × g for 3 minat 23 °C (brake low). After discarding supernatant, cells were re-suspended in 100 μL FACS buffer supplemented with 7-AAD (1:40).Flow cytometric measurement (acquisition time: 60 s) was performedusing a Gallios flow cytometer from Beckman Coulter. For analysisCXP and Kaluza software were used (Beckman Coulter).

ELISA. To evaluate specific cytokine release of ex vivo stimulatedsplenocytes, antibodies and Ready-SET-Go! Cytokine ELISA kitswere acquired from eBioscience and R&D Systems. Experimentswere performed according to manufacturer’s instructions. Absor-bance was measured on the Synergy ELISA plate reader at 450 nmafter colorimetric reaction of TMB 2 Component Microwell Per-oxidase Substrate Kit from KPL (Medac) and 0.5 M H2SO4.

RNA Isolation and Quantitative Real-Time PCR. RNA of pretreatedcells was isolated via Qiagen RNA Isolation kit according to themanufacturer’s protocol. Quantification of nucleic acid was de-termined by using Nanodrop (Peqlab). Five hundred nanogramsof RNA was used for first-strand cDNA synthesis following man-ufacturers’ instructions of High-Capacity cDNA Reverse Tran-scription Kit (Applied Biosystems). Expression of mRNA wasquantified by real-time PCR using Fast SYBR Green Master Mix(Applied Biosystems) with the StepOne Real-Time PCR System(Applied Biosystems). Levels of target genes were normalized toHPRT and described as fold increase of unstimulated control cells.The following primer (Microsynth AG) sequences were used:HPRT forward: 5′-CGCAGTCCCAGCGTCGTG-3′, HPRTreverse: 5′-CCATCTCCTTCATGACATCTCGAG-3′, IL-2 forward:5′-TGCAACTCCTGTCTTGCATT-3′, IL-2 reverse: 5′-GCCTTC-TTGGGCATGTAAAA-3′, IFN-γ forward: 5′-TGAGCTCATTG-AATGCTTGG-3′, IFN-γ reverse: 5′-ACAGCAAGGCGAAAAA-GGAT-3′, IL-17A forward: 5′-TGAGCTTCCCAGATCACAGA-3′,IL-17A reverse: 5′-TCCAGAAGGCCCTCAGACTA-3′.

Thell et al. www.pnas.org/cgi/content/short/1519960113 2 of 12

Statistical Analysis. Statistical significance of data was calculated byuse of GraphPad Prism software (GraphPad Software). Two-wayANOVA analyzes were used to analyze two groups over time. Twogroups were compared by using unpaired two-tailed Student’s t test.

Survival was determined by log-rank test comparing indicated groups.Results are presented as the mean ± SEM. The P values <0.05 wereconsidered statistically significant and are expressed in the figures asfollows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Fig. S1. Quality control of peptide synthesis and derivatization. (A) Native folded peptide [T20K]kB1 was purified with preparative and semipreparative HPLCand final peptide was evaluated for purity by analytical HPLC. A chromatogram with the corresponding A280 trace is shown indicating a purity ≥95%.(B) Peptide [T20K]kB1 was evaluated via MALDI-MS for identity and a representative spectrum is shown indicating the mass 2,918.09 m/z [M+H]+. (C) Labeled[T20K-VivoTag]kB1 was purified from nonlabeled peptide, and excess of labeling reagent by semipreparative HPLC. Purity of ≥95% is indicated by an analyticalHPLC chromatogram showing the A280 trace of the analysis. (D) Purified peptide [T20K-VivoTag]kB1 was analyzed with MALDI-TOF in the negative ionizationmode. Mass signals for the [M-H]− ion (4,150.47 m/z) and for the [M-2H]2- ion (2,074.77 m/z) were recorded. The spectrum shows several degradation artifactsindicated by asterisks due to the laser-induced desorption.

