immunology.sciencemag.org/cgi/content/full/3/28/eaat6975/DC1

Supplementary Materials for

Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of

Foxp3+ regulatory T cells

Ravi Verma, Changhon Lee, Eun-Ji Jeun, Jaeu Yi, Kwang Soon Kim, Ambarnil Ghosh, Seohyun Byun, Choong-Gu Lee, Hye-Ji Kang, Gi Cheon Kim, Chang-Duk Jun, Gwenaël Jan, Chang Hee Suh, Ju-Yang Jung, Jonathan Sprent,

Dipayan Rudra, Cristina De Castro, Antonio Molinaro, Charles D. Surh, Sin-Hyeog Im*

*Corresponding author. Email: iimsh@postech.ac.kr

Published 19 October 2018, Sci. Immunol. 3, eaat6975 (2018)

DOI: 10.1126/sciimmunol.aat6975

The PDF file includes:

Materials and Methods Fig. S1. Identification of Bb as Treg-inducing bacteria. Fig. S2. Effect of Bb monocolonization on cytokine levels in Treg and non-Treg cells. Fig. S3. Bb monocolonization facilitates de novo generation of pTreg cells. Fig. S4. Bb colonization induces dietary Ag– and/or microbiota-reactive Treg cells. Fig. S5. Effect of Bb monocolonization on the TCR repertoire of Treg cells. Fig. S6. Effect of Bb monocolonization on phenotypes and population of cLP-DC subtypes. Fig. S7. Effect of Bb monocolonization on phenotypes and population of DC subtypes in mLN and siLP. Fig. S8. CSGG of the Bb enhances Treg cell induction. Fig. S9. Role of DC subtypes in inducing Bb/CSGG-mediated iTreg cells. Fig. S10. CSGG facilitates iTreg induction through TLR2-mediated generation of regulatory DCs. Fig. S11. CSGG-induced iTreg cells are capable of suppressing intestinal inflammation. Fig. S12. Confirmation of Bf monocolonization by DNA sequencing. Table S1. Peptide sequences of α-chain CDR3 region of Treg cells sorted from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice. Table S2. Peptide sequences of β-chain CDR3 region of Treg cells sorted from colon, mLN, and spleen of Bb-monocolonized mice compared with GF mice. References (49–55)

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/3/28/eaat6975/DC1)

Table S3. Raw data (Excel file).

Supplementary Materials and Methods

Mouse strains

Mice were maintained in the animal facility of POSTECH Biotech Center and all the

experimental procedures were approved by the POSTECH Institutional Animal Care and Use

Committee. A colony of germ free (GF) C57BL/6 (B6) mice was established at POSTECH from

breeders obtained from Dr. Andrew Macpherson (Bern Univ., Switzerland) and Dr. David Artis

(Univ. Pennsylvania, currently at Cornell University, USA) and maintained in sterile flexible

film isolators (Class Biological Clean Ltd., USA). GF status was monitored monthly by culture

of cecal contents. Dec1-/-

and Dec2-/-

mice were kindly provided by Dr. Yoichiro Iwakura (Tokyo

University of Science, Japan). Foxp3-eGFP, Tlr2-/-

, Tlr4-/-

, Tlr6-/-

and MyD88-/-

animals were

obtained from the Jackson Laboratory. C57BL/6-CD45a(Ly5a)-Rag1-/-

TCR OT-II (Rag1-/-

OT-

II TCR transgenic) and Rag1-/-

mice were obtained through Taconic. CBir mouse was a gift by

Charles O. Elson, University of Alabama at Birmingham. Gender- and age-matched mice

between 6-12 weeks old were used.

Data availability for RNA-seq

Total RNA was extracted from the splenic CD11c+ DCs stimulated with mock or CSGG (100

g/ml) for 4 hrs and purified with RibospinTMII (GeneAll biotechnology). RNA quantitation

and quality control was performed by NanoDrop 2000™ (Thermo Fisher Scientific, Wilmington,

DE). Library preparation was performed by TruSeq Stranded mRNA Sample Preparation Kit

(Illumina, San Diego, CA), and RNA-sequencing was performed by NextSeq 500 Sequencing

System. The RNA-seq data was deposited in the Gene Expression Omnibus (NCBI) data

repository under accession number GEO: RNA-seq data: GSE98947.

T cell transfer model of colitis

Experimental colitis induced by adoptive transfer of naïve T cell transfer was performed

following a previously reported method (49, 50). In brief, sorted naïve

CD4+CD62L

hiCD44

loFoxp3

GFP- T cells (>99% pure, 1x10

6) were transferred intravenously to

congenic Rag1-/-

mice together with iTreg cells (2x105) induced by either B. bifidum PRI1 (Bb),

CSGG or mock treatment. Progression of colitis was monitored by measuring body weight two

times a week, and experimental endpoint was determined when mice have lost around 25% of

their initial body weight. As a positive control of Treg cells, ex vivo isolated CD4+ Foxp3

GFP+

Treg cells (2x105) were co-transferred together with naïve T cells into the Rag1

-/- mice. At the

end of the experiment, disease severity of colitis was determined by measuring colon lengths and

histological assessment. Level of Treg cells and cytokine were also analyzed.

Analysis of bacterial colonization by fluorescent in situ hybridization (FISH)

Three weeks after mono-colonization with B. bifidum, mice were sacrificed and the intestine was

divided into the duodenum (within 1-3 cm distal to the pylorus), jejunum (within 2-4 cm distal to

the ligament of Treitz), ileum (within 1-3 cm proximal to the ileocecal valve) and the colon.

