Extrathymically generated regulatory T cells control mucosal TH2 inflammation.

Abstract

A balance between pro- and anti-inflammatory mechanisms at mucosal interfaces, which are sites of constitutive exposure to microbes and non-microbial foreign substances, allows for efficient protection against pathogens yet prevents adverse inflammatory responses associated with allergy, asthma and intestinal inflammation. Regulatory T (T(reg)) cells prevent systemic and tissue-specific autoimmunity and inflammatory lesions at mucosal interfaces. These cells are generated in the thymus (tT(reg) cells) and in the periphery (induced (i)T(reg) cells), and their dual origin implies a division of labour between tT(reg) and iT(reg) cells in immune homeostasis. Here we show that a highly selective blockage in differentiation of iT(reg) cells in mice did not lead to unprovoked multi-organ autoimmunity, exacerbation of induced tissue-specific autoimmune pathology, or increased pro-inflammatory responses of T helper 1 (T(H)1) and T(H)17 cells. However, mice deficient in iT(reg) cells spontaneously developed pronounced T(H)2-type pathologies at mucosal sites--in the gastrointestinal tract and lungs--with hallmarks of allergic inflammation and asthma. Furthermore, iT(reg)-cell deficiency altered gut microbial communities. These results suggest that whereas T(reg) cells generated in the thymus appear sufficient for control of systemic and tissue-specific autoimmunity, extrathymic differentiation of T(reg) cells affects commensal microbiota composition and serves a distinct, essential function in restraint of allergic-type inflammation at mucosal interfaces.

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LETTER
doi:10.1038/nature10772
Extrathymically generated regulatory T cells control
mucosal TH2 inflammation
Steven Z. Josefowicz1,2*, Rachel E. Niec1*, Hye Young Kim3, Piper Treuting4, Takatoshi Chinen1,5, Ye Zheng6, Dale T. Umetsu3
& Alexander Y. Rudensky1
A balance between pro- and anti-inflammatory mechanisms at
mucosal interfaces, which are sites of constitutive exposure to
microbesandnon-microbialforeignsubstances,allowsforefficient
protection against pathogens yet prevents adverse inflammatory
responses associated with allergy, asthma and intestinal inflam-
mation1. Regulatory T (Treg) cells prevent systemic and tissue-
specific autoimmunity and inflammatory lesions at mucosal inter-
faces. These cells are generated in the thymus (tTregcells) and in
the periphery (induced (i)Tregcells), and their dual origin implies
a division of labour between tTreg and iTreg cells in immune
homeostasis. Here we show that a highly selective blockage in dif-
ferentiationofiTregcellsinmicedidnotlead tounprovokedmulti-
organ autoimmunity, exacerbation of induced tissue-specific
autoimmune pathology, or increased pro-inflammatory responses
ofThelper1(TH1)andTH17cells. However,micedeficientiniTreg
cells spontaneously developed pronounced TH2-type pathologies
at mucosal sites—in the gastrointestinal tract and lungs—with
hallmarks of allergic inflammation and asthma. Furthermore,
iTreg-cell deficiency altered gut microbial communities. These
resultssuggestthatwhereasTregcellsgeneratedinthethymusappear
sufficient for control of systemic and tissue-specific autoimmunity,
extrathymic differentiation of Tregcells affects commensal micro-
biota composition and serves a distinct, essential function in
restraint of allergic-type inflammation at mucosal interfaces.
Exquisitelybalancedcontrolmechanismsoperatingatmucosalsites
are able to accommodate potent immune defences and the need to
prevent tissue damage resulting from inflammatory responses caused
by commensal microorganisms, food and environmental antigens,
allergens, and noxious substances1.
Prominent among multiple regulatory lymphoid and myeloid cell
subsets operating at environmental interfaces are Foxp31Tregcells.
Genetic deficiency in Foxp3 (forkhead box P3, a key transcription
factor specifying Tregcell differentiation) leads to paucity of Foxp31
Tregcellsandconsequentgeneralizedlympho-andmyelo-proliferative
syndrome, featuring sharply augmented serum IgE levels, production
of TH1, TH2 and TH17 cytokines, and widespread tissue inflam-
mation2. Foxp3 can be induced in thymocytes in response to T-cell
receptor (TCR) and CD28 stimulation, and IL-2. In addition, Foxp3
canbeupregulateduponTCRstimulationofmatureperipheralCD41
T cells inthepresence of tumour growthfactor b (TGFb) in a manner
dependent on an intronic Foxp3 enhancer CNS1 (refs 3–5). Inflam-
matory cytokines and potent co-stimulatory signals antagonize the
peripheral induction of Foxp3, and retinoic acid augments Foxp3
induction through mitigating inflammatory cytokine production
and through cell intrinsic mechanisms1,6–8. Although differing in their
sitesofgeneration,tTregandiTregcellsare comingledinthesecondary
lymphoid organs and non-lymphoid tissues once mature, and their
relative contributions to the total population of Tregcells and their
specific roles in control of various aspects of immune homeostasis and
microbial colonization in normal animals has remained unexplored.
