doi:1.138/nture11813 NK cells NKp46 + RORγt - 5.4 RORγt + ILCs SSC-A FSC-H Linege 22.4 98.2 28.8 NKp46 1.2 15.4 NKp46 NKp46 + RORγt + ILCs NKp46 - RORγt + ILCs FSC-A FSC-A CD45 RORγt RORγt CCR6 + T-et - CCR6 - T-et +/- Supplementry Figure 1: Gting strtegy for intestinl RORγt + ILCs Lmin propri lymphocytes were stined nd nlyzed y flow cytometry. Gting ws performed on lymphocytes nd doulets were excluded. In the next step, non-hemtopoietic nd Lin + cells (CD3 + CD8α + CD19 + Gr-1 + ) were excluded. Lin - CD45 + cells were then nlyzed for the expression of RORγt nd NKp46 llowing for the discrimintion of the vrious ILC susets: NKp46 + RORγt - ( NK cells ), NKp46 + RORγt + ILCs nd NKp46 - RORγt + ILCs. RORγt + ILC susets were then further nlyzed for the expression of CCR6 nd T-et or other cell surfce mrkers. WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION 4.1.1 Eomes 61.3 RORγt 34.6 Supplementry Figure 2: RORγt + ILCs do not express Eomes Lin - CD45 + lmin propri lymphocytes cells were nlyzed for expression of Eomes nd RORγt y intrcellulr stining nd flow cytometry nlysis. 2 WWW.NATURE.COM/NATURE
RESEARCH NKp46 + RORγt + 9.1 9.3 5.2 8.3 CD9 Sc1..6 7.2 18.8 3.1 7.9 4.5 86.5 Kit CD25.3 14.2 4.6 38.1 1.4 56. 2.6 97. Reltive to Hprt1 1 5 Kit 2 1 Ccr6 RORγt + ILCs NKp46 + NKp46 - CD4 - CD4 + CCR6 - T-et + CCR6 + T-et - CCR7.7 8.3 CD127.4. 3.1 3.2 7. 92.2 CCR6.8 T-et 93.1 IL-12Rβ1.6.2 c Reltive to Hprt1 1..5 Tx21 1..5 Fsl.5.25 Prf1.5.25 Gzm.5.25 Cxcr3 1..5 Ccl3 3. 1.5 Ccl4 RORγt + ILCs NKp46 + NKp46 - CD4 - CD4 + CCR6 - T-et + CCR6 + T-et - d 5 *** NKp46 + RORγt - 4 NKp46 + RORγt + T-et (MFI) 3 2 *** *** CCR6 - CCR6 + NKp46 - RORγt + 1 Supplementry Figure 3: T-et trget genes re expressed in T-et + RORγt + ILCs, Coexpression of T-et with the indicted cell surfce mrkers in NKp46 + RORγt + ILCs. Numers represent percentge of cells per qudrnt.,c, Expression of the indicted genes in highly purified susets of RORγt + ILCs s determined y qrt-pcr. d, Men fluoresecence intensity (MFI ± s.d.; n=7) of T-et in the indicted ILC susets. ***p <.1. WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION Reltive to Hprt1.2.1 Lt 2 1 Lt 1 5 Tnfsf14 (Light) RORγt + ILCs NKp46 + CCR6 - NKp46 - CD4 - CCR6 CD4 + + Reltive to Hprt1 3 2 1 1. Il22 Ifng 2. 1. 7.5 Il17f Il17 c IL-22 + RORγt + ILCs (%) 3 2 1 CCR6 + T-et - CCR6 - T-et +.5 5. 2.5 Supplementry Figure 4: CCR6 + nd CCR6 - RORγt + ILCs express LTi genes nd IL-22,, Expression of the indicted genes in highly purified susets of RORγt + ILCs s determined y qrt-pcr. c, Percentge (men ± s.d.; n=3) of IL-22-expressing cells within the T-et - nd T-et + frctions of NKp46 - RORγt + ILCs. One representtive experiment of three is shown. 4 WWW.NATURE.COM/NATURE
RESEARCH CD45.1 Tx21 +/+ CD45.1 Tx21+/+ c NKp46 + RORγt + ILCs (x1 4 ) 6 3 *** CD45.2 Tx21 +/+ 1.7 14.6 NKp46 2. 9.3 CD45.2 Tx21 -/- CD45.1 2.3 8.8.3 CD45.2 8.5 NKp46 + cells mong CD45.2 RORγt + ILCs (%) 2 1 *** RORγt d e Tx21 +/+ Tx21 -/- RORγt + ILCs 23.5 27.5 CCR6 - CCR6 + CXCR3 IL-12Rβ1 Tx21 +/+ Tx21 -/- IFN-γ TNF IL-17 IL-22 27. 16.7 1.1 32.8 8.72.8 CD25 Supplementry Figure 5: T-et regultes phenotype nd function of CCR6 - RORγt + ILCs, Asolute numers (men ± s.d.; n=12) of NKp46 + RORγt + ILCs in the smll intestine of Tx21 +/+ nd Tx21 -/- mice.,c, Groups (n=5) of lethlly irrdited C57BL/6 mice (CD45.2) were reconstituted either with mixed one mrrow (rtio 1:1) from wildtype (CD45.1) nd Tx21 +/+ mice (CD45.2) or wildtype (CD45.1) nd Tx21 -/- mice (CD45.2). After 8 weeks, NKp46 nd RORγt expression y CD45.1 + nd CD45.2 + cells within Lin - cells of the smll intestinl lmin propri ws nlyzed y flow cytometry. Numers represent percent of cells WWW.NATURE.COM/NATURE 5
RESEARCH SUPPLEMENTARY INFORMATION in the indicted gtes. A representtive dot plot () or percentge (men ± s.d.; n=5) of NKp46 + cells mong CD45.2 + RORγt + ILCs (c) is shown. d, Flow cytometry nlysis of CXCR3 nd IL-12Rβ1 expression y CCR6 - or CCR6 + NKp46 - RORγt + ILCs from Tx21 +/+ or Tx21 -/- mice. Cxcr3 nd Il12r1 re cnonicl T-et trget genes. e, Lmin propri lymphocytes from Tx21 +/+ nd Tx21 -/- mice were incuted for 4h in medium contining PMA, ionomycin nd refeldin A. Expression of the indicted cytokines in NKp46 - RORγt + ILCs ws determined y intrcellulr cytokine stining nd flow cytometry nlysis. Numers represent percent cells in the indicted gtes. ***p <.1. 6 WWW.NATURE.COM/NATURE
RESEARCH Ki67 NKp46 - RORγt + 2.6 5.3 64.2 T-et 28. NKp46 + NKp46 + RORγt + RORγt - 1. 15.1.4 1.4 7. 76.9 1.8 87.5 Ki67 + cells (%) 2 1 CD4 + RORγt + *** *** CD4 - RORγt + NKp46 - RORγt + ** NKp46 + RORγt + NKp46 + RORγt - CCR6 + CCR6 - Supplementry Figure 6: CCR6 - RORγt + ILCs proliferte, Flow cytometry nlysis of Ki67 nd T-et expression y the indicted ILC susets in the lmin propri of the smll intestine of 8-week old mice. Numers represent percentge of cells per qudrnt., Percentge (men ± s.d.; n=5) of Ki67 + cells mong the indicted ILC susets. Dt shown re representtive of 5 independent experiments. **p <.1 nd ***p <.1. WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION CCR6 + RORγt + CCR6 + RORγt + ILCs CCR6 - RORγt + ILCs CD4 - CD4 +.5... CD4 - CD4 +. 5.6. 2.9 NKp46 - NKp46 + 33.5 24.1 62.3 33.9 NKp46 96.8 T-et 2. 97. 3. T-et 21. RORγt 73.4 2.6 76.5 17.7 24.7 3.3.4 Supplementry Figure 7: CCR6 + RORγt + ILCs constitute stle ILC linege, Highly purified CD4 - or CD4 + CCR6 + RORγt + ILCs isolted from Rorc(γt) GFP/+ mice (H-2 ) were doptively trnsferred into lymphoid BALB/c Rg2 -/- Il2rg -/- mice (H-2 d ). Three weeks fter doptive trnsfer, donor-derived cells were nlyzed for the expression of NKp46 nd T- et., The indicted highly purified RORγt + ILC popultions from Rorc(γt) GFP/+ mice were cultured in medium contining SCF, IL-7 nd IL-2. After 14 dys, cells were nlyzed for the expression of T-et nd RORγt (GFP) y flow cytometry. Numers represent percentge of cells in qudrnts. Dt re representtive of 3 independent experiments. 8 WWW.NATURE.COM/NATURE
RESEARCH Tx21 +/+ Tx21 -/- 43.7 NKp46 - RORγt + ILCs CCR6 + CCR6 - T-et + Kit CCR6 - CCR6 12.2 35.3 T-et Tx21 +/+ CCR6 - T-et - CCR6 + Tx21 -/- CD127 Kit Supplementry Figure 8: T-et represses Kit nd CD127 expression y RORγt + ILCs, Flow cytometry nlysis of Kit (top) nd CD127 (ottom) expression y NKp46 - CCR6 - RORγt + ILCs (CCR6 - ) nd NKp46 - CCR6 + RORγt + ILCs (CCR6 + ) from the smll intestine of Tx21 +/+ or Tx21 -/- mice., Flow cytometry nlysis of Kit expression y NKp46 - CCR6 - T-et + RORγt + ILCs (CCR6 - T-et + ), NKp46 - CCR6 - T-et - RORγt + ILCs (CCR6 - T-et - ) nd NKp46 - CCR6 + RORγt + ILCs (CCR6 + ) from the smll intestine of Tx21 +/+ or Tx21 -/- mice. Top pnel depicts the gting strtegy to identify the vrious susets of NKp46 - RORγt + ILCs. WWW.NATURE.COM/NATURE 9
RESEARCH SUPPLEMENTARY INFORMATION CCR6 Myd88 f/f 34.1 3.9 14.6 47.4 T-et Myd88 ΔLTi,T 33.8 3.3 14.7 48.2 CCR6 - T-et + cells (%) 6 4 2 n.s. c CCR6 - T-et + cells (x 1 3 ) 16 12 8 4 n.s. Myd88 f/f Myd88 ΔLTi,T Supplementry Figure 9: Genetic deletion of Myd88 in RORγt + ILCs does not interfere with the induction of T-et expression, Flow cytometry nlysis of T-et nd CCR6 expression y electroniclly gted NKp46 - RORγt + ILCs from the smll intestine of the indicted mouse strins., Percentge (men ± s.d.; n=3) of CCR6 - T-et + lymphocytes mong NKp46 - RORγt + ILCs from the indicted mouse strins. c, Asolute cell numers (men ± s.d.; n=3) of CCR6 - T-et + lymphocytes mong NKp46 - RORγt + ILCs. n.s., not significnt. 1 WWW.NATURE.COM/NATURE
RESEARCH NKp46 Il12r2 +/+ 4.5 11.5 RORγt 4.4 4.8 Il12r2 -/- 6.8 11.2 43 2.8 CCR6 - T-et + cells (%) 5 25 n.s. Il12r2 +/+ Il12r2 -/- CCR6 17. 37.9 19.8 34.4 T-et c NKp46 - NKp46 + NKp46 + RORγt + RORγt + RORγt - d NKp46 9. 13.3 1.1 7.1 14.3 8.5 CCR6 57.7 16.5 2.2 23.7 58.9 19.3 2.3 19.6 4.7 25.5.4 69.4 2.3 3.2.2 67.4 1.9.9 1.5 95.6.7 1..8 97.5 Stt4 +/+ Stt4 -/- CCR6 - T-et + cells mong RORγt + ILC (%) 3 2 1 n.s. Stt4 +/+ Stt4 -/- RORγt T-et e NKp46 + RORγt - cells (%) 8 6 4 2 n.s. NKp46 + RORγt - cells (x1 3 ) 1 75 5 25 n.s. Il23p19 +/+ Il23p19 -/- Supplementry Figure 1: STAT4 nd IL-12 signling re dispensle for the induction of T-et expression in CCR6 - RORγt + ILCs, Flow cytometry nlysis of NKp46 nd RORγt expression of Lin - lmin propri lymphocytes from the smll intestine of Il12r2 -/- or control mice (upper pnels). Numers represent percentge of cells in gtes or qudrnts. CCR6 nd T-et expression of electoniclly gted NKp46 - RORγt + ILCs is shown in the lower pnels., Percentge (men ± s.d.; n=5) of CCR6 - T-et + lymphocytes mong NKp46 - RORγt + ILCs from the indicted mouse strins. c, NKp46 - RORγt + ILCs (left) from Stt4 +/+ nd Stt4 -/- mice were nlyzed for WWW.NATURE.COM/NATURE 11
RESEARCH SUPPLEMENTARY INFORMATION CCR6 nd T-et expression (right). Numers represent percentge of cells in gtes (left) or per qudrnt (right). d, Percentge (men ± s.d.; n=3) of CCR6 - T-et + cells mong NKp46 - RORγt + ILCs in the lmin propri of the smll intestine of the indicted mouse strins. e, Percentge (men ± s.d.; n=3) nd solute numers (men ± s.d.; n=3) of NKp46 + RORγt - cells (i.e., NK cells ) from Il23p19 -/- nd control mice. Dt re representtive of three independent experiments. n.s., not significnt. 12 WWW.NATURE.COM/NATURE
RESEARCH Il23 c Reltive to Hprt1 (x1-2 ) 1.5 1..5 CD4 proximl 16.4 2.2 67.1 T-et 14.4 middle 13.4 2.5 56.9 27.3 distl 9.5 2.7 54. 33.8 T-et + mong RORγt + cells (%) 5 25 * * d NKp46 + RORγt + cells mong Lin - CD45 + cells (%) 2 1 *** * Supplementry Figure 11: T-et + RORγt + ILCs re etter represented in the distl prts of the smll intestine, Il23 expression in cells purified from the indicted prts of the smll intestine s determined y qrt-pcr., Flow cytometry nlysis of CD4 nd T-et expression y NKp46 - RORγt + ILCs isolted from the proximl, middle nd distl third of the smll intestine. Numers represent percentge of cells per qudrnt. c, Percentge (men ± s.d.; n=8) of T-et + cells within NKp46 - RORγt + ILCs in the proximl, middle nd distl third of the smll intestine. d, Percentge (men ± s.d., n=5) of NKp46 + RORγt + ILCs mong Lin - CD45 + cells of the proximl, middle nd distl prt (equl thirds) of the smll intestine. *p <.5 nd ***p <.1. WWW.NATURE.COM/NATURE 13
RESEARCH SUPPLEMENTARY INFORMATION d IL-22 Tx21 +/+ Tx21 -/- 29.2 3.8 41.4.5 56. 11. 57. 1.1 IFN-γ Reltive to Hprt1 3 2 1 Reg3g n.s. c Body weight (%) 2 15 1 5-5 Tx21 +/+ Tx21 -/- 1 2 3 4 5 6 7 8 Dys post infection Mesenteric LN Spleen Liver Feces CFU/g 1 6 1 4 1 2 1 4 1 2 1 1 4 1 2 1 1 1 1 5 1 Tx21 +/+ Tx21 -/- Supplementry Figure 12: Tx21 -/- mice control Citrocter rodentium infection, Lmin propri lymphocytes from Tx21 +/+ or Tx21 -/- mice were incuted for 4h in medium contining PMA, ionomycin nd refeldin A. IFN-γ nd IL-22 production y electroniclly gted RORγt + ILCs ws determined y flow cytometry. Numers represent percentge of cells per qudrnt., Reg3g expression y smll intestinl epithelil cells isolted from Tx21 +/+ or Tx21 -/- mice. c, Weight s the percentge (men ± s.d.; n=5) of ody weight t dy of C. rodentium infection. d, Bcteril titers per grm of indicted orgn eight dys fter infection with C. rodentium. Lines represent the men. n.s., not significnt. 14 WWW.NATURE.COM/NATURE
RESEARCH Phylum Bcteroidetes Phylum Firmicutes Totl cteril lod Mouse intestinl Bcteroides Bcteroides species Lctocillus species Segmented filmentous cteri 1 18 1 1 1 12 1 15 1 8 1 1 1 4 16S rdna copies /ng DNA 1 12 1 6 1 24 Tx21 +/+ Tx21 -/- 1 6 1 4 1 12 1 1 1 8 1 6 1 4 1 17 1 1 1 5 1 2 1 2 1 1 6 1 4 Smll intestine 1 17 1 8 1 11 1 15 1 2 Colon 1 1 1 6 1 5 1 1 1 Enterococcus Enteroctericee Aneroes 1 6 1 6 1 8 Bcteri /ng intestinl content 1 7 1 5 1 5 Tx21 +/+ 1 6 1 4 Tx21 -/- 1 5 1 4 1 3 1 4 1 7 1 6 1 6 1 6 1 5 1 5 Smll intestine Colon 1 5 1 4 1 4 Supplementry Figure 13: Norml microiot in Tx21 -/- mice, Quntifiction of totl cteri lod or the indicted cteril species s mesured y 16S rdna qpcr., Quntifiction of the indicted cteril species y culture in the indicted orgns. WWW.NATURE.COM/NATURE 15
RESEARCH SUPPLEMENTARY INFORMATION Tnf c Reltive to Hprt1.4.3.2.1 n.s. Tx21 +/+ Tx21 -/- 38.5 37.1 TNF TNF + mong colonic MHCII + CD11 + DC (%) 5 3 1 n.s. Supplementry Figure 14: Norml TNF response in Tx21 -/- mice, Expression of Tnf in cells purified from the colon of Tx21 +/+ or Tx21 -/- mice s determined y qrt-pcr., TNF production y colonic myeloid cells (CD11 + MHCII + intestinl mononucler phgocytes) s determined y flow cytometry. c, Percentge (men ± s.d.; n=6) of TNF-producing cells mong CD11 + MHCII + intestinl mononucler phgocytes. n.s., not significnt. 16 WWW.NATURE.COM/NATURE
RESEARCH c Infected IFN-γ + CD127 + mong NKp46 + cells (%) 6 5 4 3 2 1 Shm Dy 2 Dy 4 Smll intestine Cecum Colon Tx21 +/+ Tx21 +/+ Tx21 -/- Infected Tx21 +/+ Tx21 +/+ Tx21 -/- 3. 5.5 2.9 17.9. 3.5 IFN-γ 12.3 79.2 1.4 68.7 14.8 81.7 CD127 d Body weight (%) -1-2 -3 Dys post infection 1 2 3 4 Tx21 +/+ ** Tx21 -/- * e Tx21 +/+ Infected Tx21 +/+ Tx21 -/- f Cecum length (cm) 3 2 1 ** Tx21 +/+ Tx21 +/+ Infected Tx21 -/- Supplementry Figure 15: IFN-γ-producing NKp46 + CCR6 - T-et + RORγt + ILCs control mucus relese nd inflmmtion following Slmonell infection, Groups of Rg2 -/- mice were infected with S. typhimurium. Control mice received only streptomycin tretment (shm tretment). Lmin propri lymphocytes from smll intestine, cecum nd colon were purified t the indicted time points nd incuted for 4h in medium contining refeldin A. Percentge (men ± s.d.; n=3) of IFN-γ-producing CD127 + cells mong NKp46 + cells ws determined y intrcellulr cytokine stining nd flow cytometry nlysis., Groups of mice from the indicted strins were infected with S. typhimurium. Two dys lter, lmin propri lymphocytes from the smll intestine were isolted nd WWW.NATURE.COM/NATURE 17
RESEARCH SUPPLEMENTARY INFORMATION incuted for 4h medium contining refeldin A nd IL-12 (1ng/ml). Expression of CD127 nd IFN-γ y electroniclly gted NKp46 + ILCs. One representtive experiment out of two experiments with 3-5 mice per group is shown. c, Top: Representtive photomicrogrphs of cecum of uninfected Tx21 +/+ mice or Tx21 +/+ nd Tx21 -/- mice 48h fter infection with S. typhimurium (r, 1 µm, mgnifiction, x1). Middle: Representtive photomicrogrphs showing the filure of Tx21 -/- mice to relese mucus from Golet cells fter infection with S. typhimurium (r, 1 µm). Bottom: Digitl mgnifiction of selected regions, rrows identify mucus-filled Golet cells in infected Tx21 -/- mice. d, Weight s the percentge (men ± s.d.; n=5) of ody weight t dy of S. typhimurium infection. e, Representtive pictures of cec from the indicted mouse strins 48h fter infection. f, Cecum length (men ± s.d.; n=5) 48h fter infection. *p <.5 nd **p <.1. 18 WWW.NATURE.COM/NATURE
RESEARCH Infected Rg2 -/- Rg2 -/- Rg2 -/- Rg2 -/- + α-silo-gm1 + α-thy-1 Rg2 -/- Il2rg -/- Dys post infection c Body weight (%) 5 1 2 3 4-5 -1-15 -2 * -25 * -3 PBS α Thy1 α silo-gm1 Rg2 -/- Il2rg -/- Rg2 -/- PBS Rg2 -/- α-thy1 d 3.5 * ** e Cecum length (cm) 3. 2.5 2. 1.5 1..5 1-Rg2 -/- 2-Rg2 -/- 3-Rg2 -/- α Thy1 4-Rg2 -/- α-silo-gm1 5 Rg2 -/- Il2rg -/- Infected Ifngr1 +/+ Ifngr1 -/- 1 2 3 4 5 f g Body weight (%) 5-5 -1 Dys post infection 1 2 Ifngr1 +/+ Ifngr1 -/- * Cecum length (cm) 2.5 2. 1.5 1..5 *** Supplemetry Figure 16: Depletion of ILCs meliortes Slmonell-induced enterocolitis -d, Groups of Rg2 -/- or Rg2 -/- Il2rg -/- mice were infected with S. typhimurium (dy ) nd received the indicted depleting ntiodies t dy -2, dy nd dy +2. Control groups received injections of PBS. Representtive photomicrogrph of H&E stined cecum sections WWW.NATURE.COM/NATURE 19
RESEARCH SUPPLEMENTARY INFORMATION of the indicted mouse strins 96h fter infection (). Weight s the percentge (men ± s.d.; n=5) of ody weight t the dy of S. typhimurium infection (). Representtive pictures of cec from the indicted tretment groups 96h fter infection (c). Cecum length (men ± s.d.; n=5) 96h fter infection (d). e-g, Groups of Ifngr1 -/- nd control mice were infected with S. typhimurium. Representtive photomicrogrphs of H&E stined cec (e) of Ifngr1 +/+ nd Ifngr1 -/- mice 48h fter infection (r, 1 µm, mgnifiction, x1). Weight s the percentge (men ± s.d.; n=5) of ody weight t the dy of S. typhimurium infection (f). Cecum length (men ± s.d.; n=5) 48h fter infection (g). *p <.5; **p <.1 nd ***p <.1. 2 WWW.NATURE.COM/NATURE
RESEARCH T-et - CCR6 + (Fetl) CD4 + CD4 - NKp46 - Kit hi IL-17 IL-22 T-et - T-et + CCR6 - (Postntl) CD4 - NKp46 - NKp46 - Kit hi AhR Microiot Kit lo IL-23 T-et NKp46 + Kit lo IL-22 IFN-γ TNF Supplementry Figure 17: Model of RORγt + ILC lineges CCR6 + RORγt + ILCs seed the intestine efore irth nd express high levels of the receptor tyrosine kinse Kit. They re composed of CD4 + nd CD4 - suset nd they re potent producers of IL-22, IL17A nd IL-17F. CCR6 + RORγt + ILCs do not cquire NKp46 expression, do not express T-et nd their pool size is not sustntilly ffected y Ahr deficiency. CCR6 - RORγt + ILCs emerge postntlly (week 2 onwrds) s lrgely T-et - popultion. Expnsion of CCR6 - RORγt + ILCs requires AhR expression. CCR6 - RORγt + ILCs up-regulte T-et expression under the prtil control of signls from the commensl microiot nd IL-23. T-et represses Kit nd controls IFN-γ nd TNF expression. CCR6 - T- et + RORγt + ILCs differentite into NKp46 + cells, process strictly dependent on the presence of T-et. WWW.NATURE.COM/NATURE 21