Each section (1 cm) was immediately placed in Carnoy’s fixative for histological analysis. 2 ml

of Carnoy’s fixative solution (3:1 of Methanol: Glacial Acetic Acid) was added drop wise and

the cell suspension was centrifuged and pellets were re-suspended in Carnoy’s solution (51).

Paraffin-embedded fixed sections were dewaxed and hybridized with the Cy5 labelled

fluorescence probe specific for B. bifidum 16S rRNA (Bbif, 5′- CCACAATCACAT-

GCGATCATG -3′; 100 ng) (52) in 20 μl of hybridization buffer (20 mM Tris, pH 7.2. 0.9 M

NaCl, 0.1% sodium dodecyl sulphate) at 45°C for 16 h in a humidified chamber. Slides were

washed in 100 ml of preheated (37°C) hybridization buffer for 15 min and subsequently washed

again in 10 ml of preheated (37°C) washing solution (100 mM Tris, pH 7.2, 0.9 M NaCl) for 15

min (53). DAPI (4′,6-diamidino-2-phenylindole) staining was performed to detect DNA. Slides

were rinsed in water and air-dried. Images were obtained with a fluorescence microscope (IX70,

Olympus, Tokyo, Japan).

In vivo adoptive transfer experiments

Sorted naïve polyclonal CD4+CD62L

hiCD44

loFoxp3

GFP- T cells (>99.5% pure) (1x10

6) were

transferred intravenously to GF C57BL/6 mice, and single strain of bacteria was administered

orally. Mice were sacrificed 3 weeks after adoptive transfer. For the analysis of TCR specificity

of Treg cells reactive to dietary Ag, naïve T cells (1x106) isolated from the OT-II

CD4+CD62L

hiCD44

loFoxp3

GFP- T cells were transferred intravenously to the GF C57BL/6 mice

that were mono-associated with B. bifidum PRI1 for 2 weeks before adoptive transfer and fed

with OVA (20mg/dose/mice) every other day for 7 days. For the analysis of TCR specificity of

Treg cells reactive to commensal bacteria, sorted (>99.5% pure) naïve CBir

CD4+CD62L

hiCD44

loFoxp3

GFP- T cells (1x10

6) were transferred intravenously to the C57BL/6

mice maintained in SPF conditions and fed with B. bifidum PRI1 (5x108 cfu/dose) 3 times a

week till the end of the experiments. Mice were sacrificed 3 weeks after adoptive transfer.

DC-dependent in vitro iTreg cell differentiation assay

Colonic LP DCs (MHCII+CD11c

+CD11b

+CD103

+F4/80

-), splenic total DCs (tDC;

MHCII+CD11c

+), splenic CD8α

+ (MHCII

+CD11c

+CD11b

-) and splenic CD8α

-

(MHCII+CD11c

+CD11b

+) DCs, splenic pDC (MHCII

+CD11c

loBst2

+), cDC

(MHCII+CD11c

+CD11b

+), CX3CR1

+ (MHCII

+CD11c

+CD11b

+CX3CR1

+) and CX3CR1

-

(MHCII+CD11c

+CD11b

+CX3CR1

-) were purified by cell sorting or CD11c magnetic beads

(Miltenyi Biotech), respectively. Isolated DCs were cultured in complete RPMI 1640 media

containing 10% FBS, 5% penicillin/ streptomycin, 2mM L-glutamine, 1mM sodium pyruvate,

non-essential amino acids and β-ME. Splenic DCs (2x104) were seeded into a 96 well plate for 2

hours, then incubated with indicated bacteria, differential cellular fractionates of B. bifidum PRI1

or purified cell wall polysaccharides of B. bifidum PRI1 for 10-12 hrs. Cells were washed and

co-cultured with naïve CD4+ T cells (2x10

5) in sub-optimal Treg inducing condition (0.1μg/ml of

anti-CD3, 100U IL-2 and 0.1ng/ml of TGF-β) for three days. Level of cytokines or Treg cells

were determined by ELISA and flow cytometry, respectively.

Human samples for DC differentiation and iTreg cell induction by CSGG treatment.

Institutional Review Board of Ajou University Hospital approved this study (AJIRB-BMR-SMP-

17-155). Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor

through Ficoll-Hypaque gradients (Lymphoprep, Nycomed, Norway). Monocytes were isolated

from PBMCs (CD14+ > 95%) by positive selection using a CD14 microbeads kit (Miltenyi

biotech). 5 ×105 monocytes/ml were cultured in 24-well plates for 6 days (37

oC, 5% CO2) in

IMDM medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics in

presence of rhIL-4 (100 ng/ml) and GM-CSF (100 ng/ml) (Peprotech, USA). On days 2 and 5,

0.5 ml of the medium was replaced with freshly medium containing GM-CSF and IL-4. On day 6,

immature DCs were recovered, pre-treated with mock or different amounts of CSGG for 14 h.

Then they were co-cultured with naïve CD4+ T cells isolated from human PBMC in sub-optimal

Treg inducing condition (0.1μg/ml of anti-CD3, 100U IL-2 and 0.1ng/ml of TGFβ) for three

days. Level of Treg cells were determined by flow cytometry.

RNA isolation and real-time quantitative RT-PCR

Total RNAs isolated from colon and cLP DCs by using TRIzol reagent (Invitrogen) were reverse

transcribed to prepare cDNAs using M-MLV reverse transcriptase (Promega, Madison,WI).

