Article https://doi.org/10.

1038/s41586-019-0878-z

A defined commensal consortium elicits

CD8 T cells and anti-cancer immunity
­Takeshi Tanoue1,2,3,16, Satoru Morita1,16, Damian R. Plichta4, Ashwin N. Skelly1, Wataru Suda3,5,6, Yuki Sugiura7,
Seiko Narushima1,3, Hera Vlamakis4, Iori Motoo3, Kayoko Sugita1, Atsushi Shiota1,2, Kozue Takeshita1, Keiko Yasuma-Mitobe1,
Dieter Riethmacher8, Tsuneyasu Kaisho9, Jason M. Norman10, Daniel Mucida11, Makoto Suematsu7, Tomonori Yaguchi12,
Vanni Bucci13, Takashi Inoue14, Yutaka Kawakami12, Bernat Olle10, Bruce Roberts10, Masahira Hattori3,5,6, Ramnik J. Xavier4,15,
Koji Atarashi1,2,3 & Kenya Honda1,2,3*

There is a growing appreciation for the importance of the gut microbiota as a therapeutic target in various diseases.
However, there are only a handful of known commensal strains that can potentially be used to manipulate host
physiological functions. Here we isolate a consortium of 11 bacterial strains from healthy human donor faeces that
is capable of robustly inducing interferon-γ-producing CD8 T cells in the intestine. These 11 strains act together to
mediate the induction without causing inflammation in a manner that is dependent on CD103+ dendritic cells and
major histocompatibility (MHC) class Ia molecules. Colonization of mice with the 11-strain mixture enhances both host
resistance against Listeria monocytogenes infection and the therapeutic efficacy of immune checkpoint inhibitors in
syngeneic tumour models. The 11 strains primarily represent rare, low-abundance components of the human microbiome,
and thus have great potential as broadly effective biotherapeutics.

The gut microbiota is an attractive therapeutic target for the treatment (Extended Data Fig. 1f). Collectively, these results suggest that the fre-
and/or prevention of infectious and inflammatory disease1–4. In this quency of IFNγ+ CD8 T cells is plastic, with specific members of the
context, several reports have identified commensal bacterial strains microbiota promoting their intestinal accumulation in an inducible and
that can potently affect intestinal CD4 T cells to promote their devel- reversible manner. In this study, we aimed to identify commensal bac-
opment into T helper 17 (TH17) and regulatory T (Treg) cells5–9. In terial strains that could specifically induce the accumulation of IFNγ+
contrast to CD4 T cells, the relationship between the microbiota and CD8 T cells in the intestine.
intestinal CD8 T cells remains poorly characterized, although microbi-
ota-mediated activation of this population has been implicated in both Isolation of IFNγ+ CD8 T-cell-inducing human strains
host immune control of pathogen infection and the efficacy of cancer From a translational perspective, we mined the human, rather than
immunotherapy10–14. Interferon-γ (IFNγ)-expressing CD8 T cells were mouse, faecal microbiota (outlined in Extended Data Fig. 2a). Faecal
constitutively present at high frequencies in the intestinal lamina pro- samples from six healthy volunteers (donors A to F) were each orally
pria compared to other organs in specific pathogen-free (SPF) mice administered to germ-free mice housed in separate gnotobiotic isola-
(Fig. 1a). A large proportion of colonic IFNγ+ CD8 T cells expressed tors. The extent of colonic IFNγ+ CD8 T cell induction varied greatly
the αβ T cell receptor (TCR), CD8α/β, CD44 and T-bet (Extended Data between samples (Fig. 1c and Extended Data Fig. 2b), with faeces from
Fig. 1a), indicating that they were primarily activated or memory cells donor B eliciting the strongest induction, to a degree comparable to
differentiated from conventional CD8 T cells. The IFNγ+ CD8 T cells that observed in SPF mice (compare Fig. 1b and c). We then selected
contained subsets that expressed CD103, a marker of tissue-resident the mouse that exhibited the strongest induction (‘mouse B5’) (Fig. 1c)
memory T cells15, and granzyme B (GrB), a key effector molecule of and gavaged a suspension of its caecal contents into germ-free mice
cytotoxic T cells (Extended Data Fig. 1a, b). The frequency and number (GF+B5 mice). These mice were treated with ampicillin, metronida-
of intestinal IFNγ+ CD8 T cells were markedly decreased in germ-free zole, streptomycin, tylosin or a vehicle control via the drinking water.
mice in comparison with SPF mice (Fig. 1b and Extended Data Fig. 1c). Chloroform-treated caecal contents of mouse B5 were inoculated
Similarly, the treatment of adult SPF mice with an antibiotic cocktail into an additional group of germ-free mice (GF+CHL B5 mice) to
significantly reduced the frequency of IFNγ+ CD8 T cells (Extended examine the effects of spore-forming bacteria5. As expected, GF+B5
Data Fig. 1d). Conversely, oral administration of faecal suspensions mice showed a significant increase in the frequency of colonic IFNγ+
from SPF mice ameliorated the deficit in germ-free mice (Extended CD8 T cells (Fig. 1d). Treatment with metronidazole, streptomycin or
Data Fig. 1e). Furthermore, the relative abundance of this cell pop- tylosin, however, resulted in a substantial decrease, and chloroform
ulation in SPF mice varied with housing conditions, and co-housing treatment abrogated induction altogether. By contrast, treatment with
resulted in all mice ultimately displaying a high-frequency phenotype ampicillin enhanced the observed induction (Fig. 1d). We followed up
1
Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan. 2JSR-Keio University Medical and Chemical Innovation Center, Tokyo, Japan. 3RIKEN Center for
Integrative Medical Sciences, Yokohama, Japan. 4Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA. 5Cooperative Major in Advanced Health
Science, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan. 6Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan. 7Department of
Biochemistry, Keio University School of Medicine, Tokyo, Japan. 8Department of Biomedical Sciences, Nazarbayev University School of Medicine, Astana, Kazakhstan. 9Department of Immunology,
Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan. 10Vedanta Biosciences, Cambridge, MA, USA. 11Laboratory of Mucosal Immunology, The Rockefeller University,
New York, NY, USA. 12Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan. 13Department of Bioengineering, Program in
Biotechnology and Biomedical Engineering, University of Massachusetts Dartmouth, North Dartmouth, MA, USA. 14Marmoset Research Department, Central Institute for Experimental Animals,
Kawasaki, Japan. 15Center for Computational and Integrative Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. 16These authors contributed equally:
Takeshi Tanoue, Satoru Morita. *e-mail: kenya@keio.jp

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 Article RESEARCH

e f Closest species/strain % Strain ID

100 Clostridium innocuum 99.9 81A1
Ruminococcaceae bacterium GD1 99.5 81F3
90 Clostridium lavalense 99.4 81E6
Hungatella hathewayi 99.6 81G1
a b Clostridium sp. AT5 94.5 83F2
NS 80
80
*** 60 *** *** Clostridium sp. AUH-JLC39 98.2 82D2
Parabacteroides goldsteinii 99.8 82E8
Bacteroides thetaiotaomicron 99.9 81H8

Relative abundance of OTUs

IFNγ+ CD8 T (%)
70
IFNγ+ CD8 T (%)

60 Bacteroides salyersiae 99.7 82A3

40 Bacteroides cellulosilyticus 99.6 82B7

n=6
60 Bacteroides fragilis 10-mix

n=6
40 99.9 82A12
Bacteroides eggerthii 99.7 82B11
20 Anaerostipes caccae 99.5 81B4
20 50
Bacteroides uniformis st.mat-281 99.9 81A2
SPF n = 7

SPF n = 7 Bacteroides clarus 99.5 82C1

0 0 40 Parabacteroides distasonis 99.3 82G9
Colon
Lung
iLN
Spleen

Liver

SI

GF

GF
Parabacteroides gordonii 97.4 81H9 21-mix
Alistipes senegalensis 99.5 81E7
Colon SI 30 Parabacteroides johnsonii 99.9 82F11 7-mix
SPF Paraprevotella xylaniphila 99.2 82A6
(n = 4) 20 Bacteroides dorei 99.7 81B11 11-mix
Bacteroides uniformis JCM 5828 99.7 82G1
Eubacterium limosum 99.9 81C1
10
c *** d ***
**
Ruminococcaceae bacterium cv2 99.8 82B1
*** * Phascolarctobacterium faecium 99.8 82G5 4-mix
*** ** *** * Fusobacterium ulcerans 99.6 81A6
60 *** *** 60 B5-AMP2, 0
B5 * CHL.3
CHL.5
CHL.4
CHL.1
CHL.2
TYL.5
TYL.4
TYL.3
TYL.1
TYL.2
MNZ5
MNZ4
MNZ1
MNZ2
MNZ3
STM2
STM3
STM4
STM1
NoABX4
NoABX3
NoABX2
NoABX1
AMP4
AMP1
AMP3
AMP2
AMP2
OTUs corresponding to the 26 strains
B5-AMP3
IFNγ+ CD8 T (%)

CD8 T (%)

OTUs detected in +CHL B5 mice

n=5

40 40
n=5

GF OTUs not correlated with percentage IFNγ+ CD8 T

+TYL +MNZ +STM – +AMP
n=4
n=5

+CHL B5 OTUs positively correlated with percentage IFNγ+ CD8 T

GF+B5
n=9

n=4
n=5

n=5

20 20 g h i j
IFNγ+

n=4

n=4
60 80 ***
n=4
n=4

n=4
CD103+ IFNγ+ CD8 T (%)
60 ***
n=5
n=6

n=6

n=4

n=4

n=4
60

n=4
60 ** *** ***

n=4
NS
0 0

IFNγ+ CD8 T (%)

NS
IFNγ+ CD8 T (%)

IFNγ+ CD8 T (%)

IFNγ+ CD8 T (%)

60

n=4
No ABX
+AMP
GF
GF

GF+CHL B5
+MNZ

+TYL
+STM

40 *** **
C
D
B
A

E
F

40 40 40
GF+ 40
GF+B5 20 20 20
20 20

n=3
GF+11-mix n = 10
GF+21-mix n = 6

GF+10-mix n = 5

n=3

GF+4-mix n = 6
GF+7-mix n = 6
GF+11-mix n = 6
GF n = 4
GF n = 3
GF+21-mix n = 5

GF n = 6

0 0 0 0 0
0 1 2 3 4 0 1 2 3 4
Weeks after 11-mix inoculation

Fig. 1 | Isolation of 11 IFNγ+CD8 T cell-inducing bacterial strains frequency of IFNγ+ CD8 T cells are marked in red, those detected in
from a healthy human microbiota. a, b, Percentage of IFNγ+ cells chloroform-treated B5 mice are in blue, and those not significantly
among CD8 T cells in the indicated organs (iLN, inguinal lymph nodes; correlated are in grey. OTUs corresponding to the 26 isolated strains are
SI, small intestine) of SPF (a) and germ-free (GF) (b) mice. c, Percentage marked in yellow. f, List of the 26 strains isolated from mice B5-AMP2
of colonic IFNγ+ CD8 T cells from germ-free mice orally inoculated with and B5-AMP3. Their closest species or strain and percentage similarity
one of six (A–F) healthy human faecal samples. Arrow indicates mouse (%) in the Ribosomal Database Project (RDP) database are indicated.
B5, which was chosen for follow-up analysis. d, Percentage of IFNγ+ CD8 g, h, j, Percentage of IFNγ+ CD8 T cells from germ-free mice inoculated
T cells from germ-free mice inoculated with caecal contents from mouse with the indicated mixture of bacterial strains. i, Percentages of CD8
B5 and treated with or without the indicated antibiotics via the drinking T cells that are IFNγ+, and of IFNγ+CD8 T cells that are CD103+, over
water, or from germ-free mice inoculated with chloroform-treated (CHL) time in germ-free mice inoculated with the 11-mix. Each circle in a–d and
caecal contents from mouse B5. Arrow indicates mice B5-AMP2 and g–j represents an individual animal. The number of mice in each group is
B5-AMP3. ABX, antibiotics; AMP, ampicillin; MNZ, metronidazole; shown. Data are mean and s.d. ***P < 0.001; **P < 0.01; *P < 0.05; one-
STM, streptomycin; TYL, tylosin. e, Caecal microbiota composition of way ANOVA with Tukey’s test (a–d, h, j) or two-tailed unpaired t-test (g).
each mouse was determined by 16S rRNA gene sequencing. Operational NS, not significant. See Source Data for exact P values.
taxonomic units (OTUs) significantly positively correlated with the

on the two ampicillin-treated mice exhibiting the strongest IFNγ+ CD8 the efficacy of the 11 versus 10 strains in inducing colonic IFNγ+ CD8
T cell induction (denoted B5-AMP2 and B5-AMP3, respectively) and T cells. Germ-free mice inoculated with the 11-mixture (GF+11-mix
cultured their caecal contents. We picked 206 distinct colonies and mice) exhibited a robust induction, to the same extent as GF+21-mix
analysed them by 16S ribosomal RNA gene sequencing to elaborate a mice, whereas GF+10-mix mice did not (Fig. 1h). The 11-mix-mediated
consortium of 26 unique strains (Fig. 1f). increase in IFNγ+ CD8 T cells was observed in mice of various genetic
Analysis of the composition of caecal microbiota revealed that backgrounds, although the baseline and induction magnitude varied
the 26 isolated strains together roughly recapitulate the microbiota substantially between groups (Extended Data Fig. 3a). IFNγ+ CD8
of mice B5-AMP2 and B5-AMP3 (Fig. 1e, f). From these 26 strains, T cells induced by the 11-mix were largely PD-1intermediate, CD44+ and
we first excluded 5 detected in the microbiome of GF+CHL B5 mice T-bet+, and a small subset was GrB+ (Extended Data Fig. 3b, c). Time-
(blue in Fig. 1e, f) and selected the remaining 21 for further analy- course analysis of GF+11-mix mice revealed that the marked increase
sis. These 21 strains were cultured individually and introduced as a in IFNγ+ CD8 T cells occurred within a one-week timeframe (Fig. 1i).
mixture into germ-free mice (GF+21-mix mice). We observed a very These incipient IFNγ+ CD8 T cells were primarily CD103−, although
strong induction of IFNγ+ CD8 T cells in the colonic lamina pro- the CD103+ tissue-resident memory T cell phenotype subset gradu-
pria of GF+21-mix mice, with a magnitude comparable to that in ally increased in frequency thereafter (Fig. 1i), suggesting a stepwise
SPF mice (compare Fig. 1b and g). We then performed a Spearman’s differentiation pattern. IFNγ+ CD8 T cells persisted stably for at least
rank correlation test that compared the relative abundances of the 6 months, and there were no gross, histological or transcriptional signs
21 strains with the frequency of IFNγ+ CD8 T cells. Among the of colonic inflammation observed in the GF+11-mix mice (Extended
21 strains, 11 were positively associated with IFNγ+ CD8 T cell fre- Data Fig. 3d–h). The induction ability of the 11-mix was not confined
quency (red in Fig. 1e, f) and 10 showed no significant association (grey to CD8 T cells, but extended to CD4 T cells as well. Indeed, a significant
in Fig. 1e, f; see also Extended Data Fig. 2c). We subsequently compared increase in the frequencies of colonic TH17 and TH1 cells was observed,

3 1 J A N U A R Y 2 0 1 9 | V O L 5 6 5 | N A T U RE | 6 0 1
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 RESEARCH Article

a b c d e n = 3 pools (3 mice in each) f NS

GF+11-mix GF 50 *** 40 40 NS
NS **

Ki67+ IFNγ + CD8 T (%)

60 60 60 *** 60 60
***

IFNγ + CD8 T (%)

NS NS
40 40 40 30
30

IFNγ + CD8 T (%)

IFNγ + CD8 T (%)
***
IFNγ+ CD8 T (%)

20 20
40 40 40 10 10 30
NS 20 20

n=8
n=3
n=2
n=6
n=3
n=4
n=3
n=2
20

n=4
n=3

n=5
n=5
n=5
n=3
n=4
NS Mucus layer *** ***

n=4
n=6
20 20 5 5 10 10

WT n = 10
20

n=4
Il1r1–/– n = 3
10
GF n = 3
+HK 11-mix n = 5
+11-mix n = 3

NS 0 0 0
0 0 0 0 0

WT
Il18–/–
Il1r1–/–

–/–
WT
Irf9 –/–
WT
Irf9 –/–

WT
Myd88–/–Trif –/–
WT
Myd88–/–Trif –/–
11 strains (EUB)

Il18
Medium
+11 st. lysate
+10 st. lysate
+PMA+Iono.

