Chemotxis (% of dded ells) PBL totl dhesion (N ells/mm 2 /1.1 6 PBL) Frequeny (% ) PBL firm dhesion Supplementry Figure 1 4 4 3 3 2 2 1.1-4 1-3 1.1.2. 1 1 8 6 4 2 Adiponetin ( g/ml) - + Adiponetin ( g/ml) 8 6 4 2 d 1 8 6 4 2 Adiponetin ( g/ml) - + - + - + - + 4 1 L 2 CXCR3 DP-2 Adiponetin ( g/ml) 1-4.1 1 e 4 3 2 1 Adiponetin ( g/ml) - + - + - + - + CXCL12 CXCL1 PGD2 Supplementry Figure 1 Adiponetin does not influene integrin expression or PBL hemotxis. -, The effets of diponetin (- µg/ml) on () (n= 3-12) () firm dhesion (n= 6) nd () totl dhesion (n=3) on TNF-α&IFN-γ stimulted endothelil ells, N= Numer. d, The expressions of the integrin α 4 β 1 (VLA-4) nd α L β 2 (LFA-1), CXCR3 (the reeptor for hemokines CXCL9, 1 nd 11) nd DP-2 (the reeptor for PGD2) were mesured y flow ytometry on PBL pre-treted with diponetin ( μg/ml), n=3. e, Chemotxis of PBL pre-treted with diponetin ( μg/ml) ws quntified using trnswell Boyden hmer ssy. The perentge of PBL migrting ross the filter towrds CXCL12 (2 ng/ml), CXCL1 (8 ng/ml) nd PGD2 (1 nm) ws mesured, n=14-18. Dt re men±s.e.m nd were nlysed using ANOVA nd (, ) Dunnett post test, or (, d, e) y pired t-test ompred to no diponetin ontrol for eh ondition, P., P.1, P.1. = not signifint. Nture Mediine: doi:1.138/nm.3842
Supplementry Figure 2 Count Isotype AdipoR1 AdipoR2 AdipoR1 AdipoR2 Supplementry Figure 2 Expression of AdipoR1 nd AdipoR2 on HUVEC. Representtive flow ytometry dt of the expression of AdipoR1 nd AdipoR2 on HUVEC, n=4. Supplementry Tle 1 Fold hnge- Adiponetin treted to untreted ontrol VCAM-1.84 ±.9 E-seletin.69 ±.1 ICAM-1.91 ±.28 CXCL9.9 ±.16 CXCL1.94 ±.8 CXCL11.94 ±.13 Supplementry Tle 1 Effet of diponetin on the expression of dhesion moleules nd hemokines in HUVEC. The gene expression of VCAM-1, E- seletin, ICAM-1 nd CXCL9 to 11 were mesured in TNF-α&IFN-γ stimulted HUVEC using rel-time PCR. HUVEC were inuted with diponetin t µg/ml for 24 hours. All dt olleted in presene of diponetin ws normlised to the untreted ontrol. No signifint differene were found etween the onditions y ANOVA nd Dunnett post test, n=3. Dt re men±sd. Supplementry Tle 2 found m/z 14.3.3.ζδ sore sore Sequene 14.3.3.ζδ protein digestion YES 774.88 444. 86.8 SVTEQGAELSNEER Supplementry Tle 2 Effet of trypsin digestion on levge from 14.3.3.ζδ protein. Reominnt14.3.3.ζδ ws digested with trypsin for one hour nd smples were purified t 9% etonitrile nd nlysed y mss spetrometry. Protein dtse serh identified with signifint sore. Nture Mediine: doi:1.138/nm.3842
quntifition (ng/1 m illion PBL) Supplementry Figure 3.4.3.2.1. min min 3min 1h 3h 24h 774.88 Supplementry Figure 3 Identifition of., PBL were treted with diponetin for,, 3, 4 minutes, 1, 3 nd 24 hours. Superntnts were olleted nd were nlysed y mss spetrometry. ws quntified using syntheti rdiolelled, n=3. Dt re men±s.e.m nd were nlysed using ANOVA nd Bonferroni post test., MS spetr of syntheti, m/z= 774.88., Comprison MS/MS of the prent ion (774.88) derived from diponetin stimulted B ells (upper pnel) nd syntheti (lower pnel). = not signifint. Nture Mediine: doi:1.138/nm.3842
