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1 doi:1.138/nture1228 Totl Cell Numer (cells/μl of lood) d Peripherl Blood Time (d) fter nti-cd3 i.p. + TCRβ + IL17A + cells (%) Totl Cell Numer (x1 3 ) % of T H 17 cells in Duodenum Time (d) fter nti-cd3 i.p. Duodenum Time (d) fter nti-cd3 i.p. e + TCRβ + IL17A + cells (cell numer x1 3 ) c + TCRβ + T cells (cell numer x1 3 ) Totl numer of T H 17 cells in Duodenum Time (d) fter nti-cd3 i.p. Duodenum Time (d) fter nti-cd3 i.p. Supplementl Fig. 1. Dynmics of T H 17 cells during CD3-specific tretment. C57BL/6 mice were treted three times with 2μg of CD3-specific ntiody (rrows) nd totl cell numer in the lood () nd in the duodenum () were counted t different time points during the tretment. The totl numer of + TCRβ + T cells (c), percentge (d) nd the totl numer (e) of T H 17 in the duodenum ws otined fter flow cytometry nlysis of IL-17A + cells gted on + TCRβ + T cells (men ± s.e.m; n=5). Representtive results from three independent experiments. 1
2 doi:1.138/nture1228 IL17 gene BmHI E1 E2 ScII UAA A 2 UA 3 E3 MluI 4 p MW +/+ +/egfp egfp/egfp Trgeting construct TK BmHI IRES GFP NEO UAA ScII A 2 UA 3 E1 E2 E3 MluI 3 p 2 p Mutted gene BmHI UAA A 2 UA 3 IRES GFP E1 E2 E3 MluI 1 K c FACS sorted IL-17A mrna (ritrry units) IL-17A mrna Levels GFP+ GFP- d IL17A-PE +/+ egfp/egfp IL17A-PE Supplementl Fig. 2. Trgeting IRES-eGFP reporter into the mouse IL-17 locus. () Mps for mouse IL-17A locus, trgeting DNA construct nd the trgeted IL-17A locus. An 8-k mouse genomic frgment, including exons 1, 2 nd 3 of IL-17A gene, ws excised y using BmHI nd MluI (Top) nd cloned into pesy-flox vector djcent to the thymindine kinse (TK) selection mrker. A cssette contining IRES-GFP nd LoxP-flnked neomycin (Neo) selection mrker ws inserted into ScII site etween the trnsltion stop codon (UGA) nd the polydenyltion signl (A2UA3) of IL-17A gene (Middle). A correctly trgeted ES cell ws used to crete chimers nd germ-line-trnsmitted mice. The Neo gene ws removed in vivo y using deletor mice trnsgenic for Cre recominse to generte mice ering trgeted IL-17A locus (Lower). () PCR genotyping IL-17A-eGFP reporter mice. Three primers were designed to genotype reporter mice. PCR yielded 22-p product for the wild-type (Wt) IL-17A llele nd 29-p product for trgeted IL-17A llele. (c) IL-17A mrna expression in egfp positive versus egfp negtive cells fter sorting. (d) Dot plots of PE-nti-IL-17A (Y xis) versus egfp (X xis) intrcellulr stining of T H 17 cells from wild-type (left) nd homozygous (right) mice. 2
3 doi:1.138/nture1228 WT IL17A Bone Mrrow e <PerCP-A> <PerCP-A> <PerCP-A> e <PerCP-A> <PerCP-A> <PerCP-A> <PerCP-A> <PerCP-A> Lymph node <PerCP-A> <PerCP-A> <PerCP-A> <PerCP-A> Thymus Spleen Sumxilr LN Lung Smll Intestine c Liver <PerCP-A> <PerCP-A> IL17A FoxP3 Supplementl Fig 3. Expression of IL-17A detected in vivo y n IL-17A-eGFP reporter mouse. Flow cytometry nlysis of IL17A + + T cells in different orgns from reporter mice nd wild-type littermtes in stedy stte conditions re shown (). Flow cytometry nlysis of IL-17A (Y xis) nd Foxp3 (X xis) on the smll intestine + T cells from x Foxp3-mRFP doule reporter mice in stedy stte conditions (). x Foxp3-mRFP doule reporter mice were treted three times with 2μg of nti-cd3 ntiody (2C11) (c). Four hours fter the lst injection nimls were scrificed, different orgns removed nd processed for FACS nlysis. Plots re gted on + nd numers in qudrnts indicte percent cells in ech. Dt represents t lest 3 different experiments. 3