Medium
+11 st. lysate
+PMA+Iono.
Ifngr1+/–
Ifngr1–/–
Ifngr1+/–
Ifngr1–/–

GF
GF+11-mix
MUC2 DAPI 25 μm
GF GF+11-mix GF GF+11-mix
GF GF+11-mix
GF GF+11-mix
g h
40
NS
40 NS 40 NS 40 i 50 NS 50
*** j 50 GF
*** *** * CD11b–CD103+ CD11b+CD103+
NS ***
** GF+11-mix

Percentage of max
***
IFNγ + CD8 T (%)

IFNγ + CD8 T (%)

IFNγ + CD8 T (%)

IFNγ + CD8 T (%)

100 100
30 30 30 30 40 40 40 **
80 80
n=6
n=5

n=5

30 30 30
n=4
n=3

n=4

n=2
n=4
n=3

n=5

* **
20 20 20 20 60 60 NS

n=5
n=3
n=3
n=4

n=5
*
n=7
n=1

40 40 20 20 20 *
10 10 * **
10 10 20 20 10 10 10

n=5
Batf3+/– n = 6

WT n = 3

n=5
Xcr1+/+ ; DTA n = 8

Irf4f/f n = 4

= 9

0 0
n

0 0 0 0 0 103 104 105 0 103 104 105 0 0 0

Batf3+/–
Batf3–/–

Batf3–/–

Irf4ΔDC

Irf4ΔDC

Notch2ΔDC

Notch2ΔDC
Irf4f/f

Notch2f/f

f/f

WT
KbDb–/–

KbDb–/–

H2-M3+/–
H2-M3 –/–
+/–
–/–
Xcr1cre/+ ; DTA

Xcr1cre/+ ; DTA
Xcr1+/+ ; DTA

H2-Kb(PB)

SI
Caecum
Colon
cIEL
MLN
iLN
Liver
Lung
Spleen
PBL
Notch2

H2-M3
H2-M3
GF GF+11-mix
GF GF GF GF+11-mix
GF GF +11-mix n = 3 (except GF caecum and PBL; n = 4)
+11-mix GF GF GF GF+11-mix
+11-mix
GF GF+11-mix

Fig. 2 | Mechanisms of 11-strain-mediated IFNγ+ CD8 T cell ionomycin; PMA, phorbol 12-myristate 13-acetate. h, H2-Kb expression
accumulation. a, Percentage of colonic IFNγ+ cells among CD8 T cells by the indicated dendritic cell subset from germ-free mice with or without
from germ-free mice orally administered live or heat-killed (HK) 11-mix. the 11-mix (1 week after colonization). j, Percentage of IFNγ+ CD8 T cells
b, Fluorescence in situ hybridization staining with DAPI (blue), EUB338 in the indicated organs of germ-free mice 1 week after colonization with
probe (green) and mucin 2 (MUC2, red) of the proximal colon from the 11-mix. cIEL, colonic intraepithelial lymphocytes; MLN, mesenteric
germ-free mice inoculated with the 11-mix. c, f, g, i, Percentage of colonic lymph nodes; PBL, peripheral blood lymphocytes; st., strains. Each circle
IFNγ+ CD8 T cells from the indicated mice colonized with or without the represents an individual mouse (a, c, d, f, g, i, j) or a pool of mice (e). Data
11-mix for 1 (c) or 4 (f, g, i) weeks. d, e, Germ-free mice were colonized are mean and s.d. ***P < 0.001; **P < 0.01; *P < 0.05; one-way ANOVA
with the 11-mix for 1 week or left uncolonized. Percentages of Ki67+ cells with Tukey’s test (a, c, e–g, i) or two-tailed unpaired t-test (d, j). See
among the IFNγ+ CD8 T cell population (d) or IFNγ+ cells among the Source Data for exact P values.
CD8 T cell population after the indicated ex vivo stimulation (e). Iono.,

although these responses were significantly weaker than those induced feed-forward loop that promotes the recruitment and accumulation
by 20 previously reported TH17 cell-inducing strains (TH17 20-mix)8 of IFNγ+ CD8 T cells. Furthermore, approximately half of the IFNγ+
and by a reported TH1 cell-inducing Klebsiella pneumoniae strain CD8 T cells were undergoing active proliferation one week after col-
(Kp2H7)16, respectively (Extended Data Fig. 3i). These data suggest onization, as evidenced by their Ki67 expression (Fig. 2d), suggesting
that the 11 strains have non-inflammatory immunomodulatory activity that cellular expansion is also partially responsible for the observed
that is relatively specific to CD8 T cells. accumulation. In addition, IFNγ+ CD8 T cells from GF+11-mix mice
We scoured the genomes of the 11 strains and found no prominent showed a relative enrichment for the TCR Vβ6+ and Vβ8+ subsets, at
virulence factors or toxins (Supplementary Table 1). Although tetra- the expense of the Vβ5+ subset (Extended Data Fig. 4d). By contrast,
cycline-resistance and β-lactamase genes were present in most of the there was no difference in TCR Vβ usage by IFNγ-negative CD8 T cells
genomes, no strains were multidrug-resistant (Supplementary Tables 2 between germ-free and GF+11-mix mice (Extended Data Fig. 4d). We
and 3). A phylogenetic comparison revealed that the 11 strains con- also probed the antigen specificity of the induced colonic IFNγ+ CD8
sisted of 7 Bacteroidales and 4 non-Bacteroidales species (Fig. 1f and T cells, and found that a substantial fraction isolated from GF+11-mix
Extended Data Fig. 4a, b). When inoculated into germ-free mice, the but not germ-free mice recognized bacterial antigens derived from the
7 Bacteroidales-mix (7-mix) failed to induce IFNγ+ CD8 T cells, 11 strains (Fig. 2e). Together, these data suggest that the accumula-
whereas the 4 non-Bacteroidales-mix (4-mix) displayed a significantly tion of IFNγ+ CD8 T cells is probably due to the cumulative effects of
better induction capacity. However, the 4-mix alone was not sufficient colonic recruitment, cellular expansion and bacterial antigen-mediated
to achieve the full inductive effect of the 11-mix (Fig. 1j). These results differentiation.
suggest that the 11 strains act as a consortium in which the 4 non- We next examined the contributions of major innate signalling
Bacteroidales species act as effector elements and the 7 Bacteroidales pathways and dendritic cells. None of the Irf9−/−, Myd88−/−Trif−/−
have a supporting role. (Trif is also known as Ticam1), Il1r1−/− or Il18−/− GF+11-mix mice
showed any difference in IFNγ+ CD8 T cell induction capacity as com-
Mechanisms underlying IFNγ+ CD8 T cell accumulation pared to wild-type controls (Fig. 2f). By contrast, the 11-mix treat-
We then sought to determine the mechanisms that underlie 11-mix- ment failed to induce colonic IFNγ+ CD8 T cells in Batf3−/− mice
mediated IFNγ+ CD8 T cell accumulation in the colon. No accumula- and Xcr1-Cre × ROSA-DTA mice (Fig. 2g), both of which lack the
tion was observed after treating germ-free mice with heat-killed 11-mix intestinal CD11b−CD103+ dendritic cell subset17–19. Induction was
(Fig. 2a), and fluorescence in situ hybridization highlighted the ability also abrogated in CD11c-Cre × Irf4fl/fl (Irf4ΔDC) mice and CD11c-
of the strains to enter the colonic mucus layer, although no invasion into Cre × Notch2fl/fl (Notch2ΔDC) mice (Fig. 2g), which have defective
the epithelium was detected (Fig. 2b). Colonization with live 11-mix CD11b+CD103+ dendritic cell subsets19,20. Colonization with the
upregulated the expression of chemokines Cxcl9 and Cxcl10 as well as 11-mix enhanced the expression of MHC class I (H2-Kb) in both
other IFN-inducible genes in colonic epithelial cells (Extended Data CD11b−CD103+ and CD11b+CD103+ colonic lamina propria den-
Fig. 4c), and gnotobiotic Ifngr1−/− mice displayed markedly suppressed dritic cell populations in wild-type mice (Fig. 2h). In addition, IFNγ+
IFNγ+ CD8 T cell induction (Fig. 2c). These results suggest that active CD8 T cell accumulation was markedly impaired in MHC class
colonization near epithelial cells might establish an IFNγ-mediated Ia-deficient (KbDb−/−), but not MHC class Ib-deficient (H2-M3−/−),

6 0 2 | N A T U RE | V O L 5 6 5 | 3 1 J A N U A R Y 2 0 1 9
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 Article RESEARCH

a b GF (n = 6) c d
GF+11-mix (n = 6)
GF+10-mix (n = 7)
20 *** GF+11-mix (n = 8) 15 * GF+11-mix+anti-CD8 (n = 7)

Histological score

Histological score
110 105
15 ***

Body weight (%)

10

Body weight (%)

100

n=6
10 100

n=8

***
90 5 95

**
5

***

GF+11-mix n = 7
GF n = 6
GF+10-mix n = 7

NS
90
0 80 0

GF+11-mix

+anti-CD8
GF+11-mix
85
70 0 1 2 3 4 5
0 1 2 3
Days after Lm-WT gavage Days after Lm-WT gavage

e Day 3, 4, 5 f g Day 1, 2, 3, 4, 5
SPF (n = 7) SPF n = 7, 8, 8, 9, 8
SPF n = 8, 9, 8
SPF+11-mix (n = 8) SPF+11-mix n = 7, 8, 8, 9, 8
SPF+11-mix n = 8, 8, 7
SPF+11-mix+anti-CD8 n = 7, 7, 8, 9, 8
SPF+11-mix+anti-CD8 n = 7, 9, 8 SPF+11-mix+anti-CD8
(n = 8)
NS NS NS Colon MLNs

Lm CFU per mg organ

20 110 106 106
* ** * *** ***

Body weight (%)

Histological score

105 105
15 100
104 104

***
***

P = 0.135
10 90 103 103

*
**
NS

**
102 102
5 80
101 101
0 70 100 100
3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 01 2 3 4 5
Days after Lm-InlAm gavage Days after Lm-InlAm gavage

Fig. 3 | Enhanced clearance of L. monocytogenes by the 11 strains. in the indicated tissues over the course of the experiment. For depletion of
a–d, Germ-free mice were colonized with 11- or 10-mix, or left CD8 T cells, anti-CD8 antibody was administered intraperitoneally 1 day
uncolonized as a control. The mice were then orally infected with Lm-WT before the administration of the 11-mix, and every 3–4 days thereafter.
(see experimental design, Extended Data Fig. 6a). The colon histology Each circle represents an individual animal. Data are mean and s.d.
score on day 3 (a) or 5 (c) and the percentage weight change over the The number of mice in each group is shown. ***P < 0.001; **P < 0.01;
course of L. monocytogenes infection (b, d) are shown. e–g, SPF mice were *P < 0.05; one-way ANOVA with Tukey’s test (a, e), two-way ANOVA
orally administered the 11-mix, followed by oral infection with Lm-InlAm with Tukey’s (b, f) or Bonferroni’s (d) test, two-tailed unpaired t-test (c), or
(see experimental design, Extended Data Fig. 6d). e, f, Colon histology Kruskal–Wallis with Dunn’s test (g). See Source Data for exact P values.
score (e) and percentage weight change (f) are shown. g, Lm-InlAm CFU

mice (Fig. 2i). These results are consistent with the hypothesis that the protective effects of the 11-mix (Fig. 3c, d and Extended Data Fig. 6c).
11 strains activate signalling independently of major innate immune These trends were replicated under SPF conditions (SPF+11-mix
pathways, and this conditions CD103+ lamina propria dendritic cells to mice), in infection models using an invasive mutant (Lm-InlAm)21 or
induce IFNγ+ CD8 T cell accumulation in an MHC class Ia-dependent using Lm-WT (Fig. 3e–g and Extended Data Fig. 6d–j). Moreover, in an
manner. ovalbumin-expressing L. monocytogenes (Lm-OVA)22 infection model,
SPF+11-mix mice displayed enhanced induction of OVA-specific
Systemic effects of 11-mix colonization IFNγ+ CD8 T cells (Extended Data Fig. 6k). Colonization with the
The 11 strains preferentially localized to the caecum and colon 11-mix also protected mice against Listeria dissemination from the
(Extended Data Fig. 4e), which was reflected by a prominent induction intestine after oral gavage with invasive Lm-InlAm and against sys-
of IFNγ+ CD8 T cells at these sites (Fig. 2j). Nevertheless, the immu- temic infection after intraperitoneal injection of Lm-WT (Extended
nomodulatory phenotype extended well beyond the intestine, as an Data Fig. 6f, l).
increase in IFNγ+ CD8 T cell frequency was observed in several other
organs (Fig. 2j). The systemic effects of the 11-mix were probably not Enhancement of anti-tumour immunity
caused by bacterial dissemination (Extended Data Fig. 4f) or systemic IFNγ+ CD8 T cells also have a crucial role in anti-tumour immunity
circulation of gut-origin IFNγ+ CD8 T cells, as the colonic and sys- and affect immune checkpoint inhibitor (ICI) therapies12,13,23,24, so we
temic subsets were phenotypically distinct (Extended Data Figs. 5a, b accordingly examined whether colonization with the 11 strains could
and 10). Instead, an appealing hypothesis is that the systemic effects enhance ICI efficacy. Germ-free, GF+11-mix and GF+10-mix mice
may be due to circulating metabolites induced by the 11 strains. Indeed, were subcutaneously engrafted with MC38 adenocarcinoma cells,
examination of caecal contents from germ-free, GF+4-mix, GF+7- and tumour growth was monitored with or without intraperitoneal
mix, GF+11-mix and GF+10-mix mice revealed differences in the administration of an anti-PD-1 antibody (Extended Data Fig. 7a).
overall metabolomic profile of each group, although GF+11-mix and Anti-PD-1 treatment was markedly more efficacious in GF+11-mix
GF+4-mix mice clustered more tightly than other groups, consistent than in GF+10-mix or germ-free mice (Fig. 4a). This therapeutic
with their immunomodulatory phenotype (Extended Data Fig. 5c). efficacy was accompanied by an increase in the frequency of IFNγ+
Several molecules, such as mevalonate and dimethylglycine, were CD8 tumour-infiltrating lymphocytes (TILs) (Fig. 4b), and antibody-
increased in both the caecal contents and the sera of GF+11-mix mice mediated CD8 T cell depletion significantly diminished the protective
(Extended Data Fig. 5d–f), suggesting a potential mechanistic role. effects (Fig. 4c). IFNγ+ CD8 TILs in GF+11-mix+anti-PD-1 mice
were primarily PD-1highCD103− and relatively enriched for the Vβ13+
Enhancement of anti-microbial immunity subset, and thus are phenotypically distinct from the IFNγ+ CD8
Because IFNγ+ CD8 T cells are known to promote the clearance of T cells induced in the colon (Extended Data Figs. 7b, c and 10). Notably,
intracellular pathogens15,21, we next tested whether the 11 strains 11-mix colonization significantly suppressed tumour growth even
could augment host protective immunity against orally administered in the absence of anti-PD-1 treatment (Fig. 4a). Collectively, these
Listeria monocytogenes. GF+11-mix mice exhibited enhanced clear- results suggest that the 11 strains are effective in enhancing both spon-
ance of Listeria (a wild-type strain, Lm-WT), as evidenced by markedly taneous and ICI-mediated anti-tumour immunity in a CD8 T cell-
improved colon histology scores, reduced weight loss, and a decrease in dependent manner. The effect of the 11-mix was also observed within
colon tissue Listeria colony-forming units (CFU) as compared to germ- complex microbiota-colonized mice harbouring SPF (Fig. 4d, e and
free and GF+10-mix mice (Fig. 3a, b and Extended Data Fig. 6a–c). Extended Data Fig. 8a, b) or donor C human (Extended Data Fig. 8d–h)
Antibody-mediated depletion of CD8 T cells significantly reduced the microbiota. A subset of the IFNγ+ CD8 TILs from SPF+11-mix+