Prepared cDNA was subjected to quantitative real time-PCR (qRT-PCR) using Chromo-4

(Biorad) with following primer sets: HPRT, (forward) 5’-TTA TGG ACA GGA CTG AAA

GAC-3’ and (reverse) 5′-GCT TTA ATG TAA TCC AGC AGG T-3 ; IL-10, (forward) 5′-ATA

ACT GCA CCC ACT TCC CA-3′ and (reverse) 5′-TCA TTT CCG ATA AGG CTT GG-3′;

TGF-β, (forward) 5′-CTC CCG TGG CTT CTA GTG C-3′ and (reverse) 5′-GCC TTA GTT

TGG ACA GGA TCT G-3′; IL-1β, (forward) 5′-CAA CCA ACA AGT GAT ATT CTC C-3′

and (reverse) 5′-TGC CGT CTT TCA TTA CAC AG-3′; Csf2, (forward) 5′-AGG GTC TAC

GGG GCA ATT TC-3′ and (reverse) 5′-GGC AGT ATG TCT GGT AGT AGC TG-3′; PD-L1,

(forward) 5′-GCT CCA AAG GAC TTG TAC GTG-3′ and (reverse) 5′-TGA TCT GAA GGG

CAG CAT TTC-3′; IDO, (forward) 5′-GCT TTG CTC TAC CAC ATC CAC-3′ and (reverse)

5′-CAG GCG CTG TAA CCT GTG T-3′; COX2, (forward) 5′-TGG CTG CAG AAT TGA AAG

CCC T-3′ and (reverse) 5′-AAA GGT GCT CGG CTT CCA GTA T-3′; CD80, (forward) 5′-

ACC CCC AAC ATA ACT GAG TCT-3′ and (reverse) 5′-TTC CAA CCA AGA GAA GCG

AGG-3′; CD86, (forward) 5′-TGT TTC CGT GGA GAC GCA AG-3′ and (reverse) 5′-CAG

CTC ACT CAG GCT TAT GTT TT-3′; CD40, (forward) 5′-CCT TGC ACT GTG AGG AGA-3′

and (reverse) 5′-CTT CGC TTA CAA CGT GTG CT-3′. All the qRT-PCR experiments were

performed under the same condition as follows: 95°C for 5 min, 95°C for 30 s, 62°C for30 s, and

72°C for 30 s up to 40 cycles. The data was normalized to the expression level of hypoxanthine-

guanine phosphoribosyl transferase (HPRT). Results were described as relative expression levels

for each gene between the treatment groups.

Analysis of bacterial colonization by 16S rRNA gene analysis

To confirm the monocolonization, bacterial genomic DNA was isolated from fecal pellets or

luminal contents using a NeucleoSpin DNA Stool (Macherey-Nagel). The following primer sets

were used: to detect total bacteria, EUB (forward) 5′-TCC TAC GGG AGG CAG CAG T-3’ and

(reverse) 5′-GGA CTA CCA GGG TAT CTA ATC CTG TT-3’; to detect B. bifidum PRI1,

BibiF- (forward) 5′-CCA CAT GAT CGC ATG TGA TTG-3’ and (reverse) 5′-CCG AAG GCT

TGC TCC CAA A-3’. PCR analysis was carried out using a C1000 touch thermal cycler (Bio

Rad). Bacteroid fragilis monocolonization were confirmed by sequencing the bacterial genomic

DNA isolated from the Bf-monocolonized mice. NCBI BLAST analysis confirmed that

sequenced nucleotide sequences corresponds to the Bacteroid fragilis NCTC 9343

(https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Figure S12)

Purification of cell surface polysaccharides from B. bifidum

Cultured B. bifidum PRI1 were harvested and washed by PBS two times. Purification of cell

surface polysaccharide was performed as previously described (47) with minor modifications. In

brief, acidic phenol (Sigma-Aldrich, MO, USA) treatment was performed at 68oC to extract

capsular polysaccharides, and residual phenol was removed by ether treatment followed by

dialysis against distilled water for three days. To remove nucleic acids and proteins, DNase I

(Roche) and RNase (Sigma-Aldrich, MO, USA) digestion were perform overnight at 37oC

followed by Pronase (Protease from Streptomyces griseus, Sigma-Aldrich, MO, USA)

digestion at 37oC overnight. After acetic acid treatment, centrifugation was performed to remove

precipitates. Chilled ethanol was added to precipitate polysaccharides then dialyzed against

distilled water for 3 days and freeze dried. Purified polysaccharides were dissolved in water and

gel filtration was performed by HPLC column (TSKgel G5000PWXL, Tosho). Anion exchange

chromatography was performed (HiPrep Q FF 16/10, GE healthcare) to further separate neutral

and negative charge polysaccharides. Concentration of polysaccharide was determined by acid

phenol assay (47).

Isolation of lymphocytes and flow cytometry analysis

Naïve CD4+ T cells were purified from mLN, pLN and spleens using cell sorter (purity more

than 98%). Colons and small intestine were opened longitudinally and mucus was removed by

subsequent rinsing in PBS. Colons were cut into small pieces and incubated with 10mM EDTA,

20mM Hepes, 1mM sodium pyruvate, 3% FBS and PBS free of Ca2+

and Mg2+

for 20 minutes at

37ºC in a shaker. Tissue was minced with a razor blade into smaller pieces and incubated in

RPMI 1640 media containing 3% FBS, 20mM Hepes, 1mM sodium pyruvate, 0.5 mg/ml of each

Collagenase D (Roche) and DNaseI (Sigma-Aldrich, MO, USA) for 45 minutes at 37ºC.

Supernatant was filtered over a 100mm cell strainer into ice cold PBS containing 10mM EDTA.

