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1 doi:1.138/nature171 Total number of Neuropilin1- (x1 ) (x1 ) 5 P < P <.1 GF HFD LFD Total number of Neuropilin GF HFD LFD Total number of CD13- cells (x1 ) (x1 ) GF HFD LFD Total number of CD13+ cells P <.5 P < GF HFD LFD Supplementary Figure 1 The number of total regulatory T cells in colonic lamina propria. Colonic lamina propria cells were prepared from GF and mice fed with HFD and LFD. CD3ε + CD + T cells were further analyzed for expression of, Neuropilin1 and CD13. Data are representative of three independent experiments. The Error bars indicate s.d. (n = 1). P values were determined by the one-way ANOVA test followed by Tukey s test. 1

2 RESEARCH Gated on CD3ε + CD + GF LFD HFD Helios 5 P <.1 P <.1 1 P <.1 [%] of Helios P <.1 [%] of Helios P <.1 GF HFD LFD GF HFD LFD Total number of Helios- cells (x1 ) (x1 ) 6.5 P <.1 GF HFD LFD Total number of Helios+ cells GF HFD LFD Supplementary Figure Helios expression in + cells in the colonic lamina propria. Colonic lamina propria cells were prepared from GF and mice fed with HFD and LFD. CD3ε + CD + T cells were further analyzed for expression of and Helios. Data are representative of three independent experiments. The Error bars indicate s.d. (n = 1). P values were determined by the one-way ANOVA test followed by Tukey s test.

3 RESEARCH a Gated on CD3ε + CD + GF - LFD - HFD Helios CD13 b Gated on CD3ε + CD + GF - LFD - HFD Helios CD13 Supplementary Figure 3 Flow cytometric analysis of lymphocytes in spleen and mesenteric lymph node (MLN). MLN (a) and spleen (b) lymphocytes were prepared from GF and mice fed with HFD and LFD. CD3ε + CD + T cells were further analyzed for expression of the indicated markers. The Error bars indicate s.d. (n = 5). Data are representative of three independent experiments. 3

4 RESEARCH a LFD HFD Firmicutes Tenericutes Others b % 5% 1% LFD HFD Clostridia Mollicutes Bacilli Others % 5% 1% c LFD HFD Clostridiales Unclassified Clostridia Anaeroplasmatales Turicibacterales % 5% 1% Others d LFD HFD % 5% 1% Lachnospiraceae; Unclassified Ruminococcaceae; Oscillospira Ruminococcaceae; Unclassified Ruminococcaceae; Ruminococcus Peptostreptococcaceae; Unclassified Unclassified; Unclassified Others Supplementary Figure 16S rrna sequence-based analysis of the cecal microbiota of associated mice fed with LFD or HFD. a-d, 16S rrna gene was PCR amplified and sequenced on the 5 platform (n = ). Phylum (a), class (b), order (c) and family; genus in Clostridiales (d) are shown.