4 doi:1.138/nture1228 LPL IEL LPL IEL c control GFP-A: il GFP-A: il RFP-A: foxp RFP-A: foxp Anti-CD3 GFP-A: il GFP-A: il RFP-A: foxp RFP-A: foxp3 Supplementl Fig. 4. Smll Intestine locliztion of T H 17 cells during CD3-specific ntiody tretment. () Sections demonstrting tht egfp + + T cells cn e locted in the lmin propri comprtment (left) nd in the intrepithelil comprtment (right) of the smll intestine. Originl mgnifiction 4x. (lue, dpi; red, ; green, IL-17A-eGFP). () 3D reconstruction of smll intestine from knock-in mice treted with CD3-specific ntiody otined y multiphoton microscopy nlysis. (lue, second hrmonics; green, IL-17A-eGFP). (c) Flow cytometry nlysis of IL-17A (Y xis) nd Foxp3 (X xis) on the intestinl LPL (lmin propri lymphocytes) nd IEL (intestinl intrepithelil lymphocytes) + T cells from x Foxp3-mRFP doule reporter mice fter CD3-specific ntiody tretment. All nimls were nlyzed 1h fter the first injection of CD3-specific ntiody. 4
5 doi:1.138/nture1228 control Intct nti-cd3 (2C11) Non mitogenic nti-cd3 (JAB261) Foxp3-mRFP Foxp3-mRFP Foxp3-mRFP 2D2 control 2D2 + OVA 2D2 + MOG Supplementl Fig. 5. T H 17 cells re incresed in the smll intestine during non mitogenic nti-cd3 tretment () or during ntigen specific immuniztion (). () Flow cytometry nlysis of IL-17A nd Foxp3 on intestinl + T cells from x Foxp3-mRFP doule reporter mice 1h fter the first injection of CD3-specific ntiody or fter non-mitogenic nti-cd3 tretment. () MOG-TCR trnsgenic mice (2D2 mice) crossed with the IL-17AeGFP knock-in mice were treted three times with PBS, OVA or MOG peptide (15 mg/i.p. dose; injections on dys,3 nd 6). Four hours fter the lst injection the presence of T H 17 in the duodenum of the treted mice ws evluted y flow cytometry nlysis. (n=4). Experiments were repeted twice with similr results. 5
6 doi:1.138/nture1228 Il6 +/+ Il6 -/ PBS Clodronte IL17A-PE c cell numers X PBS Clodronte d ng/ml PBS Clodronte Th17 Mcrophges IL-6 IL-17A Supplementl Fig. 6. IL-6 secreted y ntigen-presenting cells (APCs) is criticl for the genertion of T H 17 cells during CD3-specific ntiody tretment. () Flow cytometry nlysis of IL-17A intrcellulr expression in + T cells from duodenum of wild type nd IL-6 -/- mice treted with CD3-specific ntiody.(n=5). Experiment ws performed twice with similr results. () IL-17A-eGFP reporter mice were treted with clodronte-loded liposomes or with PBS-loded liposomes one dy efore the tretment with CD3-specific ntiody. 1h fter the first injection T H 17 cells from the duodenum were nlyzed y flow cytometry. (n=5). Dt represents three independent experiments. (c) Totl + T cells, T H 17 cells nd Mcrophges (CD11 + CD11c - ) from experiment s in () were quntified. (d) Plsm levels of IL-6 nd IL-17A in these nimls were mesured y CBA (men ± s.e.m; n=5). 6
7 doi:1.138/nture CCL2 (pg/ml) n.d. Duodenum Jejunum Ileum Colon Supplementl Fig. 7. CCL2 protein expression in the gstrointestinl trct. Different prts of the intestine were cultured for two dys. CCL2 ws mesured in the superntnts using ELISA (men +/- s.e.m.). 7