Cells were put over a PercollTM

(GE Healthcare) gradient (40% percoll on top, 75% percoll on

the bottom) and spun at 2000 rpm for 20 minutes with no brake. The cells at the interface of the

40% and 75% layer were taken and washed twice with supplemented RPMI media (Hyclone

SH30027.01, supplemented with 10% FBS (Hyclone), Pen/Strep, -ME, sodium pyruvate,

2.05mM L-glutamine) and used for FACS staining. Cell suspensions were first stained with

Live/dead staining in PBS using the LIVE/DEAD fixable viable dye (Life Technologies).

Further staining procedures were carried out in the buffer containing 1% FBS (Gibco) and EDTA

(Sigma-Aldrich, MO, USA). Surface staining was performed for 20 min on ice in PBS

containing 0.2% bovine serum albumin. For intracellular transcription factor staining, cells were

fixed in Fixation/Permeabilization buffer (eBioscience) and stained in 1x permeabilization/wash

buffer (eBioscience). For intracellular cytokine analysis, purified lamina propria lymphocytes

were restimulated with 500ng/ml of ionomycin (Calbiochem) and 100 ng/ml PMA (Calbiochem)

with GolgiPlug (BD biosciences, 0.5μL/ sample) for 4-5 hours at 37C. Cells were fixed in

intracellular fixation buffer (IC, eBioscience), permeabilized in permeabilization/ wash buffer

(eBioscience) and stained using IL-10, IL-13, IFN-γ and IL-17A antibodies. The following

antibody clones were used: CD4 (RM4-5), CD44 (IM7), CD62L (MEL-14), CD45 (30-F11),

CD45.1 (A20), CD45.2 (104), CD90.1 (Thy1.1) (OX-7), CD90.2 (Thy1.2) (30-H12), CD103

(2E7), Vα2 (B20.1), Foxp3 (FJK-16s), CTLA4 (UC10-4B9), Nrp1 (3E12), Helios (22F6), T-bet

(4B10), RORγt (AFKJS-9), GATA-3 (16E10A23), IL-10 (JES5-16E3), IL-13 (eBio13A), IFN-γ

(XMG1.2), IL-17A (17B7), CD11c (N418), CD11b (M1/70), F4/80 (BM8), MHCII

(M5/114.15.2), CX3CR1 (SA011F11), Bst2 (927). For flow cytometric sorting, dead cells were

excluded and the populations were gated as follows: LP DCs (CD45+MHCII

+CD11c

+CD11b

+

CD103+ F4/80

-), SP DCs (MHCII

+CD11c

+CD11b

+CD8α

+ or MHCII

+CD11c

+CD11b

+CD8α

-),

naïve CD4+T cells (CD4

+CD45.1

+CD62L

hiCD44

loFoxp3

GFP-), naive OT-II TCR transgenic T

cells (CD4+CD90.1

+OT-II

+CD62L

hiCD44

loFoxp3

GFP-), CBir TCR transgenic naïve T cells

(CD4+CD45.1

+CD62L

hiCD44

loFoxp3

GFP-). Cells sorting performed by using Moflo-XDP and

flow cytometry analysis was performed by using LSR Fortessa flow cytometer analyzer

equipped with 5 lasers (BD Biosciences). Data was analysed by using FACSDiva software (BD

Biosciences) and FlowJo software.

TCR repertoire analysis using CDR3 high-throughput sequencing.

Total RNA was extracted from sorted CD4+Foxp3

GFP+ Treg cells from the GF mice or mice

monocolonized with B. bifidum PRI1 for three weeks. Dead cells were excluded using FVD

staining by ISOGEN (Nippon Gene). Next-generation sequencing was conducted with an

unbiased TCR repertoire analysis technology by Repertoire Genesis Inc. Adaptor-ligation PCR

was performed as described previously (54). In brief, first double-stranded cDNA was

synthesized by Superscript III reverse transcriptase (Invitrogen), and ligated with a 5’ adaptor

oligonucleotide, and then PCR-amplified by using primers specific for the adaptor and TCR α

constant region or TCR β constant region. After the amplification of TCR α and TCR β cDNA,

and then index (barcode) sequences was added using a Nextera XT index kit v2 setA (Illumina).

Sequencing was performed with the Illumina Miseq paired-end platform (2 × 300 bp). Data

processing was performed by using Repertoire Analysis software created by Repertoire Genesis,

Inc. TCR sequences were assigned by using available data set of reference sequences from the

international ImMunoGeneTics information system (IMGT) database (http://www.imgt.org).

After removal of sequences with low quality scores, TCR repertoire analysis was conducted

using bioinformatics software developed by Repertoire Genesis Incorporation (Ibaraki, Japan). A

unique sequence read (USR) were defined as sequence read do not have identity with the other

sequence reads. The copy number of an identical USR was automatically counted by the RG

software.

TGF-β neutralization experiment in vitro

Splenic MHCII+CD11c

+ DCs (2x10

4) were seeded into a round bottom 96 well plate for 2 hours

then incubated with B. bifidum or CSGG for additional 12 hrs. Cells were washed and co-

cultured with naïve CD4+

T cells (2x105) in the presence of 0.1μg/ml of anti-CD3 (BD

Bioscience), 100U IL-2 (Peprotech) and 0.1ng/ml of TGF-β, in the presence or absence of 15

μg/ml anti-TGF-β Ab (R&D). After 3 days of incubation, population of CD4+Foxp3

+ T cells

were analyzed by FACS.

In vitro suppression assay

In vitro generated CD45.1+CD4

+Foxp3

GFP iTreg cells were sorted and incubated with responder

cells (Thy1.1+CD4

+Foxp3

-) that were pre-pulsed with CTV (Cell Tracking Violet) for 10 minutes

at 37C. Cells were washed in PBS twice and immediately used. T cell-depleted splenocytes

(1x105 cells) were mixed with CTV-pulsed responder cells (5 x 10

4) and indicated amounts of

iTreg cells along with 0.5g/ml of anti-CD3 in a round bottom 96 well plate. Cells were cultured

for 4 days and their proliferation was analyzed by flow cytometry for determining the dilution of

CTV intensity. Suppression rate (%) was calculated as the total percentage of dividing cells by

comparing the percentage of responder’s cells alone.