3 1 J A N U A R Y 2 0 1 9 | V O L 5 6 5 | N A T U RE | 6 0 3
© 2019 Springer Nature Limited. All rights reserved.
 RESEARCH Article

***
a GF (n = 6) GF+anti-PD-1 (n = 7) b 100
*** ** c h i
* *** GF+11-mix+anti-PD-1+anti-CD8 (n = 8) *** **
GF+10-mix (n = 7) 10 * 8 *
1.5
Tumour volume (×103 mm3)

Tumour volume (×103 mm3)

80 2.0 GF (n = 8) GF+anti-PD-1 (n = 8)

H2-Kb MFI (×103)

IFNγ+ CD8 T (%)
GF+10-mix

CD11c+ DC (×105)
n=4
n=4
+anti-PD-1 (n = 7) GF+11-mix (n = 8) 8 6

MHC class II+

per g tumour
GF+11-mix (n = 7) 60 GF+11-mix
1.5
6

NS
1.0

n=7
n=6
n=7
***
GF+11-mix +anti-PD-1 (n = 8)
4

***
40

***
+anti-PD-1 (n = 7)

NS

***
4

***
1.0

***
n=3
n=4
n=3
n=5
***
20 2

***
0.5

***
2

n=7
n=6
n=7
n=8
***

n=8
***
0 0.5
Anti-PD-1 – + – + – + 0 0
*** Anti-PD-1 – + – + Anti-PD-1 – + – +
0

GF
GF+11-mix

GF+10-mix
0
0 5 10 15 20 0 5 10 15 20 SPF SPF SPF SPF
Days after MC38 injection Days after MC38 injection +11-mix +11-mix

d e f SPF+anti-PD-1 (n = 6)
SPF+11-mix+anti-PD-1 (n = 7)
g j SPF (n = 8) k SPF (n = 9)
SPF (n = 10) SPF+anti-PD-1 (n = 8)
60 *** SPF+10-mix+anti-PD-1 (n = 6) SPF+anti-CTLA-4 (n = 8)
*** *** SPF+anti-PD-1 (n = 9)

Tumour volume (×103 mm3)

3

Tumour volume (×103 mm3)

SPF+10-mix (n = 10) *** *** NS ***
***NS*** *** ***
60 25 * SPF+11-mix (n = 8) SPF+11-mix (n = 11)

GrB+ IFNγ+ CD8 T (%)

SPF+10-mix+anti-PD-1 (n = 10) * 3
IFNγ+ CD8 T (%)

1.5 40
Tumour volume (×103 mm3)

SPF+11-mix SPF+11-mix

IFNγ+ CD8 T (%)

SPF+11-mix (n = 8) 40 20 *** * 20 +anti-CTLA-4 (n = 8)
10 2 +anti-PD-1 (n = 11)
SPF+11-mix

NS
2

***
n=7

***
******
+anti-PD-1 (n = 9) 15
1.0

n=8

n=9

***
***
20
NS

5 NS NS 10 1

***
***

NSNS
**

1
***

n=6
n=6
n=6
n=6
n=7
n=6
***

* ***
***
NS

n=7
0.5 5
0
***
***
***

Anti-PD-1 – + – + – + 0 0 0 0
Anti-PD-1 – + – + 0 10 20 30 0 5 10 15 20 25
Medium
+p15E pep.
+11 st. lysate
+PMA+Iono.
SPF

SPF+11-mix
SPF+10-mix

0 Days after MC38 injection

0 5 10 15 20 SPF SPF Days after BrafV600EPten–/–
+11-mix melanoma injection
Days after MC38 injection

Fig. 4 | Enhancement of anti-tumour immunity and efficacy of ICI tumour growth data. e, g–i, Percentage of tumour-infiltrating IFNγ+ CD8
treatment by the 11 strains. a–c, Germ-free mice with or without 11-mix (e) and GrB+IFNγ+ CD8 (g) T cells, number of CD11c+ dendritic cells
or 10-mix were subjected to subcutaneous implantation of MC38 cells per gram tumour (h), and mean fluorescence intensity (MFI) of H2-Kb on
followed by intraperitoneal injection with anti-PD-1 antibody. For the dendritic cells (i), as determined by flow cytometry. f, Percentage of IFNγ+
depletion of CD8 T cells, anti-CD8 was administered intraperitoneally cells among the CD8 TIL population from MC38 tumours of the indicated
1 day before administration of 11-mix, and every 3–4 days thereafter mice after ex vivo stimulation with the indicated antigens. Each circle in
(see experimental design, Extended Data Fig. 7a). a, c, Representative b, e–i represents an individual animal. Green, red and orange asterisks
MC38 tumour growth data. b, Percentage of IFNγ+ among CD8 T cells show significance versus the 11-mix, the 11-mix plus anti-PD-1 and the
isolated from the tumours. d–k, SPF mice were subjected to subcutaneous 11-mix plus anti-CTLA-4 groups, respectively. Dotted brackets in a denote
implantation of MC38 cells (d–j) or BrafV600E Pten−/− melanoma cells (k), comparison with the 10-mix group. pep., peptide; Data are mean and s.d.
followed by repetitive oral dosing of 11- or 10-mix. Anti-PD-1 (d–i, k) ***P < 0.001; **P < 0.01; *P < 0.05; two-way ANOVA (a, c, d, j, k) or
or anti-CTLA-4 (j) antibodies were injected intraperitoneally three times one-way ANOVA (b, e–i) with Tukey’s test. See Source Data for exact
(see experimental design, Extended Data Fig. 8a). d, j, k, Representative P values.

anti-PD-1 mice expressed TCRs specific for the MC38 tumour- (Supplementary Discussion). Several reports have demonstrated
associated antigen p15E25, whereas the frequency of 11-strain-specific that the composition of the gut microbiota influences response to
TILs was not increased above baseline (Fig. 4f and Extended Data ICIs in both humans and mice, suggesting that its manipulation
Fig. 10). In addition, combination therapy resulted in an increase in holds immense therapeutic potential12,13,29–32. However, the specific
the frequency of GrB+IFNγ+ CD8 T cells and of tumour-infiltrating microbiota members associated with enhanced clinical response vary
dendritic cells that express high levels of MHC class I molecules substantially between studies. Although it is likely that various com-
(Fig. 4g–i). Supplementation with the 11-mix was similarly effective positions and subsets of the human microbiota can induce IFNγ+
when combined with anti-CTLA-4 in the MC38 model (Fig. 4j), and CD8 T cells in a manner similar to that of the 11-mix, our findings
with anti-PD-1 in the less immunogenic BrafV600E Pten−/− melanoma provide an example of rare species that can enhance anti-microbial
model26,27 (Fig. 4k). Importantly, there was no histological evidence of and anti-tumour immunity with a potentially large effect size. The
colitis in the 11-mix treatment groups—a frequently observed adverse contribution of such species may be underestimated by conventional,
effect of ICI therapy13,28 (Extended Data Fig. 8c). Collectively, these sequence-oriented microbiome analyses. Our isolated strains have
results suggest that the 11 strains have the capacity to simultaneously great biotherapeutic potential and could be broadly applicable to
enhance ICI therapies and negate their colitogenic side effects. enhancing the treatment of cancer and infectious disease alike, as
Finally, we evaluated the abundance of the 11 strains in the human they are severely underrepresented in the gut microbiota of most
gut microbiome using several metagenomic datasets. We found that individuals.
most of the 11 strains are generally rare, low-abundance components
of the human microbiota at both strain- and species-level resolution Online content
(Extended Data Fig. 9). Most of the strains were scarce even in the Any methods, additional references, Nature Research reporting summaries, source
microbiome of donor B, from which they were isolated (Extended data, statements of data availability and associated accession codes are available at
Data Fig. 9d, e). Similar low-abundance trends were observed in both https://doi.org/10.1038/s41586-019-0878-z.
responder and non-responder patients with melanoma treated with
Received: 26 March 2018; Accepted: 5 December 2018;
ICIs in the reported datasets29–31 (Extended Data Fig. 9b, c).
Published online 23 January 2019.
Discussion
1. Mimee, M., Citorik, R. J. & Lu, T. K. Microbiome therapeutics — advances and
In this study, we have identified 11 healthy human-associated bacterial challenges. Adv. Drug Deliv. Rev. 105, 44–54 (2016).
strains that act together to induce IFNγ+ CD8 T cells, confer resistance 2. Kim, S., Covington, A. & Pamer, E. G. The intestinal microbiota: antibiotics,
to the intracellular pathogen Listeria, and are effective at inhibiting colonization resistance, and enteric pathogens. Immunol. Rev. 279, 90–105
tumour growth in conjunction with ICIs. More research will be needed (2017).
3. El Hage, R., Hernandez-Sanabria, E. & Van de Wiele, T. Emerging trends in
to determine the cellular and molecular mechanism by which the 11 “smart probiotics”: functional consideration for the development of novel
strains effect the observed local and systemic immunomodulation health and industrial applications. Front. Microbiol. 8, 1889 (2017).

6 0 4 | N A T U RE | V O L 5 6 5 | 3 1 J A N U A R Y 2 0 1 9
© 2019 Springer Nature Limited. All rights reserved.