Thell et al. www.pnas.org/cgi/content/short/1519960113 3 of 12

Fig. S2. Cytokine response of EAE-induced or 2D2 isolated splenic T cells after MOG restimulation. (A) Restimulation scheme. EAE-induced antigen-presentingcells (APC) get activated in presence of MOG and stimulate T cells to secrete IL-2, which acts in an autocrine way to its IL-2 cell surface receptor (IL-2R) to induceproliferation and inflammatory cytokine response. (B) Proliferation was analyzed via flow cytometry measuring CFSE dilution of CD3-positive EAE-inducedsplenic T cells, incubated for 2 h with [T20K]kB1, restimulated (+) for 72 h with MOG (n = 3), and compared with nonstimulated cells (−). (C) Cytokine secretionfrom ex vivo MOG-restimulated splenic T cells of EAE-induced mice. Data (mean ± SEM) were normalized to the MOG control (n = 3) after treatment with[T20K]kB1, [V10K]kB1, and cyclosporine A (CsA, 5 μg/mL) and restimulation with MOG. (D) Quantification of cytokine-specific mRNA expression was performedby real-time PCR of splenic EAE T cells incubated for 2 h with [T20K]kB1 and restimulated with MOG for the indicated time period. Data (mean ± SEM) werenormalized to HPRT (n = 4, except n = 2 for IFN-γ and IL-17A: 15 min, 30 min, 1 h). (E) In vitro treatment of genetically modified 2D2 splenocytes with cyclotides.Proliferation (percent) of splenic T cells from 2D2 mice incubated for 2 h with [T20K]kB1 and restimulated for 3 d with MOG was analyzed via flow cytometrymeasuring CFSE dilution (n = 3). IL-2, IFN-γ, and IL-17A cytokine release of ex vivo MOG-restimulated (IL-2 48 h; IFN-γ and IL-17 72 h MOG stimulation) splenicT cells from 2D2 mice, after a preceding incubation for 2 h with [T20K]kB1 (measured by ELISA). Data illustrate mean ± SEM values of IL-2, IFN-γ, and IL-17Aconcentrations in picograms per milliliter (IL-2 n = 4, IFN-γ n = 3; IL-17A n = 2). Significance was calculated using Student’s t test, comparing indicated groups.

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Fig. S3. Body weight of EAE-induced and cyclotide-treated mice. Body weight of EAE-induced mice shown in percentage (100% represents the originalweight at the beginning of the experiment). Day 0 was the day of MOG immunization. Body weight of mice treated (A) at day −7 with cyclotides (Fig. 2A); (B) aweek before, a week after, and at the day of immunization (Fig. S4A); (C) one time (Fig. S4B); or (D) three times with 3-d intervals at a score of 2 with [T20K]kB1i.p. (Fig. 2F), (E) orally (p.o.) with two different doses of the active cyclotide at day −7 (Fig. 3A), (F) orally at a score of 2 with 20 mg/kg three times, in3-d intervals (Fig. 3B), (G) orally at day −7 or daily with [T20K]kB1 or fingolimod (FTY) (Fig. S9A), or (H) orally or i.p. three times at score 2 with [T20K]kB1 or FTY(Fig. S9B).