Histology and Histological scoring

Clinical conditions of experimental colitis were evaluated by histological analysis with H&E

staining. Briefly, colons were collected and fixed in 10% formaldehyde. After fixation, tissues

were embedded in paraffin blocks, sectioned at 3μm thickness and stained with Hematoxylin

(Sigma-Aldrich, MO, USA) and Eosin (Sigma-Aldrich, MO, USA). Inflammation was assessed

as previously described by Powrie and colleagues (55), each sample was graded semi-

quantitatively from 0-4 and typical features of each grade are: 0 = normal; 1 = mild epithelial

hyperplasia and mild mucosal inflammation; 2 = pronounced hyperplasia and significant

inflammatory infiltrates; 3 = severe hyperplasia and transmural infiltration with significant

decrease in goblet cells; 4 = severe hyperplasia, severe transmural inflammation, ulceration,

crypt abscesses, and substantial depletion of goblet cells. At least three separate sections from

colons were assessed separately and each sample were scored blind.

Statistical analysis

Statistical analyses were performed with GraphPad Prism software (La Jolla, CA). Differences

between control and experimental groups were evaluated using two-tailed unpaired-Student’s t-

test. Data are presented as mean ± SEM. For the analysis of in vivo stability of Treg cells and

experimental colitis, statistical analysis was performed using two-way analysis of variance with

Bonferroni’s multiple comparison test; p < 0.05 was considered to be statistically significant. For

the in vitro assays, data are representative of more than three independent experiments with

similar results.

Fig. S1. Identification of Bb as Treg-inducing bacteria.

(A and B) Selection of B. bifidum PRI1 (Bb) as the best bacterial strain that facilitates Treg

induction. Representative IL-10/IL-12 cytokine ratio (A) and Foxp3 expression among candidate

probiotic strains screened (B). Each bacterial strain with indicated CFU titres (colored bars in

green, blue and red) were cultured with total mesenteric lymph node (mLN) cells for 72hrs,

following which cytokine levels or Foxp3 expression were determined by ELISA or flow

cytometry analyses. Data are representative of more than three independent experiments with

similar results. (C) Representative cecum images (left) and cecum weight (right) in GF mice, Bb,

and Bf -monocolonized and SPF mice. Data are representative of four independent experiments

with similar results. (D) Representative flow cytometry plots for the in vivo Treg-inducing

activity in GF mice, mice monocolonized with B. bifidum (Bb) or B. fragilis (Bf). (E)

Representative flow cytometry plots CD4+Foxp3

+ T cells in the colon (cLP) of SPF, GF or

monocolonized mice with SFB or Lpa. (F) Percentage frequencies of CD4+Foxp3

+ T cells in the

small intestine (siLP), mLN, pLN and spleen of SPF, GF or monocolonized mice with Bb, SFB

or Lpa. (G and H) Representative flow cytometry plots and percentage frequencies of

CD4+CD103

+Foxp3

+ (G) and CD4

+CD44

hiCD62L

loFoxp3

+ T cells (H) in the siLP of the GF or

monocolonized mice with indicated bacteria. Numbers indicate cell percentages in the quadrants

and circles in the bar graphs represent individual mouse corresponding to each parameter,

respectively. Data are representative of three to five independent experiments with similar results

(n3 mice). B. bifidum RRI1 (Bb), B. fragilis (Bf). Lactobacillus acidophilus (Lac), Lactobacillus

casei (Lca), Lactobacillus reuteri (Lre), Lactobacillus paracasei (Lpa), Segmented filamentous

bacteria (SFB). All graph plots show the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

(Student’s t test).

Fig. S2. Effect of Bb monocolonization on cytokine levels in Treg and non-Treg cells.

(A-B) Representative flow cytometry plots and frequencies of IFNγ+, IL-17

+ and IL13

+ in Treg

and non-Treg cells from GF mice or those monocolonized with indicated bacteria. Numbers in

the quadrants represent cell percentage and circles in the graph plots represent individual mouse

corresponding to each parameter. Data are representative of three independent experiments with

similar results (n3 mice). Student’s t testAll graph plots show mean ± SEM. *p < 0.05, **p <

0.01, ***p < 0.001 (Student’s t test).

Fig. S3. Bb monocolonization facilitates de novo generation of pTreg cells.

Cells were isolated from the indicated lymphoid organs of GF, mice monocolonized with Bb

(Bb) or Lpa (Lpa) three week after colonization. (A and B) Percentage frequencies of Helios-

and Nrp1- Treg populations in the indicated lymphoid tissues. (C) Representative flow cytometry

plots and frequencies of RORt+Foxp3

+ Treg and

RORt

+Foxp3

- cells from cLP of GF mice or

mice monocolonized with Bb or Lpa. (D) Representative flow cytometry plots and frequencies of

RORt+Helios

- Treg cells from siLP of GF mice or mice monocolonized with Bb or Lpa. (E)

Experimental strategy for the analysis of de novo generation of pTreg cells after Bb

monocolonization. (F) Frequencies of Foxp3+ Treg population in indicated lymphoid organs. (G)

Naïve CD4+ Foxp3

− T cells sorted from CD45.1

+Foxp3

GFP reporter mice were transferred into

GF mice. Animals were either left GF or monocolonized with Bb for 3 weeks. Foxp3+ Treg

population was analyzed by GFP expression in the siLP. Data are representative of at least five

independent experiments with similar results (n3 mice). All bar graphs show the mean ± SEM.