4. Blander, J. M., Longman, R. S., Iliev, I. D., Sonnenberg, G. F. & Artis, D. Regulation
of inflammation by microbiota interactions with the host. Nat. Immunol. 18,
851–860 (2017).
5. Atarashi, K. et al. Treg induction by a rationally selected mixture of Clostridia
strains from the human microbiota. Nature 500, 232–236 (2013).
6. Faith, J. J., Ahern, P. P., Ridaura, V. K., Cheng, J. & Gordon, J. I. Identifying gut
microbe–host phenotype relationships using combinatorial communities in
gnotobiotic mice. Sci. Transl. Med. 6, 220ra11 (2014).
7. Sefik, E. et al. Individual intestinal symbionts induce a distinct population of
RORγ+ regulatory T cells. Science 349, 993–997 (2015).
8. Atarashi, K. et al. Th17 cell induction by adhesion of microbes to intestinal
epithelial cells. Cell 163, 367–380 (2015).
9. Tan, T. G. et al. Identifying species of symbiont bacteria from the human gut
that, alone, can induce intestinal Th17 cells in mice. Proc. Natl Acad. Sci. USA
113, E8141–E8150 (2016).
10. Ichinohe, T. et al. Microbiota regulates immune defense against respiratory
tract influenza A virus infection. Proc. Natl Acad. Sci. USA 108, 5354–5359
(2011).
11. Thackray, L. B. et al. Oral antibiotic treatment of mice exacerbates the disease
severity of multiple flavivirus infections. Cell Rep. 22, 3440–3453 (2018).
12. Spranger, S., Sivan, A., Corrales, L. & Gajewski, T. F. Tumor and host factors
controlling antitumor immunity and efficacy of cancer immunotherapy.
Adv. Immunol. 130, 75–93 (2016).
13. Bhatt, A. P., Redinbo, M. R. & Bultman, S. J. The role of the microbiome in cancer
development and therapy. CA Cancer J. Clin. 67, 326–344 (2017).
14. Zitvogel, L., Ma, Y., Raoult, D., Kroemer, G. & Gajewski, T. F. The microbiome in
cancer immunotherapy: diagnostic tools and therapeutic strategies. Science
359, 1366–1370 (2018).
15. Park, C. O. & Kupper, T. S. The emerging role of resident memory T cells in
protective immunity and inflammatory disease. Nat. Med. 21, 688–697
(2015).
16. Atarashi, K. et al. Ectopic colonization of oral bacteria in the intestine drives TH1
cell induction and inflammation. Science 358, 359–365 (2017).
17. Hildner, K. et al. Batf3 deficiency reveals a critical role for CD8α+ dendritic cells
in cytotoxic T cell immunity. Science 322, 1097–1100 (2008).
18. Ohta, T. et al. Crucial roles of XCR1-expressing dendritic cells and the
XCR1–XCL1 chemokine axis in intestinal immune homeostasis. Sci. Rep. 6,
23505 (2016).
19. Joeris, T., Müller-Luda, K., Agace, W. W. & Mowat, A. M. Diversity and functions
of intestinal mononuclear phagocytes. Mucosal Immunol. 10, 845–864
(2017).
20. Satpathy, A. T. et al. Notch2-dependent classical dendritic cells orchestrate
intestinal immunity to attaching-and-effacing bacterial pathogens. Nat.
Immunol. 14, 937–948 (2013).
21. Sheridan, B. S. et al. Oral infection drives a distinct population of intestinal
resident memory CD8+ T cells with enhanced protective function. Immunity 40,
747–757 (2014).
22. Foulds, K. E. et al. Cutting edge: CD4 and CD8 T cells are intrinsically different in
their proliferative responses. J. Immunol. 168, 1528–1532 (2002).
23. Gao, J. et al. Loss of IFN-γ pathway genes in tumor cells as a mechanism of
resistance to anti-CTLA-4 therapy. Cell 167, 397–404 (2016).
24. Wei, S. C. et al. Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1
checkpoint blockade. Cell 170, 1120–1133 (2017).
25. Yang, J. C. & Perry-Lalley, D. The envelope protein of an endogenous murine
retrovirus is a tumor-associated T-cell antigen for multiple murine tumors.
J. Immunother. 23, 177–183 (2000).
© 2019 Springer Nature Limited. All rights reserved.
Article RESEARCH
26. Cooper, Z. A. et al. Response to BRAF inhibition in melanoma is enhanced when
combined with immune checkpoint blockade. Cancer Immunol. Res. 2, 643–654
(2014).
27. Dankort, D. et al. BrafV600E cooperates with Pten loss to induce metastatic
melanoma. Nat. Genet. 41, 544–552 (2009).
28. Dubin, K. et al. Intestinal microbiome analyses identify melanoma patients at
risk for checkpoint-blockade-induced colitis. Nat. Commun. 7, 10391 (2016).
29. Routy, B. et al. Gut microbiome influences efficacy of PD-1-based
immunotherapy against epithelial tumors. Science 359, 91–97 (2018).
30. Gopalakrishnan, V. et al. Gut microbiome modulates response to anti-PD-1
immunotherapy in melanoma patients. Science 359, 97–103 (2018).
31. Matson, V. et al. The commensal microbiome is associated with anti-PD-1
efficacy in metastatic melanoma patients. Science 359, 104–108 (2018).
32. Frankel, A. E. et al. Metagenomic shotgun sequencing and unbiased
metabolomic profiling identify specific human gut microbiota and metabolites
associated with immune checkpoint therapy efficacy in melanoma patients.
Neoplasia 19, 848–855 (2017).
Acknowledgements K.H. was funded through AMED LEAP under grant
number JP18gm0010003, the Takeda Science Foundation, and the Mitsubishi
Foundation. T.T. was funded through AMED PRIME under grant number
JP18gm6010013 and the Nakajima Foundation. R.J.X. was funded through
NIH DK43351, AT009708, and the Center for Microbiome Informatics and
Therapeutics, MIT. The LC–MS platform used in this study was provided by the
JST ERATO Gas Biology Project, which was led by M.S. until March 2015. We
thank N. Palm, J. Faith and J. S. Weber for discussion, J. Baginska and O. Ohara
for their technical support, P. Burrows for comments, and RIKEN BRC and the
International Mouse Phenotyping Consortium for generating and providing the
H2-M3 knockout mice.
Reviewer information Nature thanks S. Mazmanian and the anonymous
reviewer(s) for their contribution to the peer review of this work.
Author contributions K.H. and T.T. planned experiments, analysed data, and
wrote the paper together with A.N.S., A.S., B.O. and B.R.; T.T., S.M., A.N.S., S.N.,
I.M., K.S., K.T., K.Y.-M, T.I. and K.A. performed immunological analyses and
bacterial cultures; W.S., M.H., J.M.N., V.B., H.V., D.R.P. and R.J.X. performed
bacterial sequencing and microbiome analysis; M.S. and Y.S. performed
metabolomic analysis; D.M., D.R. and T.K. provided essential materials; T.Y. and
Y.K. supervised tumour models.
Competing interests B.O., B.R. and J.M.N. are employees of Vedanta
Biosciences Inc. A.S. is an employee of JSR Corporation. K.H. is co-founder and
scientific advisor to Vedanta Biosciences since 11 August 2011.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41586-
019-0878-z.
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-019-0878-z.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Correspondence and requests for materials should be addressed to K.H.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
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 RESEARCH Article
Methods
Data reporting. No statistical methods were used to predetermine sample size. The
experiments were not randomized and investigators were not blinded to allocation
during experiments and outcome assessment.
Mice. SPF or germ-free C57BL/6, BALB/c and IQI mice of both sexes were pur-
chased from Sankyo Laboratories, Japan SLC, CLEA Japan, and Charles River
Japan. Germ-free and gnotobiotic mice were bred and maintained within the
gnotobiotic facilities of Keio University and RIKEN. Ifngr1−/−, Batf3−/−, Il18−/−,
Il1r1−/−, CD11c-Cre × Irf4fl/fl, CD11c-Cre × Notch2fl/fl and Il10−/− mice were
purchased from Jackson Laboratories. Myd88−/− Trif−/− mice were purchased from
Oriental Bio Service. Xcr1+/cre × R26:lacZbpAfloxDTA mice were generated as pre-
viously described18. Irf9−/− and H2-M3−/− mice (RBRC00916, 10034) was pro-
vided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/
AMED, Japan. KbDb−/− mice were purchased from MMRRC. Germ-free rederiva-
tion was performed at the gnotobiotic facilities of RIKEN and Keio University. All
animal experiments were approved by the Keio University Institutional Animal
Care and Use Committee and by the RIKEN Yokohama Institute. Unless otherwise
noted, all SPF mice were C57BL/6 mice obtained from CLEA Japan.
Human faecal samples, bacterial culture and generation of gnotobiotic ani-
mals. Human faecal samples were collected at the RIKEN Institute and Keio
University according to the study protocol approved by the Institutional Review
Boards (approval numbers H24-9 and 20150075, respectively). Informed consent
was obtained from each subject. Faecal samples and mouse B5 caecal contents
were suspended in an equal volume (w/v) of PBS containing 20% glycerol in PBS,
snap-frozen in liquid nitrogen, and stored at −80 °C until use. The frozen stocks
were thawed, suspended in TS broth, filtered through a 100-μm cell strainer, and
orally inoculated into germ-free mice (approximately 5–60 mg per 250 µl per
mouse). Twenty-four hours after inoculation, some mice were treated with ampi-
cillin (1 g l−1), metronidazole (1 g l−1), tylosin (0.5 g l−1) or streptomycin (0.5 g l−1)
via the drinking water. For chloroform treatment of B5 caecal contents, the caecal
suspension was mixed with chloroform (final concentration 3%) and incubated in a
shaking water bath for 60 min followed by evaporation of chloroform via bubbling
with N2 gas for 30 min. To isolate IFNγ+ CD8 T cell-inducing bacterial strains,
caecal contents from GF+B5-AMP2 and GF+B5-AMP3 mice were serially diluted
with PBS and seeded onto nonselective and selective agar plates (GAM, CM0151
and BBE). After culture under anaerobic conditions (80% N2, 10% H2, 10%
CO2) in an anaerobic chamber (Coy Laboratory Products) at 37 °C for 2–4 days,
individual colonies were picked, and the 16S rRNA gene region was amplified
with universal primers (27Fmod: 5′-AGRGTTTGATYMTGGCTCAG-3′, 1492R:
5′-GGYTACCTTGTTACGACTT-3′) and sequenced. Individual isolates in the cul-
ture collection were grouped as ‘strains’ if their 16S rRNA gene sequences had 100%
identity. The resulting strain sequences were compared to those in the Ribosomal
Database Project (RDP) database and to OTUs observed in caecal samples from
GF+B5-AMP2 and GF+B5-AMP3 to determine closely related species or strains
and their corresponding OTUs. To prepare the bacterial inocula, strains were indi-
vidually grown in GAM, CM0149 or EG broth to confluence and equal volumes of
bacterial suspensions were mixed. For culture of strain 82G5, succinate was added
to EG or CM0149 (final concentration 100 mM). The mixture of isolates was orally
administered to germ-free mice (approximately 1 × 108–2 × 108 CFU of each
strain in 250 µl medium per mouse). All mice receiving a given mixture of bacte-
rial strains were maintained in a single gnotobiotic isolator. For administration of
heat-killed bacteria, 11 strains were cultured individually for 3 days, combined,
washed with autoclaved water, heat-killed at 121 °C for 30 min, and administered
to germ-free mice via the drinking water (2 × 107–10 × 107 equivalent CFU of
each strain per 1 ml) for 4 weeks. C57BL/6 germ-free mice (6–12 weeks old) were
used and analysed 3–4 weeks after the initial gavage unless otherwise indicated.
16S rRNA gene amplicon sequencing. Frozen caecal contents and faecal pellets
from mice were thawed and suspended in 850 μl TE10 (10 mM Tris-HCl, 10 mM
EDTA) buffer containing RNase A (final concentration of 100 μg ml−1, Invitrogen)
and lysozyme (final concentration 3.0 mg ml−1, Sigma). The suspension was incu-
bated for 1 h at 37 °C with gentle mixing. Purified achromopeptidase (Wako) was
added to a final concentration of 2,000 U ml−1, and the sample was further incu-
bated for 30 min at 37 °C. Then, sodium dodecyl sulfate (final concentration 1%)
and proteinase K (final concentration 1 mg ml−1, Nacalai) were added to the sus-
pension and the mixture was incubated for 1 h at 55 °C. High-molecular mass DNA
was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), precipitated with
isopropanol, washed with 75% ethanol, and resuspended in 200 μl of TE buffer.
PCR was performed using 27Fmod 5′-AGRGTTTGATYMTGGCTCAG-3′ and
338R 5′-TGCTGCCTCCCGTAGGAGT-3′ to the V1–V2 region of the 16S rRNA
gene. Amplicons generated from each sample (~330 bp) were subsequently purified
using AMPure XP (Beckman Coulter). DNA was quantified using a Quant-iT
Picogreen dsDNA assay kit (Invitrogen) and a TBS-380 Mini-Fluorometer (Turner
Biosystems). The 16S metagenomic sequencing was performed using MiSeq
according to the Illumina protocol. Two paired-end reads were merged using the
© 2019 Springer Nature Limited. All rights reserved.
fastq-join program based on overlapping sequences. Reads with an average qual-
ity value of <25 and inexact matches to both universal primers were filtered out.
Filter-passed reads were used for further analysis after trimming off both primer
sequences. For each sample, 3,000 quality filter-passed reads were rearranged in
descending order according to the quality value and then clustered into OTUs
with a 97% pairwise-identity cutoff using the UCLUST program (Edgar 2010)
version 5.2.32 (https://www.drive5.com). Taxonomic assignment of each OTU
was made via searching by similarity against the RDP and the National Center
for Biotechnology Information genome database using the GLSEARCH program.
Bacterial genome sequencing. The genomic DNA of the 21 strains was extracted
by a method similar to that described above in the 16S rRNA gene sequencing sec-
tion. The genome sequences were determined by using a whole-genome shotgun
strategy with either the Illumina MiSeq or the MinION nanopore sequencer. The
genomic DNA was sheared by sonication to obtain DNA fragments. Template DNA
was prepared according to each supplier’s protocol. Sequences concatenated with
genes encoding 42 ribosomal proteins (large subunits L1, L3, L4, L5, L6, L7/L12,
L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L21, L23, L24, L27, L28, L29, L30,
L32 and L35, and small subunits S2, S3, S5, S6, S7, S8, S9, S10, S11, S12, S13, S15,
S16, S17, S18, S19 and S20) predicted from the genomes of each strain were used to
construct a phylogenetic tree. The calculation was performed using the MEGA v7.0
package and the neighbour-joining method with a bootstrap of 1,000 replicates.
Isolation and flow cytometric analysis of intestinal lymphocytes, dendritic
cells and epithelial cells. For analysis of intestinal lymphocytes, dendritic cells
and epithelial cells, intestines were opened longitudinally and washed with PBS to
remove luminal contents. All samples were incubated in 20 ml of Hanks’ balanced
salt solution (HBSS) containing 5 mM EDTA for 20 min at 37 °C in a shaking
water bath to remove epithelial cells. After vigorous vortexing, colonic epithe-
lial cells released into suspension were centrifuged, immediately frozen in liquid
nitrogen, and stored at −80 °C until further analysis. An aliquot of epithelial cells
was subjected to Percoll gradient separation to isolate colonic intraepithelial lym-
phocytes. After removal of the epithelial cells, the muscle layer and adipose tissue
were removed manually using forceps. The remaining lamina propria layer was
cut into small pieces and incubated in 10 ml of RPMI1640 containing 4% fetal
bovine serum, 0.5 mg ml−1 collagenase D (Roche), 0.5 mg ml−1 dispase (Gibco),
and 40 µg ml−1 DNase I (Roche) for 45 min at 37 °C in a shaking water bath.
The digested tissues were washed with 10 ml of HBSS containing 5 mM EDTA,
resuspended in 5 ml of 40% Percoll (GE Healthcare), and underlaid with 2.5 ml of
80% Percoll in a 15 ml Falcon tube. Percoll gradient separation was performed by
centrifugation at 900g for 30 min at 25 °C. The fraction containing lymphocytes
and antigen presenting cells was collected from the interface of the two layers
and washed with RPMI1640 containing 10% FBS. For dendritic cell staining, the
cells were labelled with Ghost Dye 780 (Tonbo Biosciences) and then stained
with anti-I-A/I-E (FITC, Biolegend), CD11c (APC, Biolegend), CD103 (PE/Cy7,
Biolegend), CD11b (BV605, Biolegend) and H-2Kb (PacificBlue, Biolegend). For
cytokine detection, the cells were stimulated with 50 ng ml−1 PMA and 750 ng ml−1
ionomycin (both from Sigma) in the presence of GolgiStop (BD Biosciences) at
37 °C for 3.5 h. After labelling with Ghost Dye 780, the cells were permeabilized
and stained with anti-CD3 (BV605 or BV650; Biolegend), CD4 (BV510 or FITC;
Biolegend), CD8α (APC, BV421 or PE/Cy7; Biolegend), TCRβ (PerCP-Cy5.5 or
BV711; Biolegend), CD8β (PerCP-Cy5.5), CD103 (BV421 or FITC), GranzymeB
(APC or PacificBlue; Biolegend), ICOS (PE/Dazzle594, PE or FITC; Biolegend),
CD44 (PE or BV786; Biolegend), KLRG1 (BV711 or PE/Dazzle594; Biolegend),
PD-1 (BV510; Biolegend), IFNγ (PE/Cy7 or FITC; Biolegend), IL-17A (PE; BD
Biosciences), T-bet (PE/Cy7, BV421 or BV785; Biolegend) and Ki67 (AF488;
Biolegend) using the Foxp3/Transcription Factor Staining Buffer Kit (eBioscience)
as per the manufacturer’s instructions. For analysis of TCRVβ repertoire of CD8
T cells, lymphocytes were stained using the Anti-Mouse TCRVβ Screening Panel
(BD Biosciences). All data were collected on a BD LSRFortessa or FACSAria IIIu
(BD Biosciences) instrument and analysed with Flowjo software (TreeStar). CD8
T cells were defined as the CD8α+TCRβ+CD3+ population within the live-cell
gate. For assessment of the IFNγ responses against bacterial lysate, the 11 strains
were cultured individually for 3 days, washed with PBS, heat-killed at 70 °C for
30 min, and used at a final concentration of 1 × 106–2 × 106 CFU ml−1 of each
strain. Percoll-enriched colonic lamina propria cells, which include T cells and
antigen-presenting cells, isolated from GF+11-mix mice or germ-free mice were
stimulated ex vivo with bacterial lysate for 18 h, then GolgiStop was added and the
suspension was incubated for an additional 3.5 h.
Colonization of SPF or donor C human microbiota-colonized mice with the
11-mix. SPF C57BL/6 mice (6–7 weeks old) from SLC Japan (which display a rela-
tively low-frequency IFNγ+ CD8 T cell phenotype, see Extended Data Fig. 1f) were
treated with AVMN (ampicillin (1 g l−1), vancomycin (500 mg l−1), metronidazole
(1 g l−1) and neomycin (1 g l−1)) or left untreated for 5–9 days via the drinking
water. After a one-day antibiotic washout, a faecal suspension from untreated SPF
mice (SPFfae) (SLC Japan, approximately 1 mg per mouse) was orally introduced