5 OTU 1393 OTU 1393 OTU 139 OTU 139 RESEARCH Cluster IV Cluster IV Hydrogenoanaerobacterium saccharovorans {NR 5} Hydrogenoanaerobacterium saccharovorans {NR 5} Ruminococcus bromii {DQ8869} Escherichia coli {NR 18} Clostridium leptum {AJ3538} Butyricicoccus pullicaecorum {EU1376} Escherichia coli {NR 18} Roseburia intestinalis {AY3538} Roseburia intestinalis {AJ31385} Roseburia intestinalis {NR 7557} Roseburia hominis {AJ78} Roseburia cecicola {L1676} Roseburia faecis {AY3531} Roseburia inulinivorans {NR 7} Roseburia inulinivorans {AJ773} Lachnospira pectinoschiza {AY1691} Clostridium oroticum {FR79917} Clostridium nexile {NR 98} Clostridium coccoides {EF596} Eubacterium desmolans {L3618} OTU 1653 OTU 1386 OTU 399 Clostridium hylemonae {NR 719} Clostridium clostridioforme {HM86} Clostridium bolteae {NR 5567} Clostridium lavalense {EF5678} Clostridium leptum {AJ3538} Ruminococcus bromii {DQ8869} Butyricicoccus pullicaecorum {EU1376} Roseburia intestinalis {AY3538} Roseburia intestinalis {AJ31385} Roseburia intestinalis {NR 7557} Roseburia hominis {AJ78} Roseburia cecicola {L1676} Roseburia faecis {AY3531} Roseburia inulinivorans {NR 7} Roseburia inulinivorans {AJ773} Lachnospira pectinoschiza {AY1691} Clostridium oroticum {FR79917} Clostridium nexile {NR 98} Clostridium coccoides {EF596} Eubacterium desmolans {L3618} OTU 1653 OTU 1386 OTU 399 Clostridium hylemonae {NR 719} Clostridium clostridioforme {HM86} OTU 571 Clostridium bolteae {NR 5567} OTU 93 Clostridium lavalense {EF5678} OTU 1359 OTU 571 OTU 93 OTU 88 OTU 115 OTU 11 Clostridium xylanolyticum {X71855} OTU 33 OTU 1656 OTU 33 OTU 115 OTU 1359 OTU 88 OTU 11 Clostridium xylanolyticum {X71855} Clostridium celerecrescens {NR 61} OTU 179 OTU 693 OTU 1656 Clostridium celerecrescens {NR 61} OTU 179 OTU 68 OTU 693 OTU 139 OTU 68 OTU 1578 OTU 139 OTU 1578 Ruminococcus obeum {AY16911} OTU 15 OTU 11 OTU 118 OTU 1395 Acetanaerobacterium elongatum {NR 93} Clostridium cellulosi {FJ6516} Clostridium methylpentosum {Y18181} OTU 11 OTU 118 OTU 1395 OTU 163 Acetanaerobacterium elongatum {NR 93} OTU 153 Clostridium cellulosi {FJ6516} Clostridium methylpentosum {Y18181} OTU 163 Ruminococcus obeum {AY16911} OTU 153 OTU 111 OTU 15 OTU 556 OTU 187 OTU 111 OTU 556 OTU 37 Clostridium xylanovorans {NR 87} OTU 16 OTU 187 OTU 37 Eubacterium rectale {AY35311} Clostridium xylanovorans {NR 87} OTU 878 Eubacterium rectale {AJ775} OTU 16 Eubacterium rectale {AY35311} OTU 878 Eubacterium rectale {AJ775} Butyrivibrio fibrisolvens {AJ5365} Parasporobacterium paucivorans {NR 539} Butyrivibrio fibrisolvens {AJ5365} Parasporobacterium paucivorans {NR 539} OTU 198 Anaerostipes caccae {AJ787} Blautia hydrogenotrophica {NR 631} OTU 19 OTU 19 Blautia hydrogenotrophica {NR 631} OTU 198 Anaerostipes caccae {AJ787} OTU 119 Eubacterium ruminantium {NR 661} OTU 19 OTU 19 OTU 13 Clostridium aminophilum {DQ7886} OTU 119 Eubacterium ruminantium {NR 661} OTU 158 OTU 3 OTU 953 OTU 13 Clostridium aminophilum {DQ7886} OTU 1555 OTU 18 Oscillibacter valericigenes {NR 7793} OTU 158 OTU 3 OTU 953 OTU 856 OTU 656 Oscillibacter valericigenes {NR 7793} OTU 1555 OTU 18 OTU 9 OTU 856 OTU 656 OTU 11 OTU 11 OTU 9 OTU 165 OTU 11 OTU 6 OTU 11 Oscillospira guilliermondii {AB95} OTU 165 OTU 16 OTU 6 Oscillospira guilliermondii {AB95} OTU 15 OTU 77 OTU 16 OTU 15 OTU 11 OTU 77 OTU 1 OTU 11 OTU 68 OTU 1 OTU 89 OTU 181 OTU 68 OTU 89 OTU 89 OTU 33 OTU 181 OTU 199 OTU 89 OTU 1358 OTU 33 OTU 61 OTU 199 OTU 13 OTU 1358 OTU 93 OTU 61 OTU 1181 OTU 13 OTU 1519 OTU 93 OTU 65 OTU 1181 OTU 1519 OTU 65 OTU 98 OTU 3 Clostridium viride {NR 6} OTU 98 OTU 3 Clostridium viride {NR 6} OTU 6 OTU 66 Butyrivibrio proteoclasticus {U37378} OTU 6 OTU 66 Butyrivibrio proteoclasticus {U37378} OTU 95 OTU 5 OTU 95 OTU 611 OTU 611 OTU 6 OTU 5 OTU 1 OTU 1 OTU 39 OTU 6 OTU 1397 OTU 39 OTU 1397 OTU OTU OTU 161 OTU 199 OTU 16 OTU 161 OTU 199 OTU 16 OTU 11 OTU 11 OTU 6 OTU 16 OTU 16 OTU 115 OTU 6 OTU 115 OTU 168 OTU 168 OTU 183 OTU 5 OTU 59 OTU 17 Pseudoflavonifractor capillosus {NR 567} Clostridium orbiscindens {Y18187} OTU 96 OTU 17 Flavonifractor plautii {NR 31} OTU 197 OTU 15 OTU 15 OTU 18 OTU 1538 OTU 998 OTU 7 OTU 1596 OTU 513 OTU 55 Faecalibacterium prausnitzii {AJ77} Faecalibacterium prausnitzii {AJ769} Faecalibacterium prausnitzii {AY3537} Faecalibacterium prausnitzii {NR 8961} Clostridium litorale {X7785} Clostridium difficile {AB6383} Clostridium difficile {X735} Clostridium lituseburense {M5917} OTU Clostridium disporicum {Y18176} Clostridium acetobutylicum {X6818} Clostridium butyricum {FJ38369} Clostridium sporogenes {AJ57997} Clostridium tetani {NR 798} Clostridium tyrobutyricum {M59113} Clostridium peptidovorans {AF156796} Clostridium grantii {X757} Clostridium proteolyticum {X738} Clostridium histolyticum {M599} Clostridium limosum {M5996} OTU 13 OTU 1 OTU 196 Clostridium thermolacticum {X787} OTU 5 OTU 1313 Clostridium termitidis {X7185} Clostridium aldrichii {NR 699} Clostridium thermocellum {NR 769} OTU 13 OTU 979 OTU 63 Catonella morbi {NR 68} Clostridium scindens {NR 8785} OTU 173 OTU 191 OTU 61 OTU 138 OTU 183 OTU 5 OTU 59 OTU 17 Pseudoflavonifractor capillosus {NR 567} Clostridium orbiscindens {Y18187} OTU 96 OTU 17 Flavonifractor plautii {NR 31} OTU 197 OTU 15 OTU 15 OTU 18 OTU 1538 OTU 998 OTU 7 OTU 1596 OTU 513 OTU 55 Faecalibacterium prausnitzii {AJ77} Faecalibacterium prausnitzii {AJ769} Faecalibacterium prausnitzii {AY3537} Faecalibacterium prausnitzii {NR 8961} Clostridium litorale {X7785} Clostridium difficile {AB6383} Clostridium difficile {X735} Clostridium lituseburense {M5917} OTU Clostridium disporicum {Y18176} Clostridium acetobutylicum {X6818} Clostridium butyricum {FJ38369} Clostridium sporogenes {AJ57997} Clostridium tetani {NR 798} Clostridium tyrobutyricum {M59113} Clostridium peptidovorans {AF156796} Clostridium grantii {X757} Clostridium proteolyticum {X738} Clostridium histolyticum {M599} Clostridium limosum {M5996} OTU 13 OTU 1 OTU 196 Clostridium thermolacticum {X787} OTU 5 OTU 1313 Clostridium termitidis {X7185} Clostridium aldrichii {NR 699} Clostridium thermocellum {NR 769} OTU 13 OTU 979 OTU 63 Catonella morbi {NR 68} Clostridium scindens {NR 8785} OTU 173 OTU 191 OTU 61 OTU 138 Cluster XI Cluster XI Cluster I Cluster I Cluster II Cluster II Cluster Cluster III III Cluster XIVa Supplementary Supplementary Figure Figure 5 5 Phylogenetic Phylogenetic distribution of of operational taxonomic units units (OTUs) (OTUs) obtained obtained from from 16S 16S rrna rrna sequences sequences in in order order Clostriales Clostriales colonized colonized in in mice. mice. 1, filter-passed reads of 16S rrna sequences in mice were used for OTUs and a phylogenetic tree 1, filter-passed reads of 16S rrna sequences in mice were used for OTUs and a phylogenetic tree was constructed based on the -derived 11 OTUs which were categorized into Clostridiales by RDPclassifier (v.5) and 69 reference 16S rrna sequences of Clostridiales obtained from the GenBank database. was constructed based on the -derived 11 OTUs which were categorized into Clostridiales by RDPclassifier (v.5) and 69 reference 16S rrna sequences of Clostridiales obtained from the GenBank database. The 16S rrna sequence of Escherichia coli was used as the outgroup. The phylogenetic tree was constructed The based 16S rrna on the sequence neighbor-joining of Escherichia method, coli with was 1 used bootstrap as the iterations outgroup. (bootstrap The phylogenetic values > 7 tree are was shown constructed on each based branch). on the The neighbor-joining scale bar indicates method, an estimated with 1.1 bootstrap change per iterations nucleotide (bootstrap position. values Colors > configuring 7 are shown a on each branch). doughnut The shape scale bar represent indicates the branches estimated consisting.1 change of the per distinct nucleotide clusters position. of Clostridiales. Colors configuring The reference a 16S doughnut rrna sequences shape represent from spore the branches forming, consisting non-spore forming, of the distinct and unknown clusters spore of Clostridiales. forming capacity The reference bacteria are 16S rrna shown sequences in blue, from purple spore and black, forming, respectively. non-spore The forming, outside and red unknown and green spore bars indicate forming the capacity relative bacteria number are of shown sequence in blue, reads purple in each and OTU black, from respectively. -HFD The and outside -LFD, red and respectively. green bars Most indicate of OTUs the both relative in -HFD number of sequence and -LFD reads in were each clustered OTU from into -HFD Clostridium and clusters -LFD, IV and respectively. XIVa. Most of OTUs both in -HFD and -LFD were clustered into Clostridium clusters IV and XIVa. 5