Thell et al. www.pnas.org/cgi/content/short/1519960113 5 of 12

Fig. S4. Activity and therapeutic effect of cyclotides. (A) EAE clinical disease score (mean ± SEM) of MOG-immunized mice [n = 10; T20K (day 7) n = 9] treatedwith [T20K]kB1 at day −7 (red sphere), day 0 (violet square), day 7 (blue diamond), or untreated control (black triangle, dashed line). (B) EAE clinical score ofmice treated at a score of 2, once with 10 mg/kg [T20K]kB1 i.p. (red sphere, n = 7) or left untreated (control group; black triangle, dashed line; n = 6). Sig-nificance was determined by two-way ANOVA comparing indicated groups at the day of the treatment. (C) H&E and LFB staining of spinal cord sections fromEAE-induced animals. Bar diagrams represent inflammatory index and percentage of demyelinated area. (Scale bars: 500 μm.) (D–F) Quantitative cytokinerelease of splenic T cells from indicated EAE mice after ex vivo ± MOG restimulation (ELISA). (D) Cytokine response from spleens isolated from mice treated atday −7, 0, or 7 with [T20K]kB1 (Fig. S4A); (E) from [T20K]kB1-treated and untreated MOG-immunized mice (Fig. S4B); (F) splenic T cells of three times i.p.treated mice (Fig. 2F). Data represent mean ± SEM values of IL-2, IFN-γ, and IL-17A cytokine concentrations in picograms per milliliter. Student’s t test was usedto calculate significance (comparing indicated groups).

Thell et al. www.pnas.org/cgi/content/short/1519960113 6 of 12

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Fig. S5. Effect of cyclotides on CNS T cells. (A) EAE clinical score (mean ± SEM) of mice treated i.p. at a score of 2 three times with 3-d intervals with 10 mg/kg[T20K]kB1 or left untreated (n = 4). (B) Quantification of CD3-, CD4-, or CD8-positive cells from the brain of cyclotide-treated or untreated mice (Fig. S5A) usingflow cytometric analysis. (C) CD3 immunohistochemistry staining of spinal cord sections from EAE-induced animals (Fig. 2E). Each image (white box) is illus-trated with higher magnification (right side). Bar diagrams represent number of counted CD3-positive cells. (Scale bars: 500 μm.) (D) Cytokine release (mean ±SEM values of IL-2 concentrations in picograms per milliliter) of CNS cells from indicated EAE mice (Fig. S5A) after ex vivo ± MOG restimulation (measured byELISA). Student’s t test was used to calculate significance (comparing indicated groups).

Thell et al. www.pnas.org/cgi/content/short/1519960113 7 of 12

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Fig. S6. Oral activity and therapeutic properties of cyclotides. (A) Cytokine response of ex vivo MOG-restimulated splenic T cells from orally cyclotide-treatedand untreated MOG-immunized mice measured by ELISA: restimulated for 48 h (IL-2) or for 72 h (IFN-γ and IL-17A) with MOG (Fig. S9A). Data illustrate mean ±SEM values of IL-2, IFN-γ, and IL-17A concentrations in picograms per milliliter. Significance was calculated using Student’s t test, comparing indicated groups.(B) Representative histological H&E and LFB images of spinal cord from orally cyclotide-treated or untreated MOG-immunized animals (n = 7) of two in-dependent experiments (Fig. 3A and Fig. S9A). (C) H&E and LFB images of daily [T20K]kB1 (1 mg/kg; n = 5) treated or untreated (n = 3) EAE mice (Fig. S9A).(D) H&E and LFB images from three times orally cyclotide-treated (n = 7) and control (n = 10) animals (two independent experiments Fig. 3B and Fig. S9B). Eachimage (white box) is illustrated with higher magnification (right side). Bar diagrams represent inflammatory index and percentage of demyelinated areaof three cross-sections of each animal. Statistical significance between cyclotide-treated and untreated EAE mice was analyzed using Student’s t test. (Scalebars: 500 μm.)

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Fig. S7. Biodistribution of VivoTag-labeled cyclotide. [T20K-VivoTag]kB1 (10 mg/kg or 5 mg/kg) was injected i.v. (A) or i.p. (B) into naïve mice. Biodistributionof the labeled peptide was monitored at indicated time points (5 min, 40 min, and 4, 24, and 72 h) by using the IVIS. (C) VivoTag alone was either applied i.v. orby oral gavage (p.o.) and monitored as described above. (D) After 72 h organs of euthanized [T20K-VivoTag]kB1 or VivoTag-alone treated mice were scannedfor fluorescence intensity. Quantification was calculated using the IVIS Living Image software.