Our recent investigation5showed that CNS1, which contains bind-
ing sites for transcription factors (NFAT, Smad3 and RAR/RXR)
downstream of three signalling pathways implicated in iTregcell
generation4,8(Supplementary Fig. 1), is critical for TGFb-dependent
inductionofFoxp3,buthasnoapparentroleintTregdifferentiationor
maintenance of Foxp3 expression. This observation suggested that
CNS1 activity represents a dedicated genetic determinant for the dif-
ferentiation of iTregcells, and its deficiency in mice provides a unique
means to evaluate the function of these cells in vivo. Our initial char-
acterization of CNS12mice and littermates maintained on a 129/B6
geneticbackgroundfailedtorevealdiseasephenotypes.Becausemixed
genetic backgrounds frequently mask adverse phenotypes or make
them highly variable, to understand iTregfunction in vivo we back-
crossed CNS1 mice onto the B6 background (Supplementary Fig. 2).
First, we sought to ascertain that on the B6 genetic background
CNS1isdispensablefortTregcellgenerationbutcriticalforgeneration
of iTregcells.Tworecentstudies establisheda role forTGFb signalling
in tTreg cell differentiation in neonates9,10. Thus, to exclude the
possibility that CNS1 deficiency adversely affects generation of
Foxp31T cells in the neonatal thymus, we examined the Foxp31Treg
cell population in heterozygous female CNS1WT/2mice. As Foxp3 is
encodedontheXchromosomeandissubjecttorandomX-chromosome
inactivation,characterizationoffemaleCNS1WT/2miceallowsforcom-
parison of CNS12and CNS1WTTregcells in a competitive environ-
ment. In neonatal female CNS1WT/2mice, CNS12cells constituted,
on average, one-half of the thymic Foxp31cell population (Fig. 1a).
Additionally,neonatalCNS12hemizygousandcontrolmalesharboured
comparable numbers of Foxp31thymocytes (Supplementary Fig. 3).
Therefore, tTregdifferentiation is independent of CNS1. In contrast,
CNS12naive CD4 T cells showed severely impaired induction of
Foxp3 in vitro (Fig. 1b). Analyses of heterozygous female CNS1WT/2
mice and transfer of CNS12or CNS1WTTregcells into lymphopenic
recipients demonstrated that the ability of Tregcells to accumulate and
proliferate in various tissues was unperturbed in the absence of CNS1
(Supplementary Fig. 4). Furthermore, CNS1 deficiency did not affect
suppressor activity of tTregcells (assessed using in vitro suppression
assays and adoptive transfers of Foxp3-deficient effector T cells with
predominantly tTreg-containing Foxp31cells isolated from 4-week-old
CNS12andCNS1WTmiceintolymphopenicrecipients(Supplementary
Fig.5)).Likewise,CNS1ablationdidnotnegativelyaffectmaintenanceof
Foxp3expressionandoverallfunctionofNFAT,TGFbandretinoicacid
signalling pathways in these cells (Supplementary Fig. 5 and data not
shown). To assess how the deficiency in iTregcell generation affects
the size of the peripheral Tregcell compartment, we analysed Tregcell
frequencies in various tissues throughout the lifespan of mice. CNS12
mice failed to exhibit a progressive age-dependent increase in Foxp31
*These authors contributed equally to this work.
1HowardHughesMedicalInstituteandImmunologyProgram,SloanKetteringInstitute,NewYork,NewYork10021,USA.2LaboratoryofChromatinBiologyandEpigenetics,TheRockefellerUniversity,New
York,NewYork10065,USA.3DivisionofImmunology,Children’sHospital,HarvardMedicalSchool,Boston,Massachusetts02115,USA.4DepartmentofComparativeMedicine,andHistologyandImaging
Core, School of Medicine, University of Washington, Seattle, Washington 98195, USA.5Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.6Nomis
Foundation Laboratories for Immunobiology and Microbial Pathogenesis, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
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cell frequencies observed in wild-type littermates (Fig. 1c and Sup-
plementary Fig. 6). By 6–8 months of age, CNS12mice contained
markedly fewer Foxp31cells in comparison to control animals, with
most prominent differences in mesenteric lymph nodes, Peyer’s
patches, and small and large intestine lamina propria, sites known to
support iTregcell generation11. This trend was nottheresult of expres-
sion of a Foxp3–GFP fusion protein in CNS12mice, because age-
matched CNS1WTFoxp3–GFP and littermate control CNS1WTmice
expressingunmodifiedFoxp3proteinexhibitedsimilarage-dependent
increases in Tregcell frequencies (Supplementary Fig. 6).