along with or without the 11-mix by gavage. For human microbiota-colonized
mice, germ-free C57BL/6 mice were orally inoculated with donor C human faecal
samples (approximately 1 mg per mouse) mixed with or without 11-mix. For
repetitive administration, oral gavage of the 11-mix was performed 2 or 3 times
per week. For each inoculation, the 11 strains were grown for 2 or 3 days in GAM,
CM0149 or EG broth and approximately 1 × 108–2 × 108 CFU of each strain were
used to inoculate each mouse. Faeces or caecal contents were collected and DNA
was extracted as a part of the aforementioned 16S rRNA gene pyrosequencing.
Confirmation of colonization was achieved by quantitative PCR (qPCR) with the
following specific primers for each strain: 81A6 (5ʹ-TGCACTGTTGGATTTT
CTAAAAAGG-3ʹ, 5ʹ-ACTTTGGGCATGCTAAACCA-3ʹ); 81B11 (5ʹ-CGCCG
GATGCATATACAAGA-3ʹ, 5ʹ-TCTCGCCAATGATGTCCAAA-3ʹ); 81C1 (5ʹ-CC
GCACAAGAGAAATAAACGCCA-3ʹ, 5ʹ-TGGCAAATTCAAAGGTGAGCG
AA-3ʹ), 81E7 (5ʹ-GGGAATAAAGCTGTTCCGATATGC-3ʹ, 5ʹ-TCATGCAACATT
CTTTCGTTGG-3ʹ), 81H9 (5ʹ-TTCACCTTCTACGGCTACTACTACG-3ʹ,
5ʹ-ACATAACGATCAAGGGTGCTGAAG-3ʹ), 82A6 (5ʹ-GCTCTTTTTAGCCTGT
ATCCGGT-3ʹ, 5ʹ-ATACGATACGAACGACCAACCT-3ʹ), 82B1 (5ʹ-TACCAATG
CAAAGCGACCAA-3ʹ, 5ʹ-CGGTTTTGTTGCCGAACTCT-3ʹ), 82F11 (5ʹ-GCACA
GATTCTACACTCCCCT-3ʹ, 5ʹ-AGCAACGAAACAACCTGTGA-3ʹ), 82G1
(5ʹ-TCCATGCTGAAGCGTTGAAG-3ʹ, 5ʹ-GGACCGAACATCCCAATCA
C-3ʹ), 82G5 (5ʹ-CTGCTTCCGACAGCACACAT-3ʹ, 5ʹ-AGCTTGGCGGAGAGC
CTATT-3ʹ), 82G9 (5ʹ-AGCGCGTAAACTTAGTCAAGGA-3ʹ, 5ʹ-TAGGGCCAAAA
CCTGCATTA-3ʹ). For detection of bacteria in the MLNs, the organs were homog-
enized and total DNA was isolated according to the faecal DNA isolation proto-
col, followed by qPCR using the specific primers described above. We confirmed
the detection of bacterial DNA with this method by analysing MLNs from mice
infected with the invasive Listeria monocytogenes strain (Lm-InlAm).
qPCR analysis. Total RNA was isolated from colonic epithelial cells using the
TRIzol reagent (Invitrogen) as per the manufacturer’s instructions. For qPCR anal-
ysis, cDNA was synthesized using SuperScript VILO Master Mix (Thermo Fisher),
and qPCR was performed using the Thunderbird SYBR qPCR Mix (TOYOBO) on
a LightCycler 480 (Roche). The following primer pairs were used: Actb, 5ʹ-TTGCT-
GACAGGATGCAGAAG-3ʹ and 5ʹ-ATCCACATCTGCTGGAAGGTG-3ʹ; Gbp1
(also known as Gbp2b), 5ʹ-ATGGGCCACGTCTAAAAAGC-3ʹ and 5ʹ-TTTGCAC
TGCTGCTGAGTTC-3ʹ; Gbp6, 5ʹ-AATGCCTTGAAGCTGATCCC-3ʹ and 5ʹ-GTT
CTTTGTCATGCGTTGGC -3ʹ; Ifi44, 5ʹ-AGACAAGAGGCATTGCTGTG-3ʹ and
5ʹ-AGCAGAACTCGTGTTTGCTG-3ʹ; Ifi204, 5ʹ-AAGCAAGGGGGACATTT
GTG-3ʹ and 5ʹ-TGGTTCACACCTGACATTGG-3ʹ; Cxcl9, 5ʹ-CGAGGCACG
ATCCACTACAA-3ʹ and 5ʹ-CCGGATCTAGGCAGGTTTGA-3ʹ; Cxcl10,
5ʹ-TCCTGCCCACGTGTTGAGAT-3ʹ and 5ʹ-TGCGTGGCTTCACTCCAGTT-3ʹ;
Il6, 5ʹ-CTGCAAGAGACTTCCATCCAGTT-3ʹ and 5ʹ-AAGTAGGGAAGGCCGT
GGTT-3ʹ; Lcn2, 5ʹ-AATGTCACCTCCATCCTGGTC-3ʹ and 5ʹ-GTGGCCACTT
GCACATTGTA-3ʹ.
Listeria monocytogenes infection. SPF C57BL/6 mice (6–7 weeks old) from SLC
Japan were treated with AVMN via the drinking water, then reconstituted with
SPFfae on day −12. For the 11-mix treatment group, an initial oral administration
of the 11-mix was done in conjunction with SPFfae, followed by repetitive oral
gavage of the 11-mix alone on days −12, −11, −8 and −3. For the oral infection
group, mice were orally gavaged with approximately 5 × 109 CFU of Lm-WT
(strain EGD) or Lm-OVA, or 1 × 1011 CFU of Lm-InlAm on day 0. For quanti-
fication of the bacterial titre, colons, MLNs, spleens or livers were homogenized
in PBS, plated on CHROMagar Listeria (CHROMagar) for 2 days at 37 °C, and
colonies with white halos were counted at the indicated times after infection. For
systemic infection, Lm strain EGD (5 × 105 CFU) was injected intraperitoneally
on day 0 and the bacterial titre was quantified as described above. For the Lm-OVA
(Nanjing Sungyee Biotech)-specific IFNγ response by CD8 T cells, colonic lamina
propria lymphocytes and antigen-presenting cells were isolated and OVA-specific
IFNγ+ CD8 T cells were detected by stimulation with 5 μg ml−1 OVA-derived
peptide SIINFEKL (MBL) in the presence of GolgiStop (BD Biosciences) at 37 °C
for 5 h. For germ-free mice-based studies, C57BL/6 GF mice were colonized with
11- or 10-mix, or left untreated, on day −7. Then, the mice were orally infected
with 1 × 109 CFU of Lm-WT (strain EGD). For depletion of CD8 T cells, 200 μg
of CD8α monoclonal antibody (clone 2.43, BioXCell) was administered intra-
peritoneally 1 day before administration of 11-mix, and every 3–4 days thereafter.
Subcutaneous tumour model. The MC38 adenocarcinoma cell line was a gift
from the Surgery Branch, National Cancer Institute, National Institutes of Health
(Bethesda, MD, USA). BrafV600E Pten−/− melanoma cells were isolated and cultured
at Keio University from a tumour induced in BRafCA PtenloxP Tyr::CreERT2 mice27
(purchased from Jackson Laboratory) by treatment with 4-OH-tamoxifen on the
mouse’s clean-shaven back skin. SPF C57BL/6 mice (6–7 weeks old) from SLC
Japan were treated with AVMN via the drinking water (from day −7 to day 2),
and 3 × 105 MC38 adenocarcinoma cells or 7 × 105 BrafV600E Pten−/− melanoma
cells were subcutaneously implanted on day 0. The mice were reconstituted
with SPFfae on day 3. For the 11-mix or 10-mix treatment groups, the initial
© 2019 Springer Nature Limited. All rights reserved.
Article RESEARCH
dosing of 11-mix or 10-mix was done together with SPFfae on day 3, followed
by repetitive dosing of the 11-mix or 10-mix alone two or three times per week
until the end of the experiment. Mice were intraperitoneally injected with 200 μg
PD-1 monoclonal antibody (clone J43, BioXCell) or CTLA-4 monoclonal
antibody (clone 9H10, BioXCell) every 3 days from day 3–9. Tumour size was
measured two or three times per week and volume was determined as length ×
width2 × 0.5. Mice were euthanized either at the indicated time points or when
their tumours reached an endpoint volume of 2,000 mm3, and tumour weight
was measured. For germ-free mouse-based studies, C57BL/6 GF mice were
colonized with 11- or 10-mix, or left untreated, and were subjected to subcu-
taneous implantation of 3 × 105 MC38 adenocarcinoma cells on day 0. PD-1
monoclonal antibody injection and tumour measurement were performed as
described above in the SPF mouse-based study section. For depletion of CD8
T cells, 200 μg of CD8α monoclonal antibody (clone 2.43, BioXCell) was adminis-
tered intraperitoneally 1 day before administration of 11-mix, and every 3–4 days
thereafter. For experiments with donor C human microbiota-colonized mice,
C57BL/6 mice were orally inoculated with donor C human faecal aliquots (approx-
imately 1 mg per mouse) mixed with or without 11-mix or 10-mix on day −7, and
were subjected to subcutaneous implantation of 3 × 105 MC38 adenocarcinoma
cells on day 0 followed by repetitive dosing of 11-mix or 10-mix. PD-1 monoclonal
antibody injection and tumour measurement were performed as described above
in the SPF mouse-based study. For isolation of TILs and myeloid cells, tumours
were cut into small pieces and incubated in 10 ml of RPMI1640 containing 4% fetal
bovine serum, 0.5 mg ml−1 collagenase D (Roche), 0.5 mg ml−1 dispase (Gibco),
and 40 µg ml−1 DNase I (Roche) for 45 min at 37 °C in a shaking water bath. The
digested tumour cells were resuspended in 10 ml of HBSS containing 5 mM EDTA
and passed through a 100-μm cell strainer. Cell suspensions corresponding to 0.5 g
of the original tumour weight were used in 40–80% Percoll gradient separation as
described above. Lymphocytes and myeloid cells were collected from the interface
of the two layers, washed with RPMI1640 containing 10% FBS, and stimulated with
50 ng ml−1 PMA and 750 ng ml−1 ionomycin (both from Sigma) in the presence of
Golgistop (BD Biosciences) at 37 °C for 3.5 h. For the assessment of IFNγ response
against p15E peptide and bacterial lysate, Percoll-enriched cells, which include
T cells and antigen-presenting cells, were stimulated ex vivo with 5 μg ml−1 p15E
peptide KSPWFTTL (MBL) or bacterial lysate (1 × 106–2 × 106 CFU ml−1 of each
strain, prepared as described above) for 18 h, then GolgiStop was added and the
suspension was incubated for an additional 3.5 h. Staining and FACS analysis were
performed as described above.
Histological analysis. For fluorescence in situ hybridization (FISH) staining, colon
tissue was fixed with methanol-Carnoy’s solution and embedded in paraffin33. The
sections were treated with 0.1 M HCl and hybridized with the 5ʹ Alexa 488-labelled
EUB338 probe (5′-GCTGCCTCCCGTAGGAGT-3′) in hybridization buffer (0.9 M
NaCl, 0.02 M Tris-HCl, pH 7.6, 0.01% sodium dodecyl sulfate) at 50 °C overnight.
Slides were then incubated with FISH washing buffer (0.45 M NaCl, 0.02 M Tris-
HCl, pH 7.6, 0.01% sodium dodecyl sulfate) preheated to 50 °C for 10 min and
washed three times in PBS. For mucus visualization, sections were incubated with
blocking buffer (1% BSA, 2% FBS, 0.05% Tween20 in PBS) at room temperature for
60 min and stained with rabbit anti-MUC2 monoclonal antibody (1:1,000, Santa
Cruz Biotechnology) for 120 min, followed by Alexa 546-labelled goat anti-rabbit
IgG (1:500, Life Technologies) in blocking buffer for 60 min. All sections were
counterstained with 4,6-diamidino-2-phenylindole (DAPI, Dojindo, 1:5,000),
mounted with Fluoromount/Plus (Diagnostics BioSystems), and visualized using
a TCS SP5 confocal microscope.
To evaluate the development and severity of colitis, colons were fixed with 4%
paraformaldehyde, embedded in paraffin, sectioned, and stained with haematox-
ylin and eosin. The degree of colitis was assessed by grading each of the following
phenotypes on a scale of 0–4: inflammatory cell infiltration, mucosal thickening,
goblet cell depletion, crypt abscess formation, and destruction of architecture. The
final histological score was defined as the sum total of the individual parameter
scores.
Metagenome sequencing of faecal microbiota from healthy volunteers (A–F).
Faecal DNA from donors A–F was isolated as described above. Whole-genome
shotgun libraries were prepared using the TruSeq DNA PCR-Free Sample
Preparation kit (Illumina). 300 bp at each end of the libraries was sequenced on a
MiSeq instrument with the MiSeq Reagent kit v3 (600 cycles; Illumina).
Abundance profiling across metagenomic datasets. Draft genomes of the
11 strains were split into 1-kb regions and mapped against NCBI RefSeq (84,664
genomes) to identify regions with no or weak similarity to known isolates (iden-
tity < 80%, coverage < 85%). Of these, consecutive regions with minimum 5 kb
were defined as marker regions that are unique to each strain. A total of 3,327 gut
metagenome samples with at least 1 million quality controlled reads across various
datasets (HMP1-2, LLDeep, MetaHIT, 500FG, HMP2 (only the first time point was
used), healthy Japanese adults34, and three microbiome in cancer immunotherapy
studies29–31) were mapped to 1-kb regions in all strains (filtered by 95% mapping
 RESEARCH Article
identity). The mapped read counts were normalized to reads per kb per million
(RPKM). A strain was deemed detected if at least 95% of 1-kb regions in the marker
region were detected and the abundance was calculated as median abundance
across all marker regions in the genome. Species level abundance was calculated
as median abundance across all 1-kb regions in the genome.
Sample preparation for metabolome analysis. Metabolite extraction from serum
and caecal contents for metabolome analyses was performed as described previ-
ously35. In brief, frozen caecal contents together with internal standard compounds
(see below) were homogenized in ice-cold methanol (500 μl) using a manual
homogenizer (Finger Masher (AM79330), Sarstedt) followed by the addition of an
equal volume of chloroform and 0.4 times the volume of ultrapure water (LC/MS
grade, Wako). Forty microlitres of serum spiked with the internal standards were
added with ice-cold methanol (400 μl) followed by the addition of an equal vol-
ume of chloroform and 120 μl of ultrapure water. These suspensions were then
centrifuged at 15,000g for 15 min at 4 °C. After centrifugation, the aqueous phase
was ultrafiltered using an ultrafiltration tube (Ultrafree MC-PLHCC, Human
Metabolome Technologies). The filtrate was concentrated with a vacuum con-
centrator (SpeedVac, Thermo). The concentrated filtrate was dissolved in ultrapure
water and used for liquid chromatography–tandem mass spectrometry (LC–MS/
MS) and ion chromatography with mass spectrometry (IC–MS) analyses.
Qualification and quantification of metabolites by internal and external stand-
ards. We used both internal standards (added to the samples before extraction) and
external standard compounds to determine the m/z value and the specific retention
time for all metabolites examined. The detailed methods are as follows. Internal
standard compounds: we used 2-morpholinoethanesulfonic acid and l-methio-
nine sulfone as internal standards for anionic and cationic metabolites, respec-
tively. These compounds are not present in the samples analysed, and thus serve
as ideal standards. Loss of endogenous metabolites during sample preparation
was corrected by calculating the recovery rate (%) for each sample measurement.
External standard compounds: before sample analysis, we measured the mixture
of authentic compounds of target metabolites in ultrapure water to determine both
the m/z value and the retention time of all metabolites examined.
IC–MS/MS for anionic metabolites. Anionic metabolites were measured using
an orbitrap-type MS (Q-Exactive focus, Thermo Fisher Scientific), connected to
a high performance ion-chromatography system (ICS-5000+, Thermo Fisher
Scientific) that enables us to perform highly selective and sensitive metabolite
quantification owing to the ion chromatography separation and Fourier Transfer
MS principle36. The ion-chromatography system was equipped with an anion
electrolytic suppressor (Thermo Scientific Dionex AERS 500) to convert the
potassium hydroxide gradient into pure water before the sample enters the mass
spectrometer. The separation was performed using a Thermo Scientific Dionex
IonPac AS11-HC, 4-μm particle size column. The ion-chromatography flow rate
was 0.25 ml min−1 supplemented post-column with 0.18 ml min−1 makeup flow of
methanol. The potassium hydroxide gradient conditions for ion-chromatography
separation are as follows: from 1 mM to 100 mM (0–40 min), 100 mM
(40–50 min), and 1 mM (50.1–60 min), at a column temperature of 30 °C. The Q
Exactive focus mass spectrometer was operated under an ESI negative mode for
all detections. Full mass scan (m/z 70−900) was used at a resolution of 70,000. The
automatic gain control (AGC) target was set at 3 × 106 ions, and maximum ion
injection time was 100 ms. Source ionization parameters were optimized with the
spray voltage at 3 kV and other parameters were as follows: transfer temperature
was 320 °C, S-Lens level was 50, heater temperature was 300 °C, Sheath gas was
36, and Aux gas was 10.
LC–MS/MS for amino acid measurement. The amounts of various cationic
metabolites were quantified using LC–MS/MS37. In brief, a triple-quadrupole
mass spectrometer equipped with an electrospray ionization (ESI) ion source
(LCMS-8060, Shimadzu Corporation) was used in the positive and negative-ESI
© 2019 Springer Nature Limited. All rights reserved.
and multiple reaction monitoring (MRM) modes. The samples were resolved on
the Discovery HS F5-3 column (2.1 mm ID × 150 mm, 3-μm particle, Sigma-
Aldrich), using a step gradient with mobile phase A (0.1% formate) and mobile
phase B (0.1% acetonitrile) at ratios of 100:0 (0–5 min), 75:25 (5–11 min), 65:35
(11–15 min), 5:95 (15–20 min) and 100:0 (20–25 min), at a flow rate of 0.25 ml
min−1 and a column temperature of 40 °C.
Statistical analysis. Statistical analyses were performed using GraphPad Prism
software (GraphPad Software, Inc.) or MATLAB (for metabolomic analysis).
Specifically, the two-tailed unpaired Student’s t-test (parametric) was used for
all comparisons between two groups, except for CFU comparisons, which were
analysed using the Mann–Whitney test (nonparametric). One-way analysis of var-
iance (ANOVA) followed by Tukey’s post hoc test (parametric) was used for all
comparisons between three or more groups, except for CFU comparisons, which
were analysed using the Kruskall–Wallis test with Dunn’s multiple comparisons
(nonparametric). For time-course analysis of body weight, number of IFNγ+ CD8
T cells, and tumour volume, two-way ANOVA with Bonferroni’s test (comparisons
between two groups, parametric) or Tukey’s test (comparisons between three or
more groups, parametric) was used.
Reporting summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
Genomic sequences of the 21 strains are deposited in the European Nucleotide
Archive with accession number PRJEB29940. Microbiome data are deposited in
the DNA Data Bank of Japan with accession number DRA007611. Accession num-
bers for metagenomics datasets: NCBI BioProjects PRJNA275349 (HMP1-2)38,
PRJNA48479 (HMP1-2), PRJNA398089 (HMP2)39, PRJNA319574 (500FG)40,
PRJDB3601 (healthy Japanese adults)34 and PRJNA399742 (Gajewski)31. EBI study
accession EGAS00001001704 (LLDeep)41, PRJEB22894 (Wargo)30, PRJEB22863
(Zitvogel)29. EBI ENA ERP002061 (MetaHIT), ERP003612 (MetaHIT) and
ERP004605 (MetaHIT)42,43.
33. Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus
layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105,
15064–15069 (2008).
34. Nishijima, S. et al. The gut microbiome of healthy Japanese and its microbial
and functional uniqueness. DNA Res 23, 125–133 (2016).
35. Miyajima, M. et al. Metabolic shift induced by systemic activation of T cells in
PD-1-deficient mice perturbs brain monoamines and emotional behavior. Nat.
Immunol. 18, 1342–1352 (2017).
36. Hu, S. et al. Targeted metabolomic analysis of head and neck cancer cells using
high performance ion chromatography coupled with a Q exactive HF mass
spectrometer. Anal. Chem. 87, 6371–6379 (2015).
37. Oka, M. et al. Arl8b is required for lysosomal degradation of maternal proteins
in the visceral yolk sac endoderm of mouse embryos. J. Cell Sci. 130,
3568–3577 (2017).
38. Lloyd-Price, J. et al. Strains, functions and dynamics in the expanded Human
Microbiome Project. Nature 550, 61–66 (2017).
39. The Integrative HMP (iHMP) Research Network Consortium. The Integrative
Human Microbiome Project: dynamic analysis of microbiome-host omics
profiles during periods of human health and disease. Cell Host Microbe 16,
276–289 (2014).
40. Schirmer, M. et al. Linking the human gut microbiome to inflammatory cytokine
production capacity. Cell 167, 1897 (2016).
41. Zhernakova, A. et al. Population-based metagenomics analysis reveals
markers for gut microbiome composition and diversity. Science 352, 565–569
(2016).
42. Qin, J. et al. A human gut microbial gene catalogue established by
metagenomic sequencing. Nature 464, 59–65 (2010).
43. Li, J. et al. An integrated catalog of reference genes in the human gut
microbiome. Nat. Biotechnol. 32, 834–841 (2014).
 Article RESEARCH