*p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test).

Fig. S4. Bb colonization induces dietary Ag– and/or microbiota-reactive Treg cells. (A)

Experimental strategy to determine OVA-specific pTreg cells induced in response to dietary

antigen (OVA specific OTII T cells) in GF or Bb-monocolonized mice. (B) Experimental

strategy to determine microbiota (flagellin specific CBir T cells) specific pTreg cells induced in

Rag1-/-

SPF mice fed with PBS or Bb. (C) Analysis of RORγt, GATA3, and T-bet populations in

CD4+Foxp3

- or CD4

+Foxp3

+ T cells from cLP of CBir naïve T cells transferred into Rag1

-/- SPF

mice fed with PBS or Bb. Data are representative of three independent experiments with similar

results (n3 mice).

Fig. S5. Effect of Bb monocolonization on the TCR repertoire of Treg cells.

(A) Experimental strategy to determine Bb induced Treg proliferation in the presence of

indicated combinations of fecal Ags from GF mice or mice monocolonized with Bb or Lpa. (B)

Heat map from α-chain and β-chain showing the frequencies of top 85 dominant TCR-CDR3

regions of colonic Treg cells from the Bb-monocolonized and GF mice. CDR3 regions with

‘common’ mark (28 peptides) indicate the presence of read counts in each group. Colour shades

show the relative frequencies with which given TCRs were found.

Fig. S6. Effect of Bb monocolonization on phenotypes and population of cLP-DC subtypes.

(A) Quantification of relative mRNA expression of cytokines normalized for Hprt (plotted as

fold change) in the total colons of the GF or Bb-monocolonized mice. Data are representative of

three independent experiments with similar results. (B-C) cLP DCs from GF mice or those

monocolonized with Lpa or Bb were examined for expression of CD103, CD11b and CX3CR1,

with the numbers representing the frequency of each population as a percentage of total

CD11c+MHCII

+. (D) cLP pDC from GF mice or those monocolonized with Lpa or Bb were

examined for expression of Bst2, from the total CD11c+MHCII

+ population.

Fig. S7. Effect of Bb monocolonization on phenotypes and population of DC subtypes in

mLN and siLP.

(A-B) Expression of CD103, CD11b and CX3CR1 macrophages (M) on siLP and mLN DCs

isolated from GF mice or those monocolonized with Lpa or Bb were examined. Numbers

representing the frequency of each population as a percentage and Scatter plot shows the

absolute number of total CD11c+MHCII

+. pDC in siLP and mLN were examined for expression

of Bst2, and Scatter plot shows absolute number from total MHCII+. Data are representative of at

least three independent experiments with similar results (n3 mice). All graphs show the mean ±

SEM. *p < 0.05 (Student’s t test).

Fig. S8. CSGG of the Bb enhances Treg cell induction.

(A-B) Splenic CD11c+DCs pre-treated with indicated fractions of B.bifidum (A) or different

amounts of total cell surface polysaccharides (tCSPS) (B) were co-cultured with naïve CD4+ T

cells under suboptimal Treg inducing condition and after 3 days Foxp3+ Treg cells were analyzed

by FACS. (C) Effect of β-1,6-glucanase, β-1,4-galactanase and β-1,6- galactanase treatment on

CSGG-induced iTreg cell induction (CSGG; 5g/ml). Data are representative of more than

three independent experiments with similar results (A, B, C). (D-E) CD4+Foxp3

+ populations

generated in vitro after co-culturing of CD11c+ DCs, pre-treated as indicated bacteria or CSGG,

with naïve CD4+ T cells under suboptimal Treg inducing conditions. Comparison of cytokine

profiles in the culture supernatants and IL10+IFNγ

+ population between B. bifidum (Bb) or B.

fragilis (Bf) treated group were measured by FACS and ELISA. Data are representative of more

than four independent experiments with similar results. (F and G) GF mice were

intraperitoneally injected with CSGG (100g/dose) or PBS every other day for three weeks.

Frequencies of indicated populations of Treg cells were analyzed in lymphoid organs.

Data are representative of at least three independent experiments with similar results (n3 mice).

All graphs show the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test).

Fig. S9. Role of DC subtypes in inducing Bb/CSGG-mediated iTreg cells.

(A) Naïve CD4+ T cells were treated with CSGG (50g/ml) in the absence of DCs under

suboptimal Treg inducing conditions. (B) Experimental strategy for in vitro Treg-induction in

DC:T cell co-culture system. (C) Sorted pDCs (MHCII+CD11c

loBst2

+) and cDCs

(MHCII+CD11c

+CD11b

+) from spleen pre-treated with Bb were co-cultured with naïve CD4

+ T

cells in suboptimal Treg inducing conditions for 3 days, after which Foxp3+ Treg cells were

analyzed within live cells. Representative flow cytometric analysis and bar graph are shown. (D)

Sorted CX3CR1+ (MHCII

+CD11c

+CD11b

+CX3CR1

+)

or CX3CR1

-

(MHCII+CD11c

+CD11b

+CX3CR1

- ) cells from spleen pre-treated with Bb were co-cultured with

naïve CD4+ T cells in suboptimal Treg inducing conditions for 3 days, and Foxp3

+ Treg cells

were analyzed within live cells. Representative flow cytometric analysis and bar graph are

shown. (E) Splenic total DCs (tDC, MHCII+CD11c

+) were further fractionated into the CD8α

+

(MHCII+CD11c

+CD11b

-) or CD8α

- (MHCII

+CD11c

+CD11b

+) by cell sorting, and treated with

mock or CSGG, then co-cultured with naïve OT II+

CD4 T cells in the presence of OVA peptide

(0.05M) under suboptimal Treg inducing conditions for 3 days, following which iTreg

induction was determined by FACS. Data are representative of at least three independent

experiments with similar results. All graphs show the mean ± SEM. *p < 0.05, **p < 0.01, ***p

< 0.001 (Student’s t test).