To assess the extent of impairment of peripheral generation of Treg
cellsin vivo, weexamined Foxp3inductioninantigen-specificnaive T
cells upon exposure to ingested ‘non-self’ antigen12. Ovalbumin
(OVA)-specific OT-II1TCR-transgenic Foxp32(GFP2) Tregcells
from CNS12or Foxp3GFPmice were transferred into CD45.11
lymphoreplete recipients followed by ad libitum administration of
OVA in drinking water. We failed to detect Foxp3 induction in
CNS1-deficient cells, whereas up to 20% of transferred OT-II T cells
fromcontrolFoxp3GFPmiceinducedFoxp3uponexposuretocognate
antigenintheintestinaltract(Fig.1dandSupplementaryFig.7).These
results were in agreement with a marked impairment in Foxp3 induc-
tion in polyclonal CNS1-deficient Foxp32T cells in vitro, which was
most severe at lower, more physiologically relevant concentrations of
TGFb (Fig. 1b). Together these data indicate that iTregcells have a
stringent requirement for CNS1 for their differentiation.
Recent studies showed a limited TCR-dependent clonal niche for
tTregcell differentiation and peripheral maintenance13–15. The sus-
tained numerical impairment in the peripheral Tregcell populations
in CNS1-deficient mice suggests that tTregcells fail to fill the ‘void’ in
the peripheral Tregcell pool, left by iTregcell deficiency. This obser-
vation combined with largely non-overlapping TCR repertoires of
tTregand iTregcells suggests that iTregand tTregcells occupy distinct
‘niches’16. To test this notion we co-transferred CNS12(tTregcells) or
CNS1WTTregcells(iTreg1tTreg)fromagedmicewithCNS1-sufficient
naive CD45.11Foxp32CD41T cells into lymphopenic recipients. We
observed more efficient Foxp3 induction in CD45.11CD41T cells
upon co-transfer with CNS12Tregcells (tTregcells), indicating that in
lymphopenicrecipientsthedenovogenerationofiTregcellsismarkedly
more efficient in the absence of pre-existing iTregcells (Supplementary
Fig.8).ThesedataalsoimplytheexistenceofastableiTregcellsubsetin
normalmice.However,thedynamicsandstabilityofFoxp3expression
has been a controversial issue, with a number of studies favouring
unstable Foxp3 expression in iTregcells17–19. Thus, we next employed
genetic fate mapping using inducible Cre recombinase expressed in a
Treg-specific manner (Foxp3EGFP-CRE-ERT2) and a Rosa26–YFP recom-
binationreporterallele(R26Y)20todetermine ifiTregcells generated in
vivo are able to acquire stable Foxp3 expression and, thus, have the
capacity to contribute to the stable Tregcell compartment.
Double-sorted naive CD45.21Foxp32YFP2CD4 T cells from
Foxp3EGFP-CRE-ERT2R26Y mice were transferred together with
congenically marked CD45.12Foxp31Tregcells into lymphopenic
recipient mice. Foxp3 expression within the population of tagged
YFP1cellsgeneratedfromYFP2Foxp32precursorswasassessedfour
weeks after treatment of recipient mice with tamoxifen, which was
administered early (one week) and late (five weeks) following cell
transfer. Approximately half of the newly generated YFP-tagged
iTregcells lost Foxp3 expression, whereas ‘mature’ iTregcells tagged
at a later time point displayed remarkable stability (.90% Foxp31
cells among YFP1cells), comparable to that of transferred peripheral
Tregcells (Fig. 1e and Supplementary Fig. 9). Together these data
indicatethatiTregcellshaveastringentrequirementforCNS1fortheir
differentiation, accumulate throughout life, and occupy a sizable frac-
tion of the stable peripheral Tregcell compartment.
CNS12mice onthe B6geneticbackgrounddisplayedneitherearly-
nor late-onset systemic autoimmunity nor spontaneous widespread
tissue lesions nor severe morbidity associated with systemic Tregcell
deprivation (data not shown). However, it was possible that iTregcell
deficiency may exacerbate initial or late stages of provoked tissue-
specific autoimmune pathology directed against a self-antigen. To
addressthisquestion,weinducedexperimentalautoimmuneencepha-
lomyelitis(EAE)inCNS1-deficientorlittermatecontrolmicethrough
immunization with myelin oligodendrocyte glycoprotein (MOG)
peptide. The onset, severity and remission of disease were indistin-
guishable, and no detectable differences were observed in Tregcell
subsets in the brain in these two groups of mice (Supplementary Fig. 10).
Although it will be important to evaluate the role of iTregcells in
additionalmodelsofinducedautoimmunity,theseresultsindicatethat
tTregcells are largely sufficient for control of tolerance to self-antigens
and that the distinct functional role of iTregcells might be to control
inflammation at mucosal surfaces, which are sites of preponderant
exposure to non-self substances. This notion is consistent with data
indicating that tTregcells arise from a subset of thymocytes, which
exhibitTCRwithanincreasedaffinityforself-antigensyetinsufficient
for negative selection10,21, whereas iTregcells are efficiently generated
TGFβ (ng ml–1)
CNS1 WT
Percentage of Foxp3+
cells in CD45.2+
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Figure 1 | Impaired iTregcell generation and altered composition of the
peripheral Tregcell population in CNS1-deficient mice. a, Relative
contribution of CNS12(GFP1) and CNS1WT(GFP2) cells to the Foxp31
thymocyte subset in 4-day-old CNS1WT/2female mice. SP, single positive.