% enrihement firmly dhered B ells Totl PBL dhesion (ells/mm 2 ) PBL firm dhesion Supplementry Figure 4 f 4 3 2 1 6 4 2 R 2 =.72 EC=18.6pM -4-2 2 4 Log 1 onentrtion (ng/ml) 6.1 ng/ml 1 ng/ml 1 ng/ml 1 ng/ml d 1 8 6 4 2 g 4 3 2 1.1 ng/ml 1 ng/ml 1 ng/ml 1 ng/ml Adiponetin ( g/ml) (1 ng/ml) Srmled CTRL (1 ng/ml) - + - - - - - - - + + - e 8 6 4 2 Untreted (1 ng/ml) HUVEC HDMEC 4 3 2 2 1 on PBL/EC on PBL - + - - - - + - on EC - - - + Nive Memory Plsm Intermedite Trnsitionnl Supplementry Figure 4 regultes T ell trnsmigrtion., Trnsmigrtion of PBL preinuted with different onentrtions of (-1 ng/ml) ross TNF-α&IFN-γ treted HUVEC ws mesured in stti onditions using phse ontrst videomirosopy, n=3-4., Trnsmigrtion of PBL with diponetin ( μg/ml) or or srmled ontrol (1 ng/ml) in the sene of lumin, n=4., d, The effets of (-1 ng/ml) on () totl dhesion nd (d) firm dhesion on TNF-α&IFN-γ stimulted endothelil ells, n=4. e, Trnsmigrtion of PBL pre-treted with ws ompred on TNF-α&IFN-γ treted HUVEC nd HDMEC, n=3-4. f, Endothelil ells were ultured in low serum medium nd stimulted with TNF-α&IFN-γ for 24 hours in the sene of diponetin. 1) ws dded with the PBL to the endothelil ells or 2) the endothelil ells were pre-treted with nd PBL dded fter wshing wy the or, 3) PBL were pre-treted with, wshed nd dded to the endothelil ells, n=3. ws used t 1 ng/ml. g, The enrihment rtio of eh B ell suset on the endothelil surfe ws lulted y dividing the perentge of eh suset reruited on endothelil ells y the perentge of eh suset dded from the whole PBL (lood) stining determined y flow ytometry, n=3. Nive (mture) B ells were identified s CD38 + IgD + IgM +, memory B ells s CD38 - IgD - IgM - CD27 +, plsm ells s CD38 +++ IgD - IgM - CD27 -, intermedite B ells s CD38 ++ IgD + IgM + nd trnsitionl B ells s CD38 +++ IgD + IgM +. Dt re represented s men±s.e.m nd nlysed y () liner regression, ANOVA nd (-d, f) Dunnett or (g) Bonferroni post test or (e) 2-wy ANOVA. P., P.1, P.1. Nture Mediine: doi:1.138/nm.3842
Supplementry Tle 3 No tretment Srmled CTRL (1 ng/ml) (1 ng/ml) Memory CD4 + T-ells 2.9 ± 8.74 24.3 ± 43.8 24.8 ± 69.98 Memory CD8 + T-ells 919.37 ± 33.6 781.36 ± 377.44 749.61 ± 323.89 Neutrophils 863.3 ± 17.18 718.6 ± 91.26 711.77 ± 178.13 CD14 + CD16 - monoytes 39.8.2 ± 449.97 1233.23 ± 664.88 1316. ± 63.9 CD14 + CD16 + monoytes 84.34 ± 494.94 68.4 ± 343.39 88.94 ± 484.91 Supplementry Tle 3 Effet of on the totl dhesion of pre-sorted leukoyte susets. Dt re totl dhesion in stti trnsmigrtion ssy s numer of ells/mm 2 /1 million of ells dded. No signifint differenes were oserved etween the susets y ANOVA nd Dunnett post test, n=3-4. Dt re