Fig. S10. CSGG facilitates iTreg induction through TLR2-mediated generation of regulatory

DCs.

(A) Naïve CD4+ T cells and CD11c

+ DCs derived from indicated mice, pre-treated with mock or

CSGG, were co-cultured in suboptimal iTreg generation condition for three days, following

which CD4+Foxp3

+ Treg cell induction was determined by flow cytometry. Data are

representative of five independent experiments with similar results. (B-C) Sorted splenic CD11c+

DCs derived from WT, TLR6KO (B) or 3DKO (TLR3-/-

TLR7-/-

TLR9-/-

triple knock-out) (C)

mice were pre-treated with mock or CSGG along with combination of anti α-TLR2 blocking Ab

(α-TLR2Ab) (B), then were co-cultured with naïve CD4+ T cells in suboptimal iTreg generation

condition. CD4+Foxp3

+ iTreg population was determined by flow cytometry. Data are

representative of three independent experiments with similar results. (D) Analysis of IFNγ levels

by ELISA in the culture supernatant after naïve CD4+ T cells were co-cultured with WT or

TLR2-deficient CD11c+ DCs pre-treated with mock or CSGG. Data are representative of four

independent experiments with similar results. (E and F) Splenic CD11c+ DCs were pre-treated

with mock or CSGG in the presence of blocking Abs (C) or CD11c+ DCs from Dectin1-/-

and

Dectin2-/-

mice (D) then co-cultured with naïve CD4+ T cells. iTreg induction

was analyzed by

flow cytometry. Data are representative of four independent experiments with similar results. All

bar graphs show the mean ± SEM. *p < 0.05 (Student’s t test).

Fig. S11. CSGG-induced iTreg cells are capable of suppressing intestinal inflammation.

(A) CTV labelled naïve responder Thy1.1+CD4

+Foxp3

- T cells were co-cultured with sorted

CD45.1+CD4

+Foxp3

+ iTreg cells generated in vitro with mock or CSGG treated DCs, in the

presence of APCs purified from T cell depleted splenocytes of allelically marked CD45.2+

mice.

Responder T cell proliferation was analyzed by flow cytometry. Data are representative of four

independent experiments with similar results. (B) Colon length of adoptively transferred RAG1-/-

colitis suppressed by nTreg and mock, Bb, or CSGG induced iTreg cells generated in vitro. (C)

Colon length of RAG1-/-

colitis induced by adoptively transferred naïve T cells treated

intraperitoneally with mock (PBS) or CSGG (100g/dose). (D) CD4+CD45.1

+ Foxp3

+ Treg

populations in mLN of adoptively transferred RAG1-/-

mice that were mock treated or treated

with CSGG. Data are representative of at least two independent experiments with similar results

(n3 mice). All bar graphs show the mean ± SEM. *p < 0.05, (Student’s t test).

Fig. S12. Confirmation of Bf monocolonization by DNA sequencing.

(A-B) Fecal isolated DNAs from B. fragilis-monocolonized mice were sequenced by forward

EUB primer (A) and analyzed by blast in NCBI database (B).

Table S1. Peptide sequences of α-chain CDR3 region of Treg cells sorted from colon, mLN,

and spleen of Bb-monocolonized mice compared with GF mice.

Frequency (%)