a b
Gated on IFNγ+CD8 T cells
SPF Colon 200 200
SPF Colon
75.7 90
gated on: 150
60
150 gated on:
CD3+TCRβ+ cells 100
97.2
40
100
CD8 T cells
105 6.9 8.35 105 1.22 6.04
20 50
50

GrB(APC)
CD8α(APC)

4
10 4
10

0 0 0
0 102 103 104 105 102 103 104 105

# cells
0 10
3
10
4
10
5 0

CD8β CD44 T-bet

3
10
103

0 (PerCPCy5.5) (BV786) (BV421)

0

67.3 17.4 100 120

43.5 49.2
0 102 103 104 105

91.2
2 3 4 5 80
0 10 10 10 10

IFNγ 99.4
80
90 IFNγ
(PECy7) 60
60 48.3 (FITC)
60
40
40

30
20 20

0 0 0
0 102 103 104 105 0 103 104 105 0 103 104 105

ICOS CD103 KLRG1

(PE) (FITC) (PEDzl594)

c d e f
IFNγ+CD8 T Colon Colon Colon Colon
10 50 * 50 * 50 50
***
Cell number (x103)

% IFNγ+ in CD8 T

% IFNγ+ in CD8 T
% IFNγ+ in CD8 T

% IFNγ+ in CD8 T
*** *** ***
8 40 40 40 40 *** ***
***
6 30 30 30 30

4 20 20 20 20
n=4

10 10
n=4

2 10 10
n=5

n=5

n=3

n=3

n=4

n=4

n=4
n=4

n=4

n=3

n=3

n=4

n=4
0 0 0 0 0
AVMN

CLEA
Charles
SLC

CLEA
GF

0w
2w
6w
GF +SPF fae
SPF
GF
SPF
GF

Colon Small Charles

intestine SPF+ SPF co-housed with
CLEA
Extended Data Fig. 1 | Characterization of the intestinal IFNγ+ CD8 IFNγ+ cells among CD8 T cells was analysed. f, Left, percentage of IFNγ+
T cell population. a, b, Representative flow cytometry histograms and cells among CD8 T cells in the colonic lamina propria of SPF C57BL/6
plots showing the expression of CD8β, CD44, T-bet, ICOS, CD103, mice (10 weeks old), reared at the indicated institute or vendor. Right, SPF
KLRG1 and GrB by colonic lamina propria IFNγ+ CD8 T cells isolated C57BL/6 mice (8 weeks old) obtained from Charles River were cohoused
from SPF mice from two independent experiments. c, Number of IFNγ+ with C57BL/6 mice from CLEA for 0, 2 or 6 weeks, and the percentage of
CD8 T cells in the colonic or small intestinal lamina propria of germ-free IFNγ+ cells among colonic lamina propria CD8 T cells was analysed. Each
and SPF mice. d, Percentage of IFNγ+ cells among CD8 T cells in the colon circle represents an individual animal, and the number of mice in each
of SPF mice (between 8 and 12 weeks old) treated with or without AVMN group is shown. Data are mean and s.d. ***P < 0.001; *P < 0.05; one-way
(ampicillin, vancomycin, metronidazole and neomycin) via the drinking ANOVA with Tukey’s test (c, f) or two-tailed unpaired t-test (d, e). See
water for 4 weeks. e, Faeces from SPF mice (SPFfae) were orally inoculated Source Data for exact P values.
into germ-free mice (7 weeks old), and 4 weeks later the percentage of

© 2019 Springer Nature Limited. All rights reserved.

RESEARCH Article
Extended Data Fig. 2 | Bacteria from the healthy human gut microbiota
associated with IFNγ+ CD8 T cell induction. a, Schematic representation
of the strategy for isolating IFNγ+ CD8 T cell-inducing bacteria from the
healthy human gut microbiota. b, Representative flow cytometry plots
showing the expression of IFNγ and CD8α by colonic or small intestinal
lamina propria CD3+TCRβ+ T cells from germ-free mice orally inoculated
with healthy human faecal samples (from donors A to F). c, Spearman’s
correlation coefficient and P values for the relationship between the
number of individual bacterial 228 OTUs (among 3,000 reads) detected
© 2019 Springer Nature Limited. All rights reserved.
in mice shown in Fig. 1e and the percentage of colonic IFNγ+ CD8 T cell
population shown in Fig. 1d was calculated using GraphPad Prism. OTUs
positively and negatively correlated with the frequency of colonic IFNγ+
CD8 T cells with statistical significance (P < 0.05) are highlighted in
red and dark blue, respectively. OTUs detected in GF+CHL B5 mice are
marked in light and dark blue. OTUs showing no significant correlation
(P > 0.05) are marked in grey. The closest species/strain and individual
Rho (ρ) value for each OTU are shown.
 Article RESEARCH

a b
gated on:
60 CD3+TCRβ+ cells gated on: IFNγ+CD8 T cells
% IFNγ+ of CD8 T

***
** 10
5
11.9 1.84 20
20 52.9 36.7 5.14 25
25
8

40 22.3 20 96.9
15

91.8
20
4 15
10 15
6

76.2

GF
* 15 15
n=4

10
10 10

79.3
n=5 10
3 4
10

20
10
n=3

5
5 5

CD8α(APC)
2
0 5 5

77.8 8.47
n=4

n=4
n=6

# cells
0 0 0 0 0 0
2 3 4 5
2 3 4 5 0 10 10 10 10 2 3 4 5 0 103 104 105

0
3 4 5 0 10 10 10 10 0 10 10 10 10 2 3 4 5 3 4 5
0 10 10 10 0 10 10 10 10 0 10 10 10

10 5.69
150 60

26.4 62.2 7.01

5 100
GF+11mix
GF+11mix

GF+11mix

10 60 300
GF
GF

GF

GF+11mix
58.6 96.7
120

98.5
80
104
100 40
40
85
200 90
60

103

20
40
50
95.2 20
60

100

0 20 30

B6 IQI BALB/c 72.6

3 4
11.7
5
0 0 0 0 0 0
0 10 10 10 102 103 104 105 102 103 104 105 3 4 5

IFNγ(PECy7)
0 0 103 104 105 0 10 10 10
0 102 103 104 105 0 10
2
10
3
10
4
10
5 0

CD103 PD-1 CD44 Tbet ICOS KLRG1

(BV421) (BV510) (PE) (BV785) (PEDzl594) (BV711)

c gated on: CD8 T cells

GF GF+11mix
d e GF GF+11mix SPF
10
5
1.95 0.456 10
5
1.7 2.23

Il6 Lcn2

GrB(APC)
104 104
10 10
Relative expression

3 3
10 10

8 8
n=3
n=4
n=4

n=3
n=4
n=4

102 10
2

6 6 0 0

79.7 17.9 55.6 40.5

4 ns 4 ns 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5

IFNγ(FITC)
2 2
0 0
GF+11mix

GF+11mix
SPF

SPF
GF

GF

i IFNγ+CD4- T
TH17 TH1 (mostly CD8T)

of CD3+TCRβ+CD4- T
8 *** 60 *** 20
f g h 80 ***

% IFNγ+ of CD4+ T
% IL17+ of CD4+ T
*** *** *** ***
20
%IFNγ+ of CD8 T

6
Histological score

GF GF+11mix (4 weeks) GF+11mix (6 months) *** ** 40 *** 15

15 60 ***

n = 14
4 10

n = 10

n=3
n=3
n=3
n=3

10 40

n=6
20

% IFNγ+
2 5
20

n=4
5

n=8
n=4
n=3
ns

n=7
n=9
n=4
n=3
n=3
n=3
n=3

0 0 0 0
0 GF
GF+11mix
GF+Kp2H7
GF+ TH17 20mix

GF
GF+11mix
GF+Kp2H7
GF+ TH17 20mix

GF
GF+11mix
GF+Kp2H7
GF+ TH17 20mix
GF+11mix (6m)
GF+11mix (4w)
GF
GF+11mix (6m)
GF+11mix (4w)
GF

Extended Data Fig. 3 | Induction of IFNγ+CD8 T cells by the 11-mix photomicrographs (f), histology score (g), and percentage of IFNγ+ cells
via non-inflammatory immunomodulation. a, Percentage of IFNγ+ among CD8 T cells in the colonic lamina propria (h) of germ-free and
cells among colonic CD8 T cells from mice of the indicated genetic GF+11-mix mice (4 weeks or 6 months after colonization). Scale bar,
background, colonized with or without the 11-mix. b, c, Representative 50 μm. i, Percentages of IL-17A+ (TH17) and IFNγ+ (TH1) cells among
flow cytometry histograms and plots showing the expression of CD103, CD3+TCRβ+CD4 T cells and IFNγ+ cells among CD3+TCRβ+CD4−
PD-1, CD44, T-bet, ICOS, KLRG1 and GrB by colonic IFNγ+ CD8 T cells T cells (representing primarily CD8 T cells) in the colons of gnotobiotic
from germ-free mice colonized with or without the 11-mix for 4 weeks, mice colonized with the 11 strains, a reported TH1 cell-inducing Klebsiella
from two independent experiments. d, Expression of the indicated pro- pneumoniae strain (Kp2H7), or 20 reported TH17 cell-inducing strains
inflammatory genes in colonic epithelial cells from germ-free, GF+11-mix (TH17 20-mix). Each circle represents an individual animal. The number
(4 weeks after colonization), and SPF mice, as determined by qPCR. of mice in each group is shown. Data are mean and s.d. ***P < 0.001;
e, Representative photographs of caecums and colons from germ-free, **P < 0.01; *P < 0.05; two-tailed unpaired t-test (a) or one-way ANOVA
GF+11-mix (4 weeks after colonization) and SPF mice from several with Tukey’s test (d, g, h, i) was used. See Source Data for exact P values.
independent experiments. f–h, Haematoxylin and eosin-stained colon

© 2019 Springer Nature Limited. All rights reserved.

RESEARCH Article

Extended Data Fig. 4 | See next page for caption.

© 2019 Springer Nature Limited. All rights reserved.