8 doi:1.138/nture1228 IL-17A + Foxp3 - BrdU / 7-AAD IL17A + Foxp Duodenum IL-17A - Foxp3 - IL17A - Foxp Foxp3-mRFP BrdU 7-AAD Supplementl Fig. 8. T H 17 cells from duodenum ctively proliferte during CD3-specific ntiody tretment. Prolifertion cpcity ssessed y BrdU/7-AAD nlysis on different popultions of FACS sorted + TCRβ + T cells from the duodenum of IL-17A-eGFP x Foxp3-mRFP doule reporter mice treted with CD3-specific ntiody (clone 2C11). Cells were isolted 1h fter the first injection. Dt represents three independent experiments. 8
9 doi:1.138/nture1228 IL-17A + Foxp3 - IL-17A + Foxp3 - IL17A + Foxp3 + PI PI IL-17A - Foxp3 - IL-17A - Foxp3 + IL-17A - Foxp3 - IL17A - Foxp3 + Ki-67 PI PI Supplementl Fig. 9. T H 17 cells from duodenum ctively proliferte during CD3-specific ntiody tretment. Prolifertion cpcity ssessed y Ki-67 () or y Propidium Iodide (PI) cell cycle nlysis () on different popultions of FACS sorted + TCRβ + T cells from the duodenum of IL-17A-eGFP x Foxp3-mRFP doule reporter mice treted with CD3-specific ntiody (clone 2C11). % of cells tht re in different phses of the cell cycle re represented in (). Dt represents three independent experiments. () ws nlyzed 6h fter the first injection nd () 1h fter the first injection 9
10 doi:1.138/nture1228 1x 2x control control Anti-CD3 Anti-CD3 c d Supplementl Fig. 1. Elimintion of T H 17 cells in the intestinl lumen of mice treted with CD3-specific ntiody. () Photogrphs re of intestine from C57l/6 mice treted with 3 i.v. injections of CD3-specific ntiody (clone 2C11) or control mice. () Histologicl sections (H&E stining nd mgnifiction ws 1x or 2x) from the sme treted nd control mice shown in (). (c) Flow cytometry nlysis of the luminl content of IL-17A-eGFP x Foxp3-mRFP treted with CD3-specific ntiody. Decresed percentge of Tregs (Foxp3 + T cells) in the lumen (left) ws oserved when compred to the duodenum (LPL +IEL comprtment) (right). Gte is on the + TCRβ + T cells. Numers in qudrnts indicte percent cells in ech. (d) Immunofluorescence stining of frozen sections of duodenum from IL-17A-eGFP knock-in mice treted with CD3-specific ntiody. T H 17 cells were found in the intestinl lumen (rrows). (red); IL-17A-eGFP (green); cell nuclei (DAPI). Originl mgnifiction ws 4x. Dt re representtive of four experiments. Anlysis ws done 1h fter the first injection. 1
11 doi:1.138/nture1228 control ll ntiodies nti-il-1 nti-tgfβ1 nti-ctla-4 IL17A+ FoxP3- (TH17) control suppression ll ntiodies T H 17 (nti-cd3) T H 17 (EAE) Spleen Smll Intestine IL17A- FoxP3+ (Tregs) IL-2-PE IL1-eGFP TNFα-PE IL1-eGFP Supplementl Fig. 11. Suppressive cpcity of the rt H 17 cells is prtilly dependent on IL-1, CTLA-4 nd TGF-β signlling. () Suppression ssy performed using sorted egfp-mrfp++ (Tregs) or egfp+mrfp-+ (T H 17) cells from IL-17A-eGFP x Foxp3-mRFP doule reporter mice treted with CD3-specific ntiody. Cells were sorted from duodenum. (Br represents undivided CFSE lelled + CD25 - responder T cells). Antiodies were dded t 1 μg/ml finl concentrtion. () Expression of IL-2, TNF-α nd IL-1 ws determined y flow cytometry nlysis. Plots re gted on + IL-17A + T cells from IL-1-eGFP reporter mice with EAE or treted with CD3-specific ntiody. Numers in qudrnts indicte percent cells in ech. Dt re representtive of 3 different experiments. 11