b, Induction of Foxp3 in Foxp32TN(naive) cells FACS sorted from CNS12
(knockout, KO) or Foxp3GFPmice stimulated in vitro with TGFb, IL-2 anti-
CD3 and anti-CD28. c, Percentage of Foxp31cells (of CD41) in the spleen,
lymph node (LN), mesenteric lymph nodes (MLN), Peyer’s patches (PP) and
cellsfromthesmallandlargeintestinelaminapropria(SIandLI)of6–9month
oldCNS12orcontrol mice.d, Percentageof transferred (CD45.21) CNS12or
CNS11CD252CD44lowCD45.21OTII1cells that induced Foxp3 following
administration of OVA in water for 6 days. e, Stability of Foxp3 expression in
iTregcells.FACSsortedGFP1orGFP2cellsfromFoxp3eGFP-Cre-ERT2micewere
transferredwithGFP-orGFP1cells,respectively,fromCD45.1Foxp3GFPmice
intoTCRbd-deficientrecipients.Micereceivedtamoxifen(TMX)at1(left)or5
weeks (right) after transfer and stability of Foxp3 expression among YFP-
labelled cells was assessed after 4 weeks. All data are representative of two or
more independent experiments with n$3. Error bars, s.d.; *P,0.05,
**P,0.01, ***P,0.001, as calculated by Students’ t-test.
RESEARCH LETTER
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upon TCR engagement with a high affinity cognate ligand under
subimmunogenic conditions22,23.
The absence of iTregcell induction in response to oral antigen in
CNS12micesuggestedthattheimmunebalanceinthegastrointestinal
tract might be impaired owing to deficiency in gut antigen-specific
iTregcells. Indeed, while IL-17and IFN-c production by CD41T cells
was unaffected by iTregdeficiency in CNS12mice (Supplementary
Fig. 11), we observed markedly augmented production of the TH2
cytokines, IL-4, IL-5 and IL-13, by CD41T cells, especially in the
mesentericlymphnodes,Peyer’spatchesandintestinallaminapropria
(Fig.2aandSupplementaryFig.12).Furthermore,thevastmajorityof
CD41T cells in the lamina propria of CNS12mice expressed high
amounts of Gata3, a key TH2 differentiation factor. Increases in
Gata31CD41T cells were observed not only in gastrointestinal tract
tissues in CNS12mice but also in other lymphoid tissues, albeit to a
lesser extent (Fig. 2b and Supplementary Fig. 12). Consistent with the
sharply augmented TH2 responses at mucosal sites, CNS12mice
exhibited increasedfrequencies
(Fas1GL71) in the Peyer’spatches, butnot in the spleen or peripheral
lymph nodes (Supplementary Fig. 13), and spontaneous increases in
serum levels of IgE and IgA, but not in other Ig isotypes (Fig. 2c, and
data not shown).
The dysregulated TH2 responses were associated with a decreased
body weight (Fig. 3a and Supplementary Fig. 2) and distinct highly
penetrant pathology throughout the gastrointestinal tract (Fig. 3b and
Supplementary Fig. 14): all CNS12mice (12/12) and no CNS1WT
control littermates (0/6) were affected by gastritis and plasmacytic
enteritis characterized by increased frequencies of plasma cells in the
intestinal lamina propria and other associated lesions such as crypt
abscesses. Accordingly, serum antibodies in CNS12mice exhibited
reactivity against antigens of the small and large intestine, pancreas
andchow(SupplementaryFig.13).Notably,thepathologyobservedin
the gastrointestinal tissue of CNS12mice was markedly diminished
upon B-cell depletion, but was not ameliorated by administration of
IL-4neutralizingantibody(SupplementaryFig.15).Theinflammatory
ofgerminal centreBcells
features and lesions observed in CNS12mice were consistent with
allergic TH2-type intestinal disease (Fig. 3).
One possible explanation for the pronounced TH2 responses and
intestinal pathology associated with iTregcell deficiency is simply a
numerical decrease in Tregcells. However, we consider this possibility
unlikely, because graded depletion of Foxp31Tregcells in Foxp3DTR
mice upon administration of titrated amounts of diphtheria toxin
resulting in Tregfrequencies similar to those observed in CNS12mice
revealed augmented TH1 and TH17, but not TH2, responses24.
Alternatively,certainqualitativefeaturesofiTregcellscouldallowthem
to efficiently limit TH2 inflammation in the gut. Recent studies sug-
gested that some of the transcriptional regulators involved in a par-
ticular type of effector T-cell response facilitate the ability of Tregcells
to suppress those responses25–27. Thus, we explored the expression of
TH2-associated transcription factor Gata3 in Tregcells in CNS12and
CNS1WTmice. In contrast to a sharp increase in Gata3 expression in
effector T cells (Fig. 2b and Supplementary Fig. 12), we found its
expression markedly diminished in Tregcells in CNS12mice (Fig. 3c
and Supplementary Fig. 12). Notably, ablation of a conditional Gata3
allele in Tregcells leads to Treg cell dysfunction28,29and marked
augmentation of TH2 cytokine production by CD41T cells (D.