Extended Data Fig. 4 | Characterization of the 11 strains. a, DNA
was extracted from each of the 26 strains and 16S rRNA gene sequences
were determined by PCR and Sanger sequencing. Genome sequencing
was conducted for 21 strains using the Illumina MiSeq or the MinION
nanopore sequencer. To identify the closest reference species or strain,
16S rRNA gene and genes encoding 42 ribosomal proteins predicted from
the assembled draft genome of each strain were blasted to the RDP and
NCBI genome databases (RefSeq genome representative), respectively.
Top-hit strains were defined as those with the highest 16S rRNA sequence
similarity or those with the highest ribosomal gene sequence similarity for
the maximum percentage of the 42 queried genes (strains for which this is
less than 37 out of 42 are listed in parentheses). Percentage similarity refers
to average sequence similarity between ribosomal genes of the isolated
strain and those of the top-hit reference strain. b, A phylogenetic tree was
constructed by comparing 42 concatenated ribosomal gene sequences
of each isolate using the MEGAv7.0 package and the neighbour-joining
method with a bootstrap of 1,000 replicates. c, Relative expression of
© 2019 Springer Nature Limited. All rights reserved.
Article RESEARCH
the indicated genes in colonic epithelial cells (ECs) of germ-free mice
colonized with or without the 11-mix for 1 week, as determined by qPCR.
d, Frequencies of TCR Vβ gene usage among IFNγ+ CD8 T cells (left) and
IFNγ− CD8 T cells (right) from the colons of GF (grey) or GF+11-mix
(red) mice, as determined by flow cytometry. 2-way ANOVA interaction
p-values: 0.0001 (IFNγ+ subset), 0.31 (IFNγ− subset). e, Luminal contents
from the indicated anatomical positions of the gut as well as faecal samples
were collected from three GF+11-mix mice 3-4 weeks post-colonization.
The relative abundance of each of the 11 strains’ DNA was determined
by qPCR. f, MLNs were collected from GF+11-mix mice 1 week after
colonization and bacterial DNA was extracted. The relative abundance
of each of the 11 strains’ DNA was determined by qPCR. Each circle
represents an individual animal (c, e) or a pool of mice (d), and the height
of each bar indicates the mean, and the number of mice in each group
is shown. n.d., not detected. Error bars, s.d. ***P < 0.001; **P < 0.01;
*P < 0.05; two-tailed unpaired t-test. See Source Data for exact P values.
 RESEARCH Article

a b
IFNγ-CD8 T cells
25 25
IFNγ+CD8 T cells
gated on: gated on: IFNγ+CD8 T cells
CD3+TCRβ+CD8α+ cells 150

90.6 7.17 0.385 50

20 20
GF iLN (n = 4) GF iLN(n = 4)
800 150

14.6 120
14.7 96.2
150
98.7

%TCRVβ+
40
GF mice

GF+11mix iLN GF+11mix iLN

%TCRVβ+
600 100

59.5
90 100
iLN
30

15 (n = 4) 15 (n = 4)
100

400
60
20
50
50 50
200 30 10

0 0 0 0 0 0
10 10
0 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 10
3
10
4
10
5
0 103 104 105
600

97.5 1.96 0.168

2000
200

1500
22.1 400
14.7 600
95.3
600
99.2 5 5
iLN

150
# cells

400
300

400
65.2 400
GF+11mix mice

1000

0 0
100
200

200
200 200

Vβ 2
Vβ 3
Vβ 4
Vβ 5.1&5.2
Vβ 6
Vβ 7
Vβ 8.1&8.2
Vβ 8.3
Vβ 9
Vβ 10b
Vβ 11
Vβ 12
Vβ 13
Vβ 14
Vβ 17a

Vβ 2
Vβ 3
Vβ 4
Vβ 5.1&5.2
Vβ 6
Vβ 7
Vβ 8.1&8.2
Vβ 8.3
Vβ 9
Vβ 10b
Vβ 11
Vβ 12
Vβ 13
Vβ 14
Vβ 17a
500 50
100

0 0 0 0 0 0
0 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 103 104 105 0 103 104 105
150 60

26.4 62.2 7.01

60 100
100

40.2 58.6 80
96.7
120
98.5
80
Colon

100 40
40
60
85 90

IFNγ-CD8 T cells
60

IFNγ+CD8 T cells
40 60

25 25
40
20 50 20

20
20 30
*
0
0
2
10 10
3
10
4
10
5
0
0 102 103 104 105
0
0 102 103 104 105
0
0 10
2
10
3
10
4
10
5
0
0 103 104 105
0
0 103 104 105
20
GF+11mix colon 20
GF+11mix colon (n = 3)
IFNγ(PECy7) CD103(BV421) PD-1(BV510) CD44(PE) ICOS(PEDzl594) KLRG1(BV711) (n = 3) GF+11mix iLN (n = 4)
GF+11mix iLN

%TCRVβ+

%TCRVβ+
15 * (n = 4) 15

10 10

5 5

0 0

Vβ 2
Vβ 3
Vβ 4
Vβ 5.1&5.2
Vβ 6
Vβ 7
Vβ 8.1&8.2
Vβ 8.3
Vβ 9
Vβ 10b
Vβ 11
Vβ 12
Vβ 13
Vβ 14
Vβ 17a

Vβ 2
Vβ 3
Vβ 4
Vβ 5.1&5.2
Vβ 6
Vβ 7
Vβ 8.1&8.2
Vβ 8.3
Vβ 9
Vβ 10b
Vβ 11
Vβ 12
Vβ 13
Vβ 14
Vβ 17a
c

12 15

10
10
8

GF (n = 4) 6 GF (n = 3)
11mix (n = 4)
5
11mix (1w) (n = 3)
PC3 (11.2071%)

PC3 (8.7805%)
4

7mix (n = 4) 2 0 11mix (4w) (n = 3)

4mix (n = 4) 0 10mix (1w) (n = 4)
SPF (n = 2) -2
-5
10mix (4w) (n = 4)
-4
-10
-6

-8 -15 20
10 20 20 10
5 10 10
0 0 0
-5 -10 0 -10
-10 -20 -10 -20
PC2 (18.1795%) PC1 (30.2944%) PC2 (25.0772%) PC1 (30.5669%)

d e f
Caecal contents Caecal contents Sera
11mix

11mix 10mix 11mix 10mix

7mix
4mix
SPF
GF

GF 1w 4w 1w 4w GF 1w 4w 1w 4w
Mevalonate Mevalonate Mevalonate
Dimethylglycine Dimethylglycine Dimethylglycine
Hypoxanthine Hypoxanthine Hypoxanthine
IMP IMP IMP
2-Deoxy-glucose 6P 2-Deoxy-glucose 6P 2-Deoxy-glucose 6P
TMP TMP TMP
6-Phosphogluconate 6-Phosphogluconate 6-Phosphogluconate
Rib5P+Ru5P+Xu5P Rib5P+Ru5P+Xu5P Rib5P+Ru5P+Xu5P
GDP GDP GDP
GlcNAc 6P GlcNAc 6P GlcNAc 6P
UMP UMP UMP
GMP GMP GMP
CDP CDP CDP
Nicotinic acid mononucleotide Pyridoxine
n=3

n=3

n=3

n=4

n=4

Galactose-1P Ophthalmic acid

5''-Adenylyl sulfate CTP
dCTP GTP
4-Aminobutyric acid ATP
4-Hydroxyproline F6P
Adenylsuccinic acid 5-Methyltetrahydrofolic acid Z-score
Carbamoyl aspartic acid Adenylosuccinate
1.5
Niacin B3 G6P
Nicotinic acid 3-Hydroxyanthranilic acid 1
UDP Glycerol 3P 0.5
Picolinic acid Sedoheptulose 7P
TDP 0
2-Keto-4-methylthiobutyate
Pyridoxal-5''-P dUMP -0.5
2-Aminobutyric acid Histamine -1
Riboflavin-5''-P Valeric acid
UDP-GlcNAc 3PG + 2PG -1.5
GlcNAc 1P G1P
AMP Thymidine
3-Methylthiopropionate CMP
n=4

n=4

n=4
n=4

n=2

Dopamine
Phosphoenolpyruvate
HPO4
Allatoin
Acetylcarnitine
Thymine
n=3

n=3

n=3
n=4

n=4

Extended Data Fig. 5 | See next page for caption.

© 2019 Springer Nature Limited. All rights reserved.


Extended Data Fig. 5 | Systemic effects of 11 strain colonization.
a, Representative flow cytometry histograms showing the expression of
CD103, PD-1, CD44, ICOS and KLRG1 by inguinal lymph node (iLN) or
colonic IFNγ+ CD8 T cells from the indicated mice from two independent
experiments. b, Frequencies of TCR Vβ gene usage among IFNγ+ CD8 T
cells (left) and IFNγ− CD8 T cells (right) from the inguinal lymph nodes
of germ-free (grey) or GF+11-mix (red) mice, and those from the colons
of GF+11-mix mice (blue). c, Principal component analysis plots of the
water-soluble caecal metabolome from gnotobiotic mice colonized with
the indicated bacterial mixtures. d–f, Heat maps depicting differentially
elevated metabolites in the caecum (d, e) or serum (f) of gnotobiotic
mice colonized with the indicated bacterial mixtures. Raw metabolomic
data was screened for metabolites specifically increased in GF+11-mix
mice only (d, e) or in GF+11-mix and GF+4-mix mice only (d) with
at least fourfold enrichment as compared to GF controls and at least
twofold enrichment compared to the other gnotobiotic groups, with a
cut-off P value of 0.05. Overlapping metabolites between the two datasets
were selected (highlighted in green), and their levels in the serum were
© 2019 Springer Nature Limited. All rights reserved.
Article RESEARCH
queried (f). The most promising candidate effector molecules for the local
and systemic effects observed in GF+11-mix mice were identified by
considering the metabolomic data in light of the phenotypic
immunomodulatory data, and are highlighted in red font (d–f). For
a more detailed discussion of how these metabolites were chosen,
see Supplementary Discussion. The area under each individual metabolite
peak was normalized by dividing by the area under the peak of the
corresponding internal standard. Heat map colours represent the z-score
(red and blue indicate high and low abundance, respectively). Caecal
contents were isolated 4 weeks after gavage unless otherwise noted (1w,
1 week; 4w, 4 weeks). 2PG, 2-phospho-d-glycerate; 3PG, 3-phospho-d-
glycerate; GlcNAc 6P, N-acetyl-d-glucosamine 6-phosphate; GlcNAc 1P,
N-acetyl-d-glucosamine 1-phosphate; Rib5P, ribose-5-phosphate; Ru5P,
ribulose-5-phosphate; Xu5P, xylulose-5-phosphate. Each circle represents
an individual mouse (b, iLN, c), a pool of mice (b, colon). The number of
mice in each group is shown. Data are mean and s.d. *P < 0.05; two-tailed
unpaired t-test. See Source Data for exact P values.
 RESEARCH Article

a b
Colon
GF+11mix+αCD8
GF mice GF+Lm-WT GF+10mix+Lm-WT
104 ** c GF+11mix+Lm-WT +Lm-WT

Lm CFU / mg organ
GF *
103
GF
+11mix
or 10mix 102

GF 101

n=6
n=7
n=8
+11mix+αCD8
-7 0 Day
100

GF
GF+10mix
GF+11mix
11mix or 10mix (gavage)
Lm-WT Infection (oral)
αCD8 (ip) @day -8, -4, -1 and 2

f Lm-InlAm gavage
d e (n = 8 for each group)
SPF Liver Spleen
mice
ns
AVMN SPF+11mix SPF+11mix+αCD8 ns
SPF 107 * ** 106
SPF+Lm-InlAm +Lm-InlAm +Lm-InlAm ** **

Lm CFU / mg organ
Lm CFU / mg organ
106 10 5
SPF+11mix AVMN
105 104
SPF+11mix 104
AVMN 103
+αCD8 103
-18 -13 -12 -11 -8 -3 0 Day 102
102
SPFfae (gavage) Lm-WT, -InlAm or -OVA Infection 101 101
(oral or ip) 100
100
αCD8 (ip)

SPF
SPF+11mix
SPF+11mix
+αCD8

SPF

+αCD8
SPF+11mix
SPF+11mix
11mix (gavage)
@day-13, -10, -7, -4, -1 and 2

h k Day 1, 3, 5, 7
Day 0, 1, 3
SPF n = 3, 4, 3 SPF+11mix+Lm-OVA n = 4, 4, 5, 5

g SPF+11mix n = 3, 4, 3 SPF+Lm-OVA n = 4, 4, 5, 5 l Lm-WT i.p.

Liver Spleen
12
Number of IFNγ+CD8 T (x103)

Number of IFNγ+CD8 T (x103)

8 OVA peptide 25 PMA+Ionomycin
** 104 * 105 **
Histological score

stimulation stimulation

Lm CFU / mg organ

Lm CFU / mg organ
n = 11

n = 11
20 103
8 6 104
15 102 103
** 4
4 10 101 102
2
5 100 101
**

n = 10

n = 10
0
0 1 3 10-1
Days post Lm-WT
0 0 100

SPF

SPF
SPF
+11mix

SPF
+11mix
1 3 5 7 1 3 5 7
gavage
i j 6 OVA peptide 20 PMA+Ionomycin
SPF+11mix (n = 11) stimulation stimulation
Ki67+IFNγ+CD8 T (x103)

Ki67+IFNγ+CD8 T (x103)

SPF (n = 8)
SPF+11mix (n = 8) SPF+11mix+αCD8 (n = 10)
15
4
115 115
% initial body weight

% initial body weight

110 10
110
Number of

Number of

105 2
**

5
100 105
**

95
90
85
80
0 1 2 3 4 5
Days post Lm-WT gavage Days post Lm-WT gavage
Extended Data Fig. 6 | 11-mix-mediated enhancement of host
protection against Listeria monocytogenes infection. a, Experimental
design for germ-free mouse-based studies in b, c (and Fig. 3a–d),
C57BL/6 germ-free mice were colonized with 11- or 10-mix, or were left
uncolonized as a control. The mice were then orally infected with Lm-WT.
b, Listeria CFU in the colon on day 3 after infection. c, Representative
H&E staining on day 3 after infection. Scale bar, 50 μm. d, Experimental
design for SPF mouse-based studies in e–l (and Fig. 3e–g). C57BL/6 SPF
mice were treated with AVMN, then reconstituted with SPFfae by oral
gavage. In the 11-mix treatment group (+11mix), initial administration
of the 11-mix was done simultaneously with SPFfae, followed by repetitive
oral gavage of the 11-mix alone at the indicated time points. Mice were
then orally (e–k) or intraperitoneally (l) treated with Lm-InlAm (e, f),
Lm-WT (g–j, l) or Lm-OVA (k). e, g, Representative H&E staining on days
5 (e) and 3 (g) after infection. Scale bar, 50 μm. f, l, Listeria CFU in the
indicated tissues on day 5 (f) or 3 (l) after infection. h, Colonic histology
100 0 0
1 3 5 7 1 3 5 7
95 Days post Lm-OVA gavage
90
0 1 2 3 4 5 6
score at the indicated time points after Lm-WT infection. i, j, Percentage
weight change over the course of Lm-WT infection. k, Number of OVA
peptide SIINFEKL-specific or general (determined by PMA+ionomycin
stimulation) colonic IFNγ+ CD8 T cells induced after Lm-OVA infection,
as enumerated by flow cytometry at the indicated time points. The mean
of each group is represented by a line. Note that the expansion of general
IFNγ+ CD8 T cells occurred as early as 3 days after infection, sooner than
that of antigen-specific IFNγ+ CD8 T cells. Therefore, the 11-mix seems
to elicit both general and antigen-specific induction. To deplete CD8 T
cells, anti-CD8α antibody was administered intraperitoneally 1 day before
the administration of 11-mix, and every 3–4 days thereafter. Each circle
represents an individual animal. The number of mice in each group is
shown. Data are mean and s.d. **P < 0.01; *P < 0.05; Kruskal–Wallis with
Dunn’s test (b, f), two-tailed unpaired t-test (h), two-way ANOVA with
Bonferroni’s test (j, k), or two-tailed Mann–Whitney test (l). See Source
Data for exact P values.