6 RESEARCH a b n.s. 1 1 n.s. (%) CV 5 (%) CV 5 nd GF HFD LFD nd GF HFD LFD Supplementary Figure 6 The number of total commensal microbes and Clostridiales in GF, and mice fed with HFD and LFD. a, b, Total number of commensal microbes (a) and Clostridiales (b) were detected by qpcr with 16S rrna gene universal primers and Clostridiales-specific primers. The results were calculated as the quantity relative to the copy number detected in the feces of conventionalized mice (CV). Error bars indicate s.e.m. (n = 5). P values were determined using the Kruskal-Wallis test followed by the Scheffé test. n.s., not significantly different. n.d., not detected. 6

7 RESEARCH a -8-6 Orthogonal component LFD HFD PredicUve component b -1. Modeled correlauon Acetate Butyrate/Propionate Butyrate L- Isoleucine L- Leucine GABA Lactate UnidenUfied Others p[1] Covariance Supplementary Figure 7 Orthogonal partial least squares discriminate analysis (OPLS-DA) on the cecal metabolome data of -LFD and -HFD mice. a, Cross-validated score plots from OPLS-DA of 1 H-NMR data of -LFD and -HFD murine cecum (n = 7). The model resulted in one predictive and one orthogonal (1+1) components with the cross-validated predictive ability Q (Y) =.93 and the total explained variance R (X) =.79. The ellipse denotes the 95% significance limit of the model, as defined by Hotelling s t-test. b, S-plot for predictive component from OPLS-DA of 1 H-NMR data of -LFD and -HFD murine cecum. The S-plot visualizes the variable influence in a model and corresponds to combining the contribution or magnitude (covariance) with the effect and reliability (correlation) for the model variables with respect to model component scores. The highlighted signals ( covariance >.16 ) in the S-plot have high contribution and reliability for class separation between -LFD and -HFD mice. 7

8 RESEARCH a b c 3 L- leucine 3 L- isoeucine 3 GABA [%] of + 1 [%] of + 1 [%] of + 1 Supplementary Figure 8 Leucine, Isoleucine and GABA have no effect on the induction of + cells a, b, c, Naïve CD + T cells were cultured under Treg-inducing conditions in vitro. The cells were cultured in RPMI 16 medium, which contains.38 mm each of L-leucine and L-isoleucine, that was supplemented with L-leucine (,,1 and.6 mm) (a), L-isoleucine (,,1 and.6 mm) (b) or GABA (,,1,.1 and.1 mm) (c). Cells were further analyzed for expression of. The Error bars indicate s.d. (n = 3). 8

9 RESEARCH Gated on CD3ε + CD + CD Total number of + cells ( 1 ) P <.5 Supplementary Figure 9 The number of total + cells in colonic lamina propria of C57BL/6 mice fed with HAMS or HAM. Colonic lamina propria cells were prepared from C57BL/6 mice fed with HAMS () or HAM (). CD3ε + CD + T cells were further analyzed for expression of. Data are representative of three independent experiments. The Error bars indicate s.d. (n = 1). P values were determined by the non-parametrical Mann-Whitney U test 9

10 RESEARCH Gated on CD3ε + CD + rol EdU Supplementary Figure 1 Butyrate has no effect on proliferation of Treg cells in colonic lamina propria. a, Mice fed with HAMS (rol) or HAM () were treated with EdU, a nucleoside analog incorporated into DNA during synthesis phase of cell cycle, for days before the analysis. Colonic lamina propria cells were prepared from those mice and CD3ε + CD + T cells were further analyzed for expression of and EdU. The Error bars indicate s.d. (n = 5). 1