Rudra,R.E.N.andA.Y.R.,manuscriptinpreparation).Wehypothesized
thatincreasedGata3expressioniniTregcellsreflectstheiractivationstate
upon TCR ligation by high affinity ligands in the gut rather than an
intrinsic feature of iTregcells. In support of this idea, we found that
bothCNS12andcontrolTregcellsstimulatedinvitrothroughtheTCR
and IL-2 receptor exhibited similarly robust Gata3 induction (Sup-
plementaryFig.12).Thus,wesuggestthatincreasedGata3expression
in iTregcells, a likely consequence of their generation in response
to high affinity TCR ligands present in the gut, endows these cells
with the capacity to efficiently control spontaneous mucosal TH2
inflammation.
Certain commensal bacteria increase the frequencies of Tregcells in
thegutandprovideantigensrecognizedbyaconsiderableproportionof
iTregTCR1,16. In addition to TCR ligands the gut microbial community
0.0
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IL-5+ cells
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Spleen MLNPPSI
3 months
10 months
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Percentage of Gata3+
in Foxp3–
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** **
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*
*
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********
IgA
CNS1 WT
CNS1 KO
a
b
c
Figure 2 | Paucity of iTregcells results in TH2 inflammation in the
gastrointestinaltract. a,PercentageofCD41cellsproducingIL-4(top),IL-13
(middle) and IL-5 (bottom) in 3-month-old mice. Left, spleen, peripheral
lymph nodes (LN) and mesenteric lymph nodes (MLN); right, lamina propria
of small and large intestine (SI and LI, respectively). b, Percentage of Foxp32
CD41cells that were Gata31in 3-month old mice (PP, Peyer’s patches).
c, Concentration of IgE and IgA in serum, determined by enzyme linked
immunosorbent assay (ELISA) at 1, 3 and 10months. All data are
representative of three or more independent experiments with $3 mice per
group. Error bars, s.d.; *P,0.05, **P,0.01, ***P,0.001, as calculated by
Students’ t-test.
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cells in CD4+Foxp3+
Weight (g)
30
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*
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a
b
cd
Percentage of total
16S rRNA
Firmicutes
Bacteriodetes
Figure 3 | iTregcell deficiency leads to TH2 type gastrointestinal pathology
and altered microbial communities. a, Body weights of 9–12 (left) or 2.5-
month-old individually housed (right) CNS12(KO) and littermate control
(WT) mice (n$12). b, Plasmacytic enteritis (arrowhead) in CNS1-deficient
micerevealedbyhaematoxylinandeosinstainingofsmallintestinefrom9–12-
month-old CNS12(bottom and right) and littermate control mice (top). An
early crypt abscess is indicated (asterisk). Data are representative of $20 mice
analysed. c, Percentage of Foxp31CD41cells expressing Gata31in 3-month-
oldmice.d,Percentageoftotal16SrRNAgenesequencesoftheFirmicutesand
Bacteroidetes phyla in stool from individually housed CNS12(n59) and WT
(n56) littermate mice. All data are representative of three or more
independentexperimentswith$3micepergroup.Errorbarsshows.d.(a,c)or
s.e.m. (d). *P,0.05, **P,0.01, ***P,0.001, as calculated by Student’s
t-test. Scale bars, 150mm.
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also contributes to the local cytokine environment, which facilitates
iTregcell differentiation and maintenance in the gut1. These observa-
tions raise a question as to whether iTregcells, in turn, influence
composition of the commensal microbiota. To address this question,
we sequenced 16S ribosomal RNA coding genes from bacterial con-
tents of stool samples isolated from CNS12and CNS1WTlittermates,
which were housed individually for 5weeks after weaning.
Phylogenetic analysis revealed distinct gut microbial communities in
CNS12mice,withstatisticallysignificantenrichment ofthe candidate
phylum TM7 and the genus Bacteroidetes Alistipes (Supplementary
Fig. 16), and an overall decrease in the ratio of Firmicutes to
Bacteroidetes (2.60 in wild-type and 1.51 in knockout) (Fig. 3d).
Interestingly, an opposite trend in the Firmicutes/Bacteroidetes ratio
wascorrelatedwithobesity30,suggestingthe possibilitythatalterations
in energy harvest and metabolism (caused by inflammation or
microbe-dependent effects on energy balance) could account for the
decreasedweightobservediniTregcelldeficientmice.Thus,iTregcells
help maintain a ‘normal’ microbial community in the gut, probably
through exerting control over TH2 mucosal inflammation.