Colon mLN Spleen

CAAAPNSGTYQRF 0.216242 0.007945 0.438834

CAAEGGNYQLIW 0.219277 0.034226 0.014377

CAAETGNYKDVF 0.000759 0.000611 0.000685

CAAGGQGGSAKLIF 0.263284 0.001222 0.006846

CAAIMSNYNVLYF 0.298186 0.018947 0.000685

CAARRGSALGRLHF 0.087255 0.047673 0.009585

CAARRNNYAQGLTF 0.000759 0.004278 0.0178

CAASEDYSNNRLTL 0.000759 0.011001 0.002054

CAASGAASLGKLQF 0.043248 0.011001 0.11433

CAASGASSSFSKLVF 0.194238 0.006112 0.021223

CAASGTASLGKLQF 0.037937 0.000611 0.004792

CAASGYAQGLTF 0.083462 0.028726 0.005477

CAASKGSSGNKLIF 0.000759 0.009168 0.000685

CAASPTGANTGKLTF 0.000759 0.003667 0.002738

CALGDRGSNYNVLYF 0.01214 0.008557 0.02533

CALGDSSNNRIFF 0.003035 0.000611 0.032861

CALGPSNMGYKLTF 0.000759 0.017113 0.015746

CALSNTNAYKVIF 0.000759 0.000611 0.019169

CALSSSSGSWQLIF 0.000759 0.064786 0.030807

CAMERGSALGRLHF 0.000759 0.025059 0.019169

CAMERRGSALGRLHF 0.141885 0.03606 0.006846

CAMTGGYKVVF 0.000759 0.007945 0.013692

CAPSGGNYKPTF 0.185892 0.001834 0.001369

CASSSGSWQLIF 0.237486 0.01528 0.003423

CATDAASGSWQLIF 0.000759 0.009168 0.008215

CATDTNAYKVIF 0.000759 0.016502 0.016431

CATGASSGSWQLIF 0.000759 0.007945 0.000685

CATGRSNYNVLYF 0.069046 0.034838 0.010954

CATSGGSNAKLTF 0.000759 0.006723 0.010269

CAVLDSNYQLIW 0.000759 0.011613 0.007531

CAVPNSNNRIFF 0.039455 0.003667 0.021223

CAVSAPQGGRALIF 0.000759 0.003056 0.019854

CAVSETNTGKLTF 0.000759 0.008557 0.002054

CAVSLDSNYQLIW 0.177546 0.005501 0.0178

CAVSLPGTGSNRLTF 0.070563 0.009779 0.017115

CAVSMRGSALGRLHF 0.123675 0.004889 0.022592

CAVSPNTGYQNFYF 0.046283 0.009168 0.019169

CAVSQGGRALIF 0.000759 0.000611 0.000685

CVLGDTNAYKVIF 0.035661 0.028726 0.002054

CAAMTNSAGNKLTF 0.173752058 0.006723059 0.008215296

CAASPNTNKVVF 0.176787029 0.068452963 0.015061375

CAASANTNKVVF 0.169958345 0.019557989 0.017799807

CAAGTGGYKVVF 0.726875422 0.262810483 0.253304945

Table S2. Peptide sequences of β-chain CDR3 region of Treg cells sorted from colon, mLN,

and spleen of Bb-monocolonized mice compared with GF mice.

Frequency (%)

Colon mLN Spleen

CASGDRGGSYEQYF 0.000605 0.004368 0.015442

CASGGDNYAEQFF 0.265584 0.000624 0.021233

CASGGTANERLFF 0.000605 0.004368 0.043752

CASMDRGTERLFF 0.217791 0.001248 0.087504

CASRQGAETLYF 0.262559 0.004368 0.045039

CASRQGSGNTLYF 0.00121 0.011231 0.004504

CASSDAGGTNERLFF 0.000605 0.024334 0.006434

CASSDNSGNTLYF 0.438002 0.000624 0.011581

CASSDRGNSDYTF 0.176048 0.004992 0.007078

CASSDRGSQNTLYF 0.000605 0.002496 0.008364

CASSETANTEVFF 0.000605 0.013727 0.010295

CASSFGGNYAEQFF 0.000605 0.006863 0.007078

CASSFQNTLYF 0.095586 0.029326 0.014799

CASSFRGRQDTQYF 0.148824 0.007487 0.060481

CASSGDSSGNTLYF 0.0121 0.006863 0.000643

CASSGDWGNYAEQFF 0.000605 0.001872 0.007078

CASSGQGQDTQYF 0.021174 0.011855 0.004504

CASSLDISNERLFF 0.053843 0.003744 0.00193

CASSLDRDTEVFF 0.000605 0.009359 0.011581

CASSLDSSGNTLYF 0.033274 0.001248 0.005791

CASSLDWGQNTLYF 0.036299 0.029949 0.010938

CASSLEDTQYF 0.026619 0.042428 0.003217

CASSLGAQDTQYF 0.047793 0.012479 0.000643

CASSLGGGAETLYF 0.000605 0.028078 0.025737

CASSLGGIQDTQYF 0.000605 0.004368 0.001287

CASSLGGSDYTF 0.091956 0.021214 0.000643

CASSLGGTEVFF 0.052028 0.016847 0.008364

CASSLGQGRNTLYF 0.193592 0.001872 0.03024

CASSLGQQDTQYF 0.056263 0.008735 0.012225

CASSLGSNSDYTF 0.190567 0.001248 0.010938

CASSLGVEQYF 0.000605 0.004992 0.010295

CASSLLGGDTQYF 0.000605 0.003744 0.003217

CASSLPGSYEQYF 0.000605 0.001872 0.00386

CASSLSNSDYTF 0.065337 0.012479 0.008364

CASSLTGGNTEVFF 0.000605 0.003744 0.033458

CASSLVGYTGQLYF 0.000605 0.013727 0.00386

CASSPNANTEVFF 0.000605 0.006863 0.002574

CASSPRQNTGQLYF 0.166973 0.016223 0.005147

CASSPTGSNERLFF 0.065337 0.006239 0.014799

CASSQDAEQFF 0.000605 0.003744 0.009008

CASSQDLGGAREQYF 0.059288 0.000624 0.001287

CASSQDRGYEQYF 0.000605 0.010607 0.031527

CASSQDWGSYEQYF 0.000605 0.021214 0.025093

CASSQVGGQDTQYF 0.000605 0.004992 0.012868

CASSRDKEVFF 0.00121 0.000624 0.017372

CASSRDTEVFF 0.214766 0.014975 0.004504

CASSRGNYAEQFF 0.11434 0.016847 0.009008

CASSRQASQNTLYF 0.000605 0.004368 0.005791

CASSRQENTEVFF 0.00121 0.001872 0.009008

CASSRQGDTQYF 0.00484 0.000624 0.012225

CASSSGGGAETLYF 0.000605 0.002496 0.000643

CASSVRDRGQAPLF 0.206902 0.006863 0.00386

CASVGDGTEVFF 0.141564 0.000624 0.000643

CAWSLGYEQYF 0.079252 0.010607 0.002574

CAWTGTNERLFF 0.059893 0.009359 0.012225

CGARPGPRTLYF 0.045978 0.003744 0.020589

CGARQGNSDYTF 0.062917 0.008111 0.010295

CTCSADYGVAEQFF 0.146404 0.008735 0.000643

CTCSAEENSPLYF 0.261954 0.014351 0.00386

CTCSAENLSYNSPLYF 0.100426 0.021838 0.033458

CTCSAGTEQFF 0.010285 0.011231 0.005791