12 TNFα <APC-A>: TNF <PE-Cy7-A>: T H 17 WT T H 17 Ccr6 -/- S. Intestine spleen spleen <APC-A>: TNF <PE-Cy7-A>: <APC-A>: TNF <PE-Cy7-A>: # Cells # Cells control <CFSE-A>: CFSE 25.1 # Cells # Cells Treg <CFSE-A>: CFSE 13.6 Supplementl Fig. 12 T H 17 WT S.Intestine T H 17 WT spleen T H 17 KO spleen 36.6 # Cells c d <CFSE-A>: CFSE <CFSE-A>: CFSE <CFSE-A>: CFSE T H 17 WT S.Intestine T H 17 WT spleen T H 17 KO spleen Supplementl Fig. 12. Migrtion to the smll intestine is essentil for T H 17 cells to cquire regultory phenotype. IL-17A egfp+ cells were isolted from the smll intestine nd spleen of wild type nd Ccr6 -/- mice fter CD3-specific ntiody tretment. () Cells were restimulted nd intrcellulr cytokine stining for TNF-α performed. () Cells were cocultured with CFSE responder cells to ssess the in vitro suppressive cpcity. (c+d) Sorted IL-17A egfp+ cells were trnsferred into Rg1 -/- mice, nd the colitis development monitored using colonoscopy (men+/- s.e.m.). Results re representtive of two independent experiments. 12
13 doi:1.138/nture1228 c 4 3 Control Anti-CD3 * * * * * EAE Clinicl Score Dys post-immuniztion d T H 17 T H 17 + rt H 17 1x 4x Supplementl Fig. 13. CD3-specific ntiody redirects ntigen specific T H 17 cells to the Smll Intestine nd they cquire suppressive function in vivo. () EAE ws induced in IL-17A-eGFP knock-in mice y immuniztion with MOG35 55 in complete Freund djuvnt (CFA). A group of mice ws treted with 2 μg of CD3-specific ntiody (clone 2C11) on dys 7, 9 nd 11 fter immuniztion (rrows). The course of EAE in these mice is shown s men clinicl score ± s.e.m., n=8, *P <.5. () Flow cytometry nlysis using MOG-specific tetrmer on smll intestine + IL-17A + T cells purified from EAE-induced nimls (IL-17A-eGFP knock-in mice) four hours fter the lst injection with PBS (upper plot) or with CD3-specific ntiody (lower plot). Numers ove outlined res indicte the frequency of MOG-tetrmer + T H 17 cells. Dt re representtive of 2 different experiments. (c+d) In vitro differentited IL-17A-eGFP + Foxp3-mRFP - + T cells from 2D2 mice were either trnsferred lone (25 x 1 3 cells) or together with IL-17A-eGFP + Foxp3-mRFP - + T cells isolted from the smll intestine of CD3-specific ntiody treted 2D2 mice 1 hours fter the first injection (25 x 1 3 cells ech). Clinicl EAE score 9 weeks fter the trnsfer (c), nd representtive section of the spinl cord stined with fst luxol lue nd hemtoxylin/eosin (d) re shown. Ech dot represents one mouse, lines indicte men. 13
14 doi:1.138/nture1228 control OKT3 Teplizum h h c Supplementl Fig. 14. Anti-humn-CD3 ntiody tretment induces the recruitment of humn IL17A + nd IL1 + cells into the duodenum in humnized mouse system. Flow cytometric nlysis () nd cell counts () showing incresed percentge nd cell numers of h + h5 + T cells in the duodenum of Bl/c RAG1-/-γc-/- mice two weeks fter reconstitution with humn PBMCs. Mice were nlyzed 5h fter tretment with OKT3, Teplizum or ntiody control (1μg/mouse/i.v.). Gte is on humn 5 + T cells. Numers in qudrnts indicte percent cells in ech. (c) The presence of humn IL17, Il1 nd Ccl2 mrna in the duodenum of OKT3, Teplizum nd control mice (from the sme nimls shown in +) ws determined y RT-PCR. Dt is normlized to humn HPRT. Dt re cumultive of two experiments performed independently. 14