men±sd. Supplementry Tle 4 No tretment Adiponetin ( µg/ml) Firm dhesion Trnsmigrted totl Firm dhesion Trnsmigrted totl Memory CD4 + T-ells 298 3 4128 2379 44 3923 Memory CD8 + T-ells 2 627 1679 889 468 137 Supplementry Tle 4 Effet of diponetin on the totl dhesion of CD4 + nd CD8 + memory T-ells. Firmly dhered ells were olleted using EDTA nd trnsmigrted with utse. Both popultions were lelled with CD4, CD8 nd CD4RO nd nlysed using flow ytometry. The whole ells were quired on the flow ytometer llowing quntifition of solute numers. No signifint differenes were oserved etween the susets. Dt re men. Supplementry Tle quntifition (ng/1 ells) Nïve B-ells.71 ±.96 Memory B-ells.14 ± 7.8 Plsm ells 14.33 ± 13.37 Supplementry Tle Anlysis of the pity of B ell susets to serete. Nïve (CD38 + IgD + IgM + ) nd memory (CD38 - IgD - IgM - CD27 + ) B-ells nd plsm ells (CD38 +++ IgD - IgM - CD27 - ) were isolted using utomted ell sorting, inuted with diponetin ( μg/ml) for one hour nd superntnts were nlysed for using mss spetrometry. Quntity of sereted y eh susets ws lulted for 1, ells, n=3-6. Dt re men±sd nd were nlysed using ANOVA nd Bonferroni post test. No signifint differenes were oserved. Nture Mediine: doi:1.138/nm.3842
Response (% of Control) Supplementry Figure 12 1 8 6 4 2 Isotype ontrol Untreted Nt Biotin CDH DAPI CDH DAPI CDH / CDH DAPI Lipofetmine ontrol CDH DAPI Control sirna CDH DAPI 6 4 2 Control Control sirna Untreted (1 ng/ml) CDH -/- d RU 3. 3 2. 2 1.. -. : 24-77 µg/ml (.6- µm) -1-1. -2 2 4 6 8 Time Supplementry Figure Cdherin- (CDH) is the endothelil reeptor for., Effet of iotinyltion on the pity of (1 ng/ml) to inhiit trnsmigrtion. Biotin ws dded t the N-terminus (Nt Biotin) of, n=3., The effets of ontrol or CDH speifi sirna on the protein expression of CDH in TNFα&IFN-γ stimulted HDMEC, determined y onfol mirosopy (CDH: green, DAPI: grey). Sle rs: 3 µm., The effets of ontrol or CDH speifi sirna on T ell trnsmigrtion ross HDMEC, n=. d, Binding of on immoilised CDH ws shown using Biore tehnology. Dt re represented s men±s.e.m nd nlysed y ANOVA nd Dunnett post test, P., P.1. = not signifint. Nture Mediine: doi:1.138/nm.3842
Unstimulted No sirna CTRL SP2 fold hnge (to unstimulted ontrol) 1 SP2 kd 4 mrna reltive expression (to 18s endogenous ontrol) Supplementry Figure 6.2.2..1. d. HUVEC B ells HUVEC SPHK1 B ells SPHK2 e SP2 -/- TNF- &IFN- f TNF-α/&FN-γ 4 4 3 2 1 Untreted (1 ng/ml) 3 2 1 3 2 1 Untreted Control SP2 -/- (2 ng/ml) S1P ( nm) S1PR ntgonist (1 M) - + - + + - - + + + - - - - + (2 ng/ml) SPHK1 inhiitor ( M) SPHK/21 inhiitor ( M) - + - + + + - - - - + - - - - - - + S1P ( nm) g 4 3 2 1 (2 ng/ml) - + - - + + - + + S1P ( nm) - - + - - + - - + Control sirna SP2 -/- Supplementry Figure 6 indues S1P seretion y endothelil ells., Levels of SPHK1 nd SPHK2 mrna expression determined y rel-time PCR of RNA from B ells nd