© 2019 Springer Nature Limited. All rights reserved.

 Article RESEARCH

a GF mice tumour

GF
GF+αPD-1 11 or 10 mix (gavage)
GF+11mix or 10mix MC38 (sc) αPD-1 (ip)

GF+11mix or 10mix
+αPD-1 Day-7 0 3 6 9

b
gated on:
CD3+TCRβ+CD8α+ cells gated on: IFNγ+CD8 T cells
100 30
100
38.2 60
100 3.56 7.26 86.9 60 99.7
12.9 80
99.7
+αPD-1

80 80
20
60 70.7
GF

TIL

40 40
60 60

40
40 40
10
20 20

20 20 20

0 0 0 0 0 0
3 4 5 2 3 4 5
0 102 103 104 105 0 10 10 10 0 10 10 10 10 0 10
2
10
3
10
4
10
5
0 10
3
10
4
10
5
0 103 104 105

250
150
80
85.5 6.56 7.24 84 200 80

99.2
GF+11mix

10.1 100
200
150
+αPD-1

60 150 60
# cells

100
150
87.8
TIL

100
40 100 40
100
50

20 50 50 20
50

0 0 0 0 0 0
2 3 4 5
0 10 10 10 10 0 10
3
10
4
10
5
0 102 103 104 105 0 102 103 104 105 0 103 104 105 0 103 104 105

150 60

26.4 62.2 7.01

100
100 60
99.0
40.2
GF+11mix

120

80
60.6 80
96.7
100 40
Colon

60
40
60 85 90

60
40
40
50 20
20

20 30
20

0 0 0 0 0 0
2 3 4 5 3 4 5 3 4 5
0 10 10 10 10 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 10 10 10 0 10 10 10

IFNγ(PECy7) CD103(BV421) PD-1(BV510) CD44(PE) ICOS(PEDzl594) KLRG1(BV711)

c IFNγ+CD8 T cells IFNγ+CD8 T cells

30 GF+αPD-1 TIL (n = 4)
30
GF+11mix colon(n = 3)
GF+11mix+αPD-1 TIL GF+11mix+αPD-1 TIL (n = 3) *
(n = 3) **
%TCRVβ+
+

20 20
%TCRVβ

10 10
*

0 0
Vβ 2
Vβ 3
Vβ 4
Vβ 5.1&5.2
Vβ 6
Vβ 7
Vβ 8.1&8.2
Vβ 8.3
Vβ 9
Vβ 10b
Vβ 11
Vβ 12
Vβ 13
Vβ 14
Vβ 17a

Vβ 2
Vβ 3
Vβ 4
Vβ 5.1&5.2
Vβ 6
Vβ 7
Vβ 8.1&8.2
Vβ 8.3
Vβ 9
Vβ 10b
Vβ 11
Vβ 12
Vβ 13
Vβ 14
Vβ 17a

Extended Data Fig. 7 | Characterization of IFNγ+CD8 TILs induced c, Left, Frequencies of TCR Vβ gene usage among IFNγ+ CD8 TILs from
by the 11-mix. a, Experimental design for germ-free mouse-based studies GF+MC38+anti-PD-1 (grey) or GF+11-mix+MC38+anti-PD-1 (red)
in b and c (and Fig. 4a–c). C57BL/6 germ-free mice were colonized mice. Right, Frequencies of TCR Vβ gene usage among IFNγ+ CD8 T cells
with 11- or 10-mix on day −7, or left uncolonized, and were subjected from the colons of GF+11-mix mice (blue) or the tumours of GF+11-
to subcutaneous implantation of MC38 adenocarcinoma cells on day 0. mix+MC38+anti-PD-1 (red). Each circle represents an individual mouse
Anti-PD-1 monoclonal antibody was injected intraperitoneally every third (TIL) or a pool of mice (colon). The number of mice in each group is
day between days 3 and 9. b, Representative flow cytometry histograms shown. Data are mean and s.d. **P < 0.01; *P < 0.05; two-tailed unpaired
showing the expression of CD103, PD-1, ICOS, CD44 and KLRG1 by t-test. See Source Data for exact values.
colonic lamina propria or TIL IFNγ+ CD8 T cells from the indicated mice.

© 2019 Springer Nature Limited. All rights reserved.

RESEARCH Article

a b c SPF SPF+11mix
AVMN
SPF
SPF mice tumour

SPF SPF+αPD-1
SPF+ICI
SPF+11mix
SPF+11 or 10mix
SPF+11mix+αPD-1
SPF+11 or 10mix SPF+αPD-1 SPF+11mix+αPD-1
+ICI SPF+10mix
Day-7 0 2 3 6 9 11
SPFfae (gavage) MC38 or BRAFV600EPTEN-/- (sc) SPF+10mix+αPD-1
11 or 10mix (gavage) ICI (αPD-1 or αCTLA-4) (ip)

d e GF+C (n = 5) GF+C+11mix (n = 5) GF+C+10mix (n = 5) SPF+αCTLA-4 SPF+11mix+αCTLA-4

GF mice
104 81A6 104 81B11 104 81C1
GF
102 102 102
0 0
GF+C 10 10 100
10-2 10-2 10-2
GF+C
+11mix 10-4 10-4 10-4
or 10mix Day0 7 14 21 104 104 104 Il10-/- (SPF)
81E7 81H9 82A6 20

n=6
Histological score
2 2 2
Donor C faeces (gavage) 10 10 10
15
100 100 100
11 or 10mix (gavage) PBS (gavage)
µg DNA / g faeces

-2 -2 -2 10
10 10 10

n=8
10-4 10-4 10-4

n=8
n=8

n=8
n=4
n=5
f Colon iLN 5

40 *** 40 104 82B1 104 82F11 104 82G1

*** *** *** 0
** *** 102 102 102
% IFNγ+ of CD8 T

SPF
SPF+11mix
SPF+αPD-1
SPF+11mix+αPD-1
SPF+αCTLA-4
SPF+11mix+αCTLA-4
Il10-/- (SPF)
% IFNγ+ of CD8 T

30 30 10 0
10 0
10 0

10-2 10-2 10-2

n=3

20
n=3

20 10-4 10-4 10-4

Eubacteria (all bacteria)
104 82G5 104 82G9 105
10 10 3
10 2
10 2 10
n=5

n=5

n=5

n=5

n=5

n=5

100 100 101

0 0
10-1
GF
+C
+C+11mix
+C+10mix

GF
+C
+C+11mix
+C+10mix

10-2 10-2
10-3
10-4 10-4
0 3 7 14 20 0 3 7 14 20 0 3 7 14 20
Days post inoculation

g h GF(n = 4) GF+αPD-1(n = 4)
GF+C+10mix+αPD-1(n = 6)
Tumour volume (x103 mm3)

GF mice tumour 1.5

GF+C+αPD-1(n = 7)
GF GF+C+11mix
+αPD-1(n = 7)
GF+αPD-1 1.0
*
*
ns ns

***

GF+C+αPD-1
***

0.5
***

GF+C+11mix or 10mix
***

+αPD-1
Day-7
Donor C faeces (gavage) MC38 (sc)
11 or 10mix (gavage)
Extended Data Fig. 8 | Efficacy of 11-mix in enhancing treatment of
MC38 tumours in the context of a complex microbiota. a, Experimental
design for SPF mouse-based studies in b and c (and in Fig. 4d–k). SPF
mice were subjected to treatment with AVMN (from day −7 to day 2) and
subcutaneous implantation of MC38 adenocarcinoma or BrafV600E Pten−/−
melanoma cells on day 0. The mice were reconstituted with SPFfae on day
3. For the 11- or 10-mix treatment groups, the initial oral administration
was done simultaneously with SPFfae on day 3, followed by repetitive
dosing of the 11- or 10-mix alone, two or three times per week until
the end of the experiment. An anti-PD-1 or anti-CTLA-4 antibody was
injected intraperitoneally every third day between days 3 and 9.
b, Representative photograph of excised MC38 tumours on day 23. Scale
bar, 10 mm. c, H&E staining, along with histology score, of the colon on
day 27 (SPF, SPF+anti-PD-1, SPF+11mix, and SPF+11mix+anti-PD-1
groups) and on day 44 (SPF+anti-CTLA-4 and SPF+11mix+anti-CTLA-4
groups). Scale bar, 50 μm. For comparison, the colonic histology score
of SPF Il10−/− (colitis-prone) mice is shown. d, Experimental design for
GF+donor C human microbiota-based studies in e and f. C57BL/6 germ-
free mice were colonized with donor C human faecal microbiota or left
uncolonized on day 0. For the 11- or 10-mix treatment groups, the initial
0 3 6 9
0
0 5 10 15 20
αPD-1 (ip) Days post MC38 injection
oral administration was done simultaneously with the donor C human
faecal sample on day 0, followed by repetitive dosing of the 11- or 10-mix
alone two or three times per week until the end of the experiment.
e, Faeces were collected at the indicated time points. The relative
abundance of each of the 11 strains’ DNA was determined by qPCR.
Colonization with all 11 strains was confirmed. f, Percentage of IFNγ+
CD8 T cells in the colonic lamina propria and iLNs at day 21 was
enumerated by flow cytometry. g, h, C57BL/6 germ-free mice were
inoculated with donor C faecal samples with or without 11- or 10-
mix, following the same protocol as in d. Mice were then subjected
to subcutaneous implantation of MC38 cells on day 0. Anti-PD-1
was injected intraperitoneally every third day between days 3 and 9.
Experimental design is shown in g and tumour growth data of MC38 is
shown in h. Each circle represents an individual animal, except in
h, where each circle represents the mean. The number of mice in each
group is shown. Red, grey and brown asterisks show significance versus
the C+11-mix+anti-PD-1, C+anti-PD-1, and C+10-mix+anti-PD-1
groups, respectively. Data are mean and s.e.m. (e) or s.d. (all others).
***P < 0.001; **P < 0.01; *P < 0.05; one-way (f) or two-way (h) ANOVA
with Tukey’s test. See Source Data for exact P values.

© 2019 Springer Nature Limited. All rights reserved.


Extended Data Fig. 9 | Strain- and species-level abundance of the 11
isolates in the human gut microbiome. A total of 3,327 gut metagenome
samples with at least 1 million quality-controlled reads across various
data sets (HMP1-2, LLDeep, MetaHIT, 500FG, HMP2 (only the first
time point was used), healthy Japanese adults, and three microbiome in
cancer immunotherapy studies) were mapped to 1-kb regions in the 11
strains (filtered by 95% mapping identity). The mapped read counts were
normalized to RPKM. a, Detection and abundance of the strain-specific
marker regions. Median abundance across all marker regions was plotted
against marker gene coverage. Points with high abundance at low coverage,
for example, less than 75%, indicate limited marker region resolution and
likely represent other strains of a species. b, Among the 11 strains, 4 were
detected in 16 out of 3,327 microbiome samples evaluated. A strain was
© 2019 Springer Nature Limited. All rights reserved.
Article RESEARCH
deemed detected if at least 95% of 1-kb regions in the marker region were
detected. c, Abundance at the species level was calculated as the median
abundance across all 1-kb regions in a genome using 3,327 metagenome
samples with at least 1 million quality-controlled reads (mapped reads
were filtered at 95% mapping identity). The mapped read counts were
normalized to reads per RPKM. d, Abundance of the strain-specific
marker regions in the faecal microbiome of the six healthy Japanese
volunteers (donors A to F) was examined, as shown in a. A strain was
deemed detected if at least 95% of the 1-kb regions in the marker region
were detected. At our sequencing depth, three strains were detected in the
donor B sample, and one in that of donor A. e, Abundance at the species-
level was calculated as in c. The mapped read counts were normalized to
RPKM.
RESEARCH Article

a
SPF+MC38
GF+11mix +11mix+αPD-1
Colon (n = 3) TIL (n = 7)
60 60
40 40
**

% IFNγ+ of CD8 T

% IFNγ+ of CD8 T
20 20 ns **
10 10
10 10
ns
ns
***
***
*** ***
5 5

0 0

+11 st. lysate

Medium

+p15E pep.
+PMA+Iono.
+11 st. lysate
+10 st. lysate
Medium

+p15E pep.
+gp100 pep.
+PMA+Iono.
b
TCRVβ PCA
GF GF+11-mix
20 14

12
15
10 CLP IFNγ+ (n = 3)
-
10 8 CLP IFNγ (n = 3)
iLN IFNγ+ (n = 4)
PC3 (19.7692%)

PC3 (15.5825%)

6
5
-
4 iLN IFNγ (n = 4)
0
2 TIL(MC38+αPD-1) IFNγ+(n = 3)
-
-5 0 TIL(MC38+αPD-1) IFNγ (n = 3)
-2
-10
-4

-15 -6
10 10
-20 -10 0 -10 -5 0 0
0 10 5 10
20 30-10 15 20 -10
PC1 (33.2885%) PC2 (25.0062%) PC1 (47.0968%) PC2 (19.6346%)

Extended Data Fig. 10 | Antigen-specificity and TCR Vβ usage of IFNγ+ versus TIL IFNγ+), 0.20 (iLN IFNγ+ versus TIL IFNγ+), <0.0001
IFNγ+CD8 T cells induced by the 11-mix differ by anatomical location. (CLP IFNγ− versus iLN IFNγ−), <0.0001 (CLP IFNγ− versus TIL IFNγ−),
a, Percentage of IFNγ+ cells among CD8 T cells in either colons from 0.0024 (iLN IFNγ− versus TIL IFNγ−). GF+11-mix group: <0.0001 (CLP
GF+11-mix mice or MC38 tumours from SPF+11mix+anti-PD-1 IFNγ+ versus iLN IFNγ+), <0.0001 (CLP IFNγ+ versus TIL IFNγ+),
mice, after ex vivo stimulation with the indicated antigens. b, Principal 0.0001 (iLN IFNγ+ versus TIL IFNγ+), <0.0001 (CLP IFNγ− versus iLN
component analysis plots of TCR Vβ usage by the IFNγ+ and IFNγ- CD8 IFNγ−), <0.0001 (CLP IFNγ− versus TIL IFNγ−), 0.0009 (iLN IFNγ−
T cell subsets isolated from the indicated tissues of GF+11-mix (right) or versus TIL IFNγ−). Each circle represents an individual mouse (iLN
germ-free (left) mice. For TIL data, IFNγ+ and IFNγ− CD8 T cells were and TIL) or a pool of mice (colon). The number of mice in each group
isolated from germ-free ± 11mix+MC38+anti-PD-1 mice. Two-way is shown. Data are mean and s.d. ***P < 0.001; **P < 0.01; one-way
ANOVA interaction P values comparing two populations at a time are ANOVA with Tukey’s test. See Source Data for exact P values.
as follows. GF group: 0.006 (CLP IFNγ+ versus iLN IFNγ+), 0.50 (CLP

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nature research | reporting summary March 2018

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