15 doi:1.138/nture K control 25K SEB 2K 2K SSC-A 15K SSC-A 15K 1K 1K 5K 5K 5K 1K 15K 2K 25K FSC-A 5K 1K 15K 2K 25K FSC-A IEL+ LPL TCR Vβ8 + TCR Vβ Lumen TCR Vβ8 + TCR Vβ Control <APC-Cy7-A>: SEB SEB-flush IL17A Supplementl Fig. 15. Elimintion of T H 17 cells in the intestinl lumen of mice treted with SEB. ( nd ) Flow cytometry nlysis of the luminl content of IL-17A-eGFP mice untreted or fter tretment with 3 i.v. injections (, 48, 96 hours) with 5μg of SEB. Animls were nlyzed 4 h fter the lst injection. () SSC versus FSC plot of the luminl content of treted nd untreted mice nlyzed y FACS. () Flow cytometric nlysis showing the presence of IL-17A + + T cells in the luminl content of SEB treted nimls s well in the LPL+IEL comprtments. Gte is on the + TCRβ + T cells. Numers in qudrnts indicte percent cells in ech. 15
16 doi:1.138/nture Spleen Smll Intestine IL IL IL TNFαα Supplementl Fig. 16. Induction of rt H 17 cells in the smll intestine during sepsis. IL-1 egfp x Foxp3 mrfp doule reporter mice were treted with SEB. Cells from the spleen nd smll intestine were restimulted with PMA nd Ionomycin nd intrcellulr cytokine stining for IL-17A, IL-2, TNF-α, nd nti-gfp performed. Plots re gted on IL-17A + + events. Results re representtive of t lest two independent experiments. 16
17 doi:1.138/nture1228 Dy 3 Dy 5 Control H1N1 Control H1N LUNG <APC-Cy7-A> <APC-Cy7-A> <APC-Cy7-A> <APC-Cy7-A> S. Intestine IL17A <APC-Cy7-A> <APC-Cy7-A> <APC-Cy7-A> <APC-Cy7-A> Supplementl Fig. 17. Accumultion of T H 17 cells in the smll intestine during Influenz A (H1N1) infection. Flow cytometric nlysis showing the presence of IL-17A + + T cells in the lung nd smll intestine of Influenz A (H1N1) infected mice t indicted time points. Gte is on the Vβ8TCR + cells. Numers in qudrnts indicte percent cells in ech. Results re representtive of two independent experiments. 17
18 doi:1.138/nture1228 Supplementl Fig. 18. Scheme of the fte of T H 17 cells during tolernce induction fter polyclonl T cell ctivtion in vivo. After polyclonl T cell ctivtion vi TCR, poptotic T cells will e engulfed y phgocytes. IL-6 nd TGF-β re going to e produced y APCs nd consequently IL-17A + CCR6 + + T cells (T H 17) re generted in the periphery. T H 17-medited production of IL17 will induce upregultion of CCL2 in the smll intestine ttrcting nd trpping the pro-inflmmtory T H 17 cells. In the Smll Intestine, prt of the T H 17 cells re going to e eliminted in the lumen nd others will e reprogrmmed in rt H 17 cells with immuno-suppressor functions. 18
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doi:0.08/nture0987 SUPPLEMENTARY FIGURE Structure of rbbit Xist gene. Exons re shown in boxes with romn numbers, introns in thin lines. Arrows indicte the locliztion of primers used for mplifiction. WWW.NATURE.COM/NATURE
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doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 1 c CFU-F colonies per 1 5 stroml cells 14 12 1 8 6 4 2 Mtrigel plug Neg. MCF7/Rs MDA-MB-231 * * MCF7/Rs-Lung MDA-MB-231-Lung MCF7/Rs-Kidney
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