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Fig. S8. Evaluation of biochemical and physiological parameters of mice after oral cyclotide treatment. (A) H&E histology staining of the stomach and theintestine of [T20K]kB1-treated (20 mg/kg, orally, p.o.), fingolimod (FTY, 20 mg/kg or daily 1 mg/kg, orally), and untreated control mice (naïve) (n = 3). (Scalebars: 200 μm.) (B) Body temperatures and (C) body weight in percent monitored at day 0, 1, 3, 6, 9, 13, 17, and 20. Analysis of (D) lipid parameters (triglycerides)as well as (E) liver enzymes [i.e., aspartate (AST or GOT), alanine transaminase (ALT or GPT), and γGT] using the Reflotron Plus system at indicated time points.

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Fig. S9. Comparison of different compounds for treatment of EAE. Clinical score (mean ± SEM) of EAE-induced mice, which received either [T20K]kB1or fingolimod (FTY) at different doses (1; 20 mg/kg) and time points, that is, at day −7 and daily starting at day 0 (A) or three times at score 2 with 3-d intervals(B). Protocol for the FTY treatment schedule was adapted as described in the main text and modified according to cyclotide treatment experiments. (C) Survivalanalysis of EAE-induced animals treated with different immunosuppressive drugs or [T20K]kB1 (20 mg/kg orally at day −7). Statistical significance was cal-culated using a log-rank (Mantel–Cox) test.

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Table S1. EAE immunization scheme and EAE incidence of individual experiments

Treatment Dose Administration No. of mice/EAE incidence

In vivo activity[T20K]kB1 10 mg/kg i.p. n = 8/6V10K 10 mg/kg i.p. n = 8/6Control n = 14/13

Single-dose treatmentT20K (day −7) 10 mg/kg i.p. n = 10/2T20K (day 0) 10 mg/kg i.p. n = 10/5T20K (day 7) 10 mg/kg i.p. n = 9/6Control n = 10/9

Therapeutic treatmentT20K at score 2 10 mg/kg, 3× i.p. n = 7/*Control n = 5/*T20K at score 2† n = 4

Single-dose oral treatmentT20K 20 mg 20 mg/kg Oral n = 6/3T20K 10 mg 10 mg/kg Oral n = 6/4Control n = 5/4

Oral therapeutic treatmentT20K at score 2 20 mg/kg, 3× Oral n = 5/‡

Control n = 5/‡

Comparative fingolimod (FTY) vs. cyclotide ([T20K]kB1) treatmentT20K 20 mg (day −7) 20 mg/kg Oral n = 6/4FTY 20 mg (day −7) 20 mg/kg Oral n = 6/6T20K 1 mg daily (day 0) 1 mg/kg, daily Oral n = 6/0FTY 1 mg daily (day 0) 1 mg/kg, daily Oral n = 6/1T20K 3 × 20 mg at score 2 20 mg/kg, 3× Oral n = 6/§

FTY 3 × 20 mg at score 2 20 mg/kg, 3× Oral n = 6/§

Control n = 18/16

Treatment protocol of EAE experiments representing application type, dose, and number of animals. Incidence illustrated number of mice developing anEAE clinical score higher than 1:*incidence: 20/19–12, score 2; ‡incidence: 12/10, score 2; §incidence: 12/12, score 2; †refer to control of fingolimod comparison.

Movie S1. Clinical score and movement monitoring of cyclotide-treated mice. [T20K]kB1, [V10K]kB1, and control EAE mice were monitored during scoring(Fig. 2A). The mouse treated with [T20K]kB1 is labeled with a red tag and the one that received [V10K]kB1 with a white tag. The untreated control mouse isunlabeled. The [T20K]kB1 mouse shows a healthy behavior, meaning score 0, which is defined by the ability to move the tail tip. In comparison the [V10K]kB1and control EAE mice demonstrated a shambling walk induced by the initiating paraparesis, also assured by the tail paralysis, characterizing a score of ~1.5.

Movie S1

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