HUVEC, n=2-., mrna expression of SP2 on unstimulted nd TNF-α&IFN-γ stimulted HDMEC trnsfeted with ontrol sirna (Control) or sirna trgeting SP2, n=6., The effets of ontrol or SP2 speifi sirna on the protein expression of SP2 ws determined y Western Blot in HDMEC. d, The effet of SP2 speifi sirna on T ell trnsmigrtion in the presene of, n=3-. e, The effets of S1P t nm in presene of nd/or S1PR ntgonist (1 µm), n=-9. f, The effets of S1P t nm in presene of nd/or SPHK1 nd SPHK1&2 inhiitors ( µm), n=4-9. g, The effets of S1P t nm in presene of nd/or SP2 sirna knok-down, n=4-9. Dt re represented s men±s.e.m nd nlysed y ANOVA nd (, e-g) Dunnett or () Bonferroni post test or (d) pired t-test ompred to no ontrol for eh ondition, P., P.1, P.1. = not signifint. Nture Mediine: doi:1.138/nm.3842
PBL rolling Supplementry Figure 7 Count Isotype ontrol Untreted on ICAM CXCL1 on ICAM CXCL1 nd S1P on ICAM Cirulting Count Isotype ontrol Untreted on ICAM KIM127= 17.7% CXCL1 on ICAM KIM127= 32.6% CXCL1 nd S1P on ICAM KIM127= 23.6% Cirulting KIM127= 6.86% S1PR1 KIM127 1 Adiponetin ( g/ml) + - Supplementry Figure 7 indues up-regultion of S1P reeptors on T ells., The expression of S1PR1 on memory T ells (CD3 + CD4RO + ) in the irultion or ound to ICAM in the sene or presene of CXCL1 (8 ng/ml) ± S1P (1 µm), n=4., The expression of the intermedite ffinity site of LFA-1 (KIM127) on memory T ells (CD3 + CD4RO + ) in the irultion or ound to ICAM in the sene or presene of CXCL1 (8 ng/ml) ± S1P (1 µm), n=4., Rolling of lymphoytes on TNF-α&IFN-γ stimulted HDMEC pre-treted with diponetin ws mesured under flow onditions, n=6. Dt re represented s men±s.e.m nd nlysed y pired t-test. = not signifint. Nture Mediine: doi:1.138/nm.3842
Asolute numer of F4/8 + /podoplnin + mrophges in the peritoneum PBS Zymosn PBS Zymosn PBS Zymosn PBS Zymosn Asolute numer of CD11 + ells in the peritoneum Men free flowing ells / min Numer of CD19 + Bells Numer of B22 + B ells Men CD3 + T ells/ infetious foi Supplementry Figure 8 Srmled Control CD3 F48 CD3 F48 1 Control 2 1 d 3 2 1 e 1 8 6 4 2 f Srmled ontrol Srmled ontrol g Srmled ontrol 2,,, 1,, 1,, WT n=3-4 B ell-defiient n=4 WT n=3-4 B ell-defiient n=4 Supplementry Figure 8 inhiits T ell migrtion in vivo.,, The effet of on the reruitment of T ells into infetious foi in the liver of Slmonell infeted C7BL/6 WT mie, n 4. () Dt shown s men numer of CD3 + T ells per infetious foi, n 4. () Representtive immunohemistry setions of liver stined for CD3 + T ells. Sle rs: 1 µm., The effets of or srmled ontrol peptide in the numer of free flowing T ells in the liver sinusoids fter ishemi nd reperfusion in C7BL/6 WT mie y intrvitl mirosopy. Dt shown s numer of flowing CD3 + T ells per minute in the liver sinusoid following reperfusion t the minute time point, n=3 per group. d, Asolute numer of CD19 + B ells in the slivry glnds of C7BL/6 