Theseobservationsraisedthequestionofwhetherthealteredmicro-
biota,ratherthaniTregdeficiency,wasthedirectcauseofobservedTH2
inflammation. To equalize gut microbiota, CNS12and littermate
controls were treated with antibiotics (metronidazole and ciprofloxa-
cin) for 4weeks. Despite indistinguishable microbial communities,
antibiotic treatment did not lead to a decrease in Gata3 expression
or Th2 cytokine production by effector T cells in CNS12mice, and
characteristic histopathologic features were maintained (Supplemen-
tary Fig. 17). Furthermore, iTreg cell sufficient germ-free mice
colonized with CNS12or control microbiota exhibited a similar
spectrum of TH1, TH2 and TH17 cytokine production and eventual
normalization of microbiota (Supplementary Fig. 18 and data not
shown). These results suggest that iTregdeficiency results in immune
dysregulation and TH2 inflammation in the gut with subsequent per-
turbation of the microbial community.
AccordingtothenotionofspecializediTregcellfunctioninsuppres-
sion of TH2 responses at mucosal sites, one would expect to observe
TH2-type pathology in the lungs of CNS12mice, despite an only
modest,20–25%decrease innumbers of Tregcells inthistissuecom-
pared to littermate controls (Fig. 1c). Indeed, we discovered that
CNS12mice suffer from spontaneous TH2-type airway inflammation
(Fig. 4 and Supplementary Fig. 19). The lungs of CNS12mice were
characterized by increased infiltration by lymphocytes, plasma cells
and macrophages, and by moderate neutrophil infiltration (Fig. 4).
The consistent features of the chronic inflammatory airway disease
observed in CNS12mice include lymphocytic infiltration, narrowed
airway lumen (Fig. 4a), increased goblet cells and mucus production
(Fig. 4a and b), smooth muscle hyperplasia, and fibrosis (Fig. 4c).
Notably, 9/12 CNS12and 0/6 CNS1WTmice developed acidophilic
macrophage pneumonia (AMP) with characteristic increases in
acidophilic macrophages and both intracellular and extracellular
chitinase 3-like 3 crystals (Chi3l3, formerly Ym1), analogous to
Charcott-Lyden crystals found in asthmatic patients (Fig. 4a and e).
In addition, the prominent presence of alternatively activated macro-
phagesinthelungsofCNS12micewasconfirmedbymorphologyand
expression of arginase 1 in addition to Chi3l3 (Fig. 4d and Sup-
plementary Fig. 20). Furthermore, both young (6–8 week old) and
aged(20weekold)CNS12miceexhibitedairwayhyper-responsiveness
accompaniedbyAMP,perivascular,peribronchiolarand intramucosal
inflammation, bronchial epithelial hyperplasia, and airway narrowing
(Fig. 4f and Supplementary Fig. 21). These spontaneous lesions are
especially striking considering the TH2-resistant, TH1-prone C57BL/6
genetic background of CNS12mice. The lung pathology in CNS12
mice reflects the hallmark features of chronic allergic inflammation
and asthma.
Our results demonstrate that Tregcells of thymic and extrathymic
origin have distinct mechanistic requirements for differentiation and
exert specialized functions in immune homeostasis. The restriction of
lesions to mucosal tissues in iTregdeficient mice implies that under
steady state conditions Tregcells generated in the thymus are largely
sufficient for control of most immune responses to self-antigens.
Thesefindingssuggestthatinnormalanimals,Tregcellsgeneratedextra-
thymically in a CNS1-dependent manner play a non-redundant role in
control of mucosal allergic Th2 inflammation and asthma.
METHODS SUMMARY
The generation of the following mouse strains has been previously described5,20:
CNS12(Foxp3DCNS1), Foxp3GFPand Foxp3eGFP-Cre-ERT2R26Y. Rag12mice were
purchased from The Jackson Laboratory, and CD45.1 B6 and Tcrb/Tcrd2mice,
alongwithabovestrainsweremaintainedintheSloanKetteringInstituteResearch
Laboratoriesanimalfacilityinaccordancewithinstitutionalregulations.Tissuesfor
histologic analysis were fixed in 10% phosphate-buffered formalin and processed
routinely for staining. In vitro induction assays were performed with 53104
Foxp3–GFP2CD41T cells and 5mgml21of anti-CD3 and anti-CD28 antibody,
100Uml21IL-2, in 96-well, flat-bottom plates. For in vitro and transfer experi-
ments, CD41T cells were pre-enriched using mouse CD4 Dynabeads (L3T4,
Invitrogen)andFACSsortedonanLSR-II(BDBiosciences).Intracellularstaining
forIL-4usedCytofix/Cytoperm(BDBiosciences),andstainingforothercytokines,
Foxp3 and Gata3, used the Foxp3 staining kit (eBiosciences). For measurement of
AHR, mice were anaesthetized with pentobarbitol and AHR was assessed by
invasive measurement of airway resistance using modified version of a described
method (Buxco Electronics). 16S rRNA sequencing was performed on a 454 GS
A
A
WT
KO
KO
KO
WT
WT
c
d
Resistance (% of baseline)
0 10 20 30 40
100
150
200
250
300
0 10 20 30 40
0
20
40
60
80
100
CNS1 WT
CNS1 KO
Compliance (% of baseline)
*
*
b
e
a
f
Methacholine (mg ml–1)
KO
WT
KO
WT
Figure 4 | Unprovoked asthma-like airway pathology in CNS1-deficient
mice. a, Representative haematoxylin and eosin-stained lung sections from
CNS12(top) and WT (bottom) mice. The CNS12lung has marked
peribronchiolar inflammation (arrowhead). The reduced lumen (L) contains
mucus produced by the hyperplastic respiratory epithelium (E). Arrows
indicate reactive (top) and normal (bottom) endothelium. Bottom right hand
corner insets are higher magnification of boxed regions and bar indicates
smooth muscle thickness. Top right inset (KO) demonstrates eosinophilic
crystals. Asterisk marks acidophilic macrophages. b, Periodic acid Schiff with
Alcian Blue staining highlighting mucus-producing goblet cells (dark blue-
purple) c, Trichrome staining illustrating lung fibrosis (blue staining).
d, Arginase-1 stainingoflungsfrom CNS12andWTmice.Aindicatesairway;
anacidophiliccrystalismarkedbythearrowhead.e,Chitinase3-like3(Chi3l3)
staining of lungs from CNS12and WT mice at 103 magnification (top) and
603 magnification of lungs from CNS12mice demonstrating robust Chi3l3
expression within acidophilic macrophages (bottom). f, Lung resistance (left)
and compliance (right) of CNS12and WT littermate control mice after
exposure to methacholine. Data representative of two independent
experiments with $4 mice per group. Error bars, s.d.; *P,0.05, **P,0.01,
***P,0.001, as calculated by Students’ t-test. Scale bars, 100mm.
RESEARCH LETTER
3 9 8 | N A T U R E | V O L 4 8 2 | 1 6 F E B R U A R Y 2 0 1 2
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Page 5

FLX Titanium pyrosequencing platform following the Roche 454 recommended
procedures.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 6 July; accepted 6 December 2011.
Published online 8 February 2012.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank T. Tedder for depleting CD20 antibody, R. Tudor for
assistance interpreting lung pathology, P. DeRoos for assistance with Ig ELISA assays,
B. Johnson for immunohistochemical expertise, Y. Chen for assistance with airway
measurements, and E. Pamer, L. Lipuma, A. Gobourne and R. Khanin for help with
analysis of intestinal microbiota. This work was supported by NIH MSTP grant
GM07739 and NINDS grant 1F31NS073203-01 (R.E.N.), Strategic Young Researcher
Overseas Visits Program for Accelerating Brain Circulation from Department of
MicrobiologyandImmunology,KeioUniversitySchoolofMedicine(T.C.)andNIHgrant
R37 AI034206 (A.Y.R.). A.Y.R is an investigator with the Howard Hughes Medical
Institute.
Author Contributions S.Z.J., R.E.N. and H.Y.K. performed experiments and analysed
data, with assistance from T.C. for tissue Ig ELISA experiments, and P.T. for
immunohistochemistry and histopathology analysis. D.T.U., S.Z.J., R.E.N., H.Y.K, and
A.Y.R designed and interpreted AHR experiments. Y.Z. generated CNS1-mice. S.Z.J.,
R.E.N. and A.Y.R. designed experiments and wrote the paper.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to A.Y.R. (rudenska@mskcc.org).
LETTER RESEARCH
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©2012

Page 6

METHODS
Mice. The generation of the following mouse strains has been previously
described5,20: CNS12(Foxp3DCNS1), Foxp3GFPand Foxp3eGFP-Cre-ERT2R26Y.
Rag12mice were purchased from The Jackson Laboratory, and CD45.1 B6 and
Tcrb/Tcrd2/2mice,alongwithabovestrainsweremaintainedintheSloanKettering
Institute Research Laboratories animal facility in accordance with institutional
regulations. Mice were killed by CO2asphyxiation. EAE was induced and scored
as previously described31. For antibiotic treatment, CNS1-deficient and sufficient
mice were treated with 1gl21metronidazole (Sigma-Aldrich) and 0.2gl21
ciprofloxacin (ENZO Life Sciences International) dissolved in drinking water for
4weeks. Mouse anti-CD208(MB20-11, provided by T. Tedder) and anti-IL-4
(11b.11, NCI-Frederick) were administered weekly as intraperitoneal injections
of 50mg or 5mg, respectively, for 3weeks.
Cell isolation, transfer and FACS staining. For in vitro and in vivo transfer
experiments, CD41T cells were pre-enriched using mouse CD4 Dynabeads
(L3T4, Invitrogen) and FACS sorted on an LSR-II (BD Biosciences). Intracellular
staining forIL-4usedCytofix/Cytoperm following treatment withGolgi-Stop(BD
Biosciences),andstainingforothercytokines(followingtreatmentwithGolgi-Plug,
BD Biosciences) and Foxp3 and Gata3 used the Foxp3 staining kit (eBiosciences).
In vitro assays. In vitro induction assays were performed with 53104Foxp3–
GFP2CD41T cells and 5mgml21of anti-CD3 and anti-CD28 antibody,
100Uml21IL-2, in 96-well, flat-bottom plates. For in vitro suppression assays,
43104CD41Foxp32CD62LhighnaiveTcellsFACS purifiedfromWTmicewere
cultured with graded numbers of CD41Foxp31Tregcells FACS purified from
Foxp3DCNS1or Foxp3gfpmice in the presence of 105irradiated T cell-depleted
splenocytes and 1mgml21anti-CD3 antibody in a 96-well round-bottom plate
for 80h. Cell proliferation was assessed by [3H]thymidine incorporation during
the final 8h of culture.
Histology and immunohistochemistry. Necropsies were performed, and
sections of pancreas, stomach, heart, lungs, kidney, external ear and haired skin
were fixed in 10%phosphate-buffered formalin. Tissues were processed routinely
for staining with haematoxylin and eosin, periodic acid Schiff with Alcian blue or
Masson Trichrome if indicated. Slides were examined by an American Board of
Veterinary Practitioners-certified veterinary pathologist blinded to genotypes.
Morphologicaldiagnoseswereappliedforalltissues.Immunohistochemicalstain-
ing was performed by the University of Washington Histology and Imaging Core
using standard protocols with a Leica Bond Automated Immunostainer. Primary
antibodies: goat anti-mouse chitinase 3-like 3/ECF-L (YM1) (R&D systems, cat.
no.AF2446,lotno.UNU01),0.2mgml21;rabbitpolyclonalantiiNOS/NOSII,NT
(Millipore, cat. no. 06-573), 1mgml21; rabbit polyclonal anti arginase 1 (H-52)
(Santa Cruz, cat. no. sc-20150, lot no. K0807), 0.2mgml21. Isotype controls were
used at the same concentration as the primary antibody with all antibodies run
with Lecia Bond reagents and Bond Polymer Refine (DAB) detection with
haematoxylin counter stain.
Histologyinflammationscoring.0,None;1,focalormultifocalmildperivascular
accumulationswithminimalextensionintosurroundingadventiaorparenchyma;
2, multifocal mild or focal moderate perivascular accumulations with mild exten-
sion into surrounding parenchyma or mild to moderate parenchymal accumula-
tions; 3, grade 2 plus mild inflammation-associated parenchymal lesions such as
loss or degeneration of cells; 4, grade 2 plus moderate to severe inflammation-
associated parenchymal lesions. Inflammation in the gastrointestinal tract was
scored as described previously32.
Airway hyperresponsiveness measurements. For measurement of AHR, mice
were anaesthetized with pentobarbitol (7.5–10mg per mouse) and AHR was
assessed by invasive measurement of airway resistance using modified version
ofa describedmethod(BuxcoElectronics). Mice were ventilatedat a tidalvolume
of 0.2ml with the use of a ventilator (Harvard Apparatus) and frequency was set
around 150Hz.Baselinepulmonary mechanics and responses to ventilated saline
(0.9% NaCl) were measured, and lung resistance (RL) was measured in response
to increasing doses (0.125–40mgml21) of acetyl-b-methylcholine chloride
(methacholine; MCh) (Sigma-Aldrich).The threevaluesofRLobtainedaftereach
dose of methacholine were averaged to obtain the final values for each dose.
Results are expressed as percentage of increase of saline-baseline. Following
measurement of AHR, mouse tracheas were cannulated and the lungs were
lavaged twice with 1ml of PBS 2% FCS and the fluids were pooled. Cells in the
lavage fluid were counted using a haemocytometer, and BAL cell differential
counts were determined on slide preparations stained with DiffQuik. At least
200 cells were differentiated on stained slides by light microscopy using conven-
tional morphological criteria. For some experiments, BAL for each mouse or
grouped BAL was stained and analysed by flow cytometry.
Stool sample collection. Fresh stool samples were induced directly into sterile
collection tubes from live CNS12and control mice and snap frozen before
preparation of material for sequencing (see below).
DNA extraction. DNA extraction was performed on each fecal specimen using
phenol-chloroform extraction with mechanical disruption based on a previously
described protocol33Briefly, an aliquot (,500mg) of each sample was suspended
inasolutioncontaining500mlofextractionbuffer(200mMTris,pH8.0;200mM
NaCl; and 20mM EDTA), 210ml of 20% SDS, 500ml of phenol/chloroform/
isoamyl alcohol (25:24:1), and 500ml of 0.1-mm-diameter zirconia/silica beads
(BioSpec Products). Microbial cells were lysed by mechanical disruption with a
bead beater (BioSpec Products) for 2min, after which two rounds of phenol/
chloroform/isoamyl alcohol extraction were performed. DNA was precipitated
with ethanoland resuspended in 50ml of nuclease-free water.DNAwas subjected
to additional purification with the QIAamp DNA Mini Kit (Qