Metaoli ontrol of type regulatory () ell ifferentiation y AHR an HIF-α Ivan D. Masanfroni, Maisa C. Takenaka, Aa Yeste, Bonny Patel, Yan Wu, Jessia E. Kenison, Shafiuin Siiqui, Alexanre S. Basso, Leo E. Otterein, Drew M. Paroll, Fan Pan, Avner Priel, Clary B. Clish, Simon C. Roson, Franiso J. Quintana Center for Neurologi Diseases, Brigham an Women s Hospital, Harvar Meial Shool, 77 Avenue Louis Pasteur, Boston, MA, USA. Divisions of Gastroenterology, Hepatology an Transplantation, Beth Israel Deaoness Meial Center, Harvar Meial Shool, Blakfan Cirle, Boston, MA, USA. Department of Miroiology, Immunology an Parasitology, Esola Paulista e Meiina, Feeral University of Sao Paulo, Sao Paulo, Brazil. Immunology an Hematopoiesis Division, Department of Onology an Meiine, Johns Hopkins University Shool of Meiine, MD, USA. Department of Physis, University of Alerta, Emonton, Alerta TG J, Canaa. Center for Proteomis, The Broa Institute of MIT an Harvar, Camrige, MA, USA. These authors ontriute equally to the work Corresponing author: Franiso J. Quintana (e-mail: fquintana@ris.wh.harvar.eu) Center for Neurologi Diseases, Harvar Meial Shool, 77 Avenue Louis Pasteur, Boston, MA, USA. Supplementary figures an legens Nature Meiine: oi:.8/nm.88
f isotype g h 8 isotype anti-stat anti-ahr 8 isotype isotype anti-stat anti-ahr i mrna (fol) Maf mrna (fol) Foxp mrna (fol) Foxp mrna (fol) STAT enrihment IL SRE- SRE Stat / Stat /.... 89..9 7. 9. Gata mrna (fol) j Tx mrna (fol) % IL- + ells, 8 Ror mrna (fol) Foxp mrna (fol) AHR enrihment Entp / ENTPD mrna (fol) XRE IL- IL- Th Th 98. CD e IFN-γ Foxp,. 9. IL-. 7..7 IL- SRE- XRE XRE: responsive element SRE: STAT responsive element Start of transription Control Stat + Stat Supplementary Figure. CD9 is expresse in ells in an AHR an STAT-epenent manner. (a-) lineage-speifi transription-fator oing mrnas (a) an ytokine expression () as etermine y qpcr an flow ytometry, respetively, of T ells use for the analyses of CD9 (Entp) mrna an protein expression shown in Figs. a,. () Naive CD+ T ells were ifferentiate for 7h in the presene of IL-7, an ells were analyze as the perentage of CD + Foxp IL- + ells. () ENTPD expression in human naive T ells ifferentiate uner, an polarizing onitions (e) Shemati representation of an promoter with AHR- an STAT-ining sites (lue: XRE, green: SRE, respetively). (f) ChIP analysis of the interation of STAT in promoter from an Stat / mie after 8h of ell ifferentiation. (g) ChIP analysis of the interation of AHR in the promoter after 8 h uner or polarizing onitions. (h) Luiferase ativity in HEK9 ells transfete with an luiferase reporter, alone (Control) or with a onstrut enoing onstitutively ativate STAT or AHR. (i) expression in ell ifferentiate from or Stat / mie. (j) Foxp mrna expression etermine y qpcr in or CD9-efiient CD9. + IL-:GFP + ells use for the ell transfer experiment esrie in Figures i,j. Numers ajaent to outline areas iniate perentage of positive ells. P <.; P <.; P <.. Data are from one of more than inepenent experiments with similar results. Luiferase ativity (fol hange ompare to empty vetor) + Nature Meiine: oi:.8/nm.88
LAG- Entp /.9..9. 8..9.9.7 CD9 % CD9 + LAG + ells Entp / 7-AAD Vehile ATP (μm)..7...79.8.7. -. 9. 7.. 7... 9.9 - Annexin V IL-7 IL-. CD Vehile ATP (μm).9. 8.. % IL- + ells ATP (μm) IL- Isotype Anti-CD9 7.. CD % IL- + ells Isotype Anti-CD9 Supplementary Figure. eatp inhiits ell ifferentiation. (a) Naive CD + ells from or CD9 (Entp / ) efiient mie were ativate uner polarizing onitions an the frequeny of CD + LAG + CD9 + ells in the IL- + population was analyze y flow ytometry. () ells were ifferentiate for 7 h in the presene of eatp, an viaility was assesse y flow ytometry with Annexin V an 7-Amino-Atinomyin (7-AAD). (-) Human ells were ifferentiate in the presene of eatp () or an anti-cd9 loking antioy () an the perentage of CD + Foxp IL- + T ells was analyze. Data are representative from inepenent experiments. Nature Meiine: oi:.8/nm.88
Hifa mrna (fol) Input WB / U IP / AHR + + + ATP + (kda) 9 7 Arnt mrna (fol) U / Input 8 + ATP + ATP e WB / ARNT AHR / GAPDH Input..... Hifa IP: AHR + + + ATP + ARNT / Input.... + + + + ATP HIF α / GAPDH... Hifa WB / ARNT Input IP / HIF α + + + ATP + + + + ARNT / Input Vehile MG + ATP f AHR enrihment 8 Il promoter Isotype Anti-AHR g IL-. xia CD. xia % IL- + ells xia xia h IL- (ng/ml) IL- mrna (fol) mrna (fol) Entp mrna (fol) i IL- IFN-γ xia 9.9. 9.. xia.9... j AHR enrihment Il promoter AHR enrihment 8 Entp promoter isotype anti-ahr isotype anti-ahr Supplementary Figure. HIF-α antagonizes AHR uring ell ifferentiation. (a) Hifa as etermine y qpcr in,, Foxp + Tregs an ells. () Arnt expression presente relative Gaph. () FACS-sorte CD + T ells were ativate an ulture uner -skewing onitions in the presene of eatp for 7 h. Cell lysates were immunopreipitate with antioies to AHR or HIF-α antioies, followe y SDS-PAGE an western lotting. The ensitometri analysis of ARNT expression was alulate. () Cell lysates were immunopreipitate with antioies to AHR, followe y SDS-PAGE an western lotting. The ensitometri analysis of U expression was alulate. (e) Densitometri analysis of AHR (left) an HIF-α (right) expression in Fig. f. (f) ChIP analysis of the interation of AHR with the Il promoter uner, or pathogeni polarizing onitions. (g-h) Naive T ells were polarize into ells uner normoxi or hypoxi onitions an the frequeny of CD + Foxp IL- + T ells (g), IL- proution an the expression of Il, an Entp (h) were evaluate y FACS, ELISA an qpcr respetively. Numers ajaent to outline areas iniate perentage of positive ells. (i) Differentiation of ells uner hypoxi or normoxi onitions. Numers ajaent to outline areas iniate perentage of positive ells. (j) ChIP analysis of the interation of AHR in the Il an Entp promoter uner or skewing onitions. P <.; P <.. Data are representative (,e,h) or are the mean ± SEM (a-g,i) of three inepenent experiments. AU, aritrary units. Nature Meiine: oi:.8/nm.88
AHR ACTIN CD + IFN-γ + ells (%) Tet mrna (fol) mrna (Fol) 8 Ctrl-RV + Vehile 9.7 Hifa fl/fl Cre-RV + + + + Entp / Hifa / Entp / Hifa / CD + IL- + ells (%) Ror mrna (fol) Maf mrna (Fol) DG ( mm) Vehile DG ( mm) 7.8 AHR / ACTIN Ctrl-RV Entp / Hifa / Entp / Hifa / Cre-RV CD + Foxp + ells (%) Foxp mrna (fol) Foxp mrna (Fol).. Entp / Hifa / Entp / Hifa / Ror mrna (Fol).8.8 Foxp IL-..7 87.. CD IFN-γ Supplementary Figure. Role of glyolysis in ifferentiation. (a) Hifa fl/fl CD+ T ells were ifferentiate uner polarizing onitions, transue with a re-reominase expressing retrovirus (Cre-RV) at ifferent time points (, an h) an the expression of AHR was evaluate y western lot. (),, Entp -/- an Hifa -/- naive CD + T ells were ativate uner, an it reg polarizing onitions an T ell ifferentiation was analyze y flow ytometry an qpcr. () lineage-speifi transription-fator oing mrnas as etermine y qpcr in the,, it regs an ells. () Naive T ells were ativate uner polarizing onitions an vehile or mm of -DG an the perentage of CD + Foxp + Treg ells was evaluate y flow ytometry. Numers ajaent to outline areas iniate perentage of positive ells. P <.; P <. of one-way ANOVA. Data are representative (a,) or the mean ± SEM (a,) of three inepenent experiments. Nature Meiine: oi:.8/nm.88
HIF-AHR network moel Ext. Stim. HIF-a AHR HIF egraing enzymes Egln Egln Moel Moel Moel Moel 8.. 8 8.. 8 enrihment Egln enrihment Egln enrihment Egln Isotype Anti- Egln mrna (fol) e f My enrihment My mrna (fol) : Site isotype anti-my (h) anti-my (8h) Eno Egln mrna (fol) 8 Site Site Site isotype anti-my (h) anti-my (8h) : My enrihment Esrra mrna (fol) Egln mrna (fol) Site Lha Site Site Site Site Site Supplementary Figure. Mathematial moel of an Hifa expression. (a) Diagram of mathematial moel. () HIF-α (lue) an AHR (re) expression uring ell ifferentiation measure (full lines) an preite (ashe lines) y the moel inluing Egln or Egln expression. () ChIP analysis of the interation of AHR with the Egln, Egln an Egln promoters after 8h uner,, Foxp + it reg or polarizing onitions. () Egln, Egln an Egln expression in,, Foxp + it reg an ells after 8h of ifferentiation; expression is presente relative to Gaph. (e) My an Esrra expression relative Gaph. (f) ChIP analysis of the interation of My in the Eno an Lha promoters uner or skewing onitions. Data are representative (a) or the mean ± SEM (,f) of three inepenent experiments. Nature Meiine: oi:.8/nm.88