WT mie treted with srmled peptide or fter indution of Sjogren s syndrome y nnultion of slivry glnds nd injetion of 1 8-1 9 pfu of luiferse-enoding replition-defetive denovirus (AdV), n=7-8. e, Asolute numer of B22 + B ells in the oulr infiltrte of C7BL/6 WT mie treted with or srmled ontrol upon indution of endotoxin medited uveitis, n=9-1. f, g, The effet of or srmled ontrol peptide on the reruitment of (f) F4/8 mrophges or (g) CD11 + ells (mostly DC s fter gting on CD4 + followed y gting out CD3 + T ells nd F4/8 + /podoplnin + mrophges) into the peritoneum of BALB/ B ell-defiient mie fter indution of peritonitis using zymosn (1 mg), n=3-4 in different groups. Dt re represented s men±s.e.m nd nlysed y ANOVA nd (f-g) Bonferroni post test or (, -e) unpired t-test. = not signifint. Nture Mediine: doi:1.138/nm.3842
Supplementry Tle 6 HC T1D N 19 29 Age (yers) 3.84 ± 13.91 38.9 ± 14.11 BMI (kg.m 2 ) 23.22 ± 3.63 22.94 ± 2.36 Durtion of dietes NA 8 dys to 46 yers Supplementry Tle 6 Clinil prmeters of the sujets inluded in the T1D diponetin reeptors on B ells study. Dt re men±sd nd were nlysed using Mnn-Whitney test for ge or unpired t-test for BMI. No signifint differenes were found for ge nd BMI etween the helthy ontrols nd individuls with T1D. Supplementry Tle 7 HC T1D N 22 Age (yers) 41.46 ±.64 37. ± 14.96 BMI (kg.m 2 ) 24.98 ±4.17 2.12 ± 3.78 Durtion of dietes NA 8 dys to 46 yers Supplementry Tle 7 Clinil prmeters of the sujets inluded in the T1D trnsmigrtion study. Dt re men±sd nd were nlysed using Mnn-Whitney test. No signifint differenes were found for ge nd BMI etween the helthy ontrols nd individuls with T1D. Supplementry Tle 8 HC RA N 14 Age (yers) 2.43 ± 12.6.93 ± 13.82 Durtion of disese-medin (months) NA (2-12) Supplementry Tle 8 Clinil prmeters of the sujets inluded in the rheumtoid rthritis (RA) study. Dt re men±sd nd were nlysed using unpired t-test. No signifint differenes were found for ge etween the helthy ontrols nd individuls with rheumtoid rthritis. Nture Mediine: doi:1.138/nm.3842
Numer of B ells (% of PBL) Supplementry Figure 9 Helthy ontrol T1D Rheumtoid rthritis Isotype ontrol AdipoR1 AdipoR2 Isotype ontrol AdipoR1 AdipoR2 Isotype ontrol AdipoR1 AdipoR2 Count AdipoR1/2 Count T1D HC RA 1 CD19 HC RA T1D Supplementry Figure 9 The pthwy is ompromised in T1D nd rheumtoid rthritis., Representtive flow ytometry dt for the expression of diponetin reeptors on the B ells of helthy ontrols (HC) nd individuls with T1D or rheumtoid rthritis., Representtive flow ytometry dt for the expression of CD19 on B ells from HC, T1D nd rheumtoid rthritis. No differene in MFI were oserved., Perentge of B ells within the PBL popultion in helthy ontrols, individuls with T1D or rheumtoid rthritis (RA), n=1-. Dt re represented s men±s.e.m nd nlysed y ANOVA nd Dunnett post test. = not signifint. Nture Mediine: doi:1.138/nm.3842