11 RESEARCH The number of cells Dead Day 1 Day Day 3 Live (x1 ) (x1 ) (x1 ) Butyrate: (- ) (+) (- ) (+) (- ) (+) Supplementary Figure 11 Butyrate has no effect on cell survival under Treg-inducing conditions in vitro. The number of total cells under Treg-inducing condition cultures in the absence or presence of butyrate. Cells were cultured as described in Fig.3a. The viability was evaluated by Trypan blue dye exclusion test. The Error bars indicate s.d. (n = 5). 11

12 RESEARCH Gated on CD3ε + CD + a (x1 ) Total number of Neuropilin1- Treg cells P <.5 Neuropilin1 (x1 ) 1 P <.5 Total number of Helios- Treg cells 8 6 Helios b µmol/g Cecal SCFA µmol/g Fecal SCFA Ace Pro But Ace Pro But Supplementary Figure 1 Intestinal butyrate induces the accumulation of Neuropilin- and Heliossubsets of Treg cells in mice colonized with Bacteroides thetaiotaomicron. GF mice were colonized with B. thetaiotaomicron. B. thetaiotaomicron-associated mice were fed with HAMS () or HAM () for weeks. a, Colonic lamina propria cells were prepared and CD3ε + CD + -gated cells were further analyzed for expression of, Neuropilin and Helios. P values were determined by the nonparametrical Mann-Whitney U test. b, Cecal and fecal amount of short-chain fatty acids from B. thetaiotaomicron-associated mice fed with HAMS or HAM. Ace, acetate; Pro, propionate; But, butyrate. The Error bars indicate s.d. (n = 5). 1

13 RESEARCH a Gated on CD3ε + CD + CD [%] of n.s. Total number of + cells (x1 ) n.s. b µmol/g Ace Pro But Supplementary Figure 13 Butyrate has no effect on + induction in colonic lamina propria of GF mice fed with HAMS or HAM. a, Colonic lamina propria cells were prepared from GF mice fed with HAMS () or HAM (). CD3ε + CD + T cells were further analyzed for expression of. b, Amount of cecal short-chain fatty acids from GF mice fed with HAMS or HAM. Ace, acetate; Pro, propionate; But, butyrate. Data are representative of three independent experiments. n.s., not statistically significant. The Error bars indicate s.d. (n = 5). 13

14 RESEARCH Gated on CD3ε + CD + T- bet rol CD [%] of T- bet+ cells n.s. Total number of T- bet+ cells (x1 ) 3 1 n.s. GATA3 CD [%] of GATA3+ cells n.s. Total number of GATA3+ cells (x1 ) 8 6 n.s. RORγt [%] of RORγt+ cells 3 1 n.s. Total number of RORγt+ cells (x1 ) 3 1 n.s. CD Supplementary Figure 1 Intestinal butyrate has no effect on the accumulation of T-bet +, GATA3 + and RORγt + T cells. Colonic lamina propria cells were prepared from SPF C57BL/6 mice fed with HAMS (rol or ) or HAM (). CD3ε + CD + T cells were further analyzed for expressions of T-bet, GATA3, and RORγt, the lineage-specific transcription factors of T H 1, T H, and T H 17, respectively. The Error bars indicate s.d. (n = 1). P-values were calculated by Mann-Whitney U test. Data are representative of three independent experiments. 1

15 RESEARCH a Butyrate PolarizaUon T H 1 T H T H 17 (- ) (+) T- bet GATA3 RORγt CD CD PolarizaUon CD Butyrate T H 1 T H T H 17 (- ) (+) b CD CD CD [%] of T- bet+ 3 1 Butyrate: [%] of + P < [%] of GATA [%] of P <.1 1 (- ) (+) (- ) (+) (- ) (+) (- ) (+) (- ) (+) (- ) (+) T H 1 polarizauon T H polarizauon T H 17 polarizauon [%] of RORγt+ [%] of + Supplementary Figure 15 Butyrate increases the frequency of + T cells under T H 1 and T H 17 polarizing conditions. a, b, Naïve CD + T cells were stimulated with immobilized anti-cd3 and soluble anti- CD8 antibodies. Cells were cultured in the presence or absence of butyrate and either with ril-1 (1 ng/ml) plus anti-il- (1 mg/ml) for T H 1 polarization, with ril- (1 ng/ml) plus anti-ifnγ (1 mg/ml) for T H polarization or with ril-6 (1 ng/ml), rhtgfβ (. ng/ml), ril-1β (1 ng/ml), anti-ifnγ plus anti-il- for T H 17 polarization. ril- (1 ng/ml) was added to culture medium days later. Cells were further analyzed for expression of, T-bet, Gata3 and RORγt on day. Representative FACS plots gated on CD3ε + CD + are shown in c. The Error bars indicate s.d. (n = 3). P-values were calculated by Mann-Whitney U test. Data are representative of three independent experiments. 15

16 RESEARCH a Gated on CD3ε + CD + Helios b [%] of Helios- + cells 3 1 P <.5 Total number of Helios- + cells (x1 ) P <.5 Supplementary Figure 16 Induction of colonic Treg cells by intestinal butyrate in SPF Myd88 -/- Ticam1 -/- mice. a-b, SPF Myd88 -/- Ticam1 -/- mice were fed with HAMS () or HAM (). Representative FACS plots gated on CD3ε + CD + are shown in a. The frequency and total number of Helios - + cells among colonic CD + T cells are shown in b. Data are representative of two independent experiments. The Error bars indicate s.d. (n = ). P-values were calculated by Mann- Whitney U test. 16

17 RESEARCH a Butyrate + AcH3 H3 b Normalized read count Normalized read count rol Butyrate - - Distance to TSS (kb) Supplementary Fig. 17 Genome-wide histone H3 acetylation status after butyrate exposure. Naïve CD + T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. a, Total and acetylated histone H3 (AcH3) were measured by Western blot analysis of total cell lysates. b, ChIP-Seq analysis was performed using anti-ach3 antibody. The average values of AcH3 status around the transcription start site (TSS) over total genes are shown. 17

18 RESEARCH Tbx1/T- bet Gata3/GATA3 Rorc/RORγt Normalized read count Normalized read count Normalized read count rol Butyrate Supplementary Figure 18 Histone acetylation status of Tbx1, Gata3 and Rorc. Naïve CD + T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. Histone acetylation in the genes encoding master molecules for effector T cells was analyzed by ChIP-sequencing using anti-acetylated histone H

19 RESEARCH Normalized read count Stat5a/STAT5A Normalized read count Stat5b/STAT5B Smad3/SMAD3 Nfatc/NFAT1 Normalized read count Normalized read count Rel/c- Rel Normalized read count rol Butyrate Supplementary Figure 19 Histone acetylation status of Stat5, Smad3, Nfatc and Rel. Naïve CD + T cells were cultured under Treg-inducing conditions in the presence or absence (control) of butyrate for 1 day. Histone acetylation in the genes encoding transcriptional regulators controlling expression was analyzed by ChIP-sequencing using anti-acetylated histone H

20 RESEARCH Day 1 Day Butyrate + + STAT5 SMAD3 NFAT1 c- Rel AcH3 H3 GAPDH Supplementary Figure Expression of STAT5, SMAD3, NFAT1, and c-rel. Naïve CD + T cells were cultured under Treg-inducing conditions in the absence or presence of butyrate. Total cell lysates were subjected to western blotting analysis with antibodies described in Methods. H3 and AcH3 indicate histone H3 and acetylated histone H3, respectively. Data are representative of three independent experiments.

21 RESEARCH a b µmol/g feces Ace Pro But rol SA SP Body weight change (%) Post transfer (week) rol SA SP c rol SA SP HE AB Supplementary Figure 1 Intestinal butyrate, but not acetate or propionate, ameliorates T- cell-dependent experimental colitis. a-f, Experimental colitis was induced by adoptive transfer of CD + CD5RB hi T cells in Rag1 -/- mice fed with HAMS (rol), HAMSA (SA), HAMSP (SP) or HAM (). Fecal amount of SCFAs (a), body weight change (b), and representative colonic specimens stained with hematoxylin-eosin (HE) and Alcian blue (AB) (c) are shown. The Error bars indicate s.d. (n = 5). Scale bars: µm (c). All data are representative of two independent experiments. 1

22 RESEARCH Epigenetic modifications Naïve T cells APC Antigen stimulation Treg cells Supplementary Figure A schematic diagram of epigenetic modifications by commensal microbial fermentation product. Clostridiales abundantly produce butyrate, which promotes histone H3 lysine acetylation in gene locus of CD + T cells, and eventually facilitates induction of Treg cells. APC: antigen-presenting cells.

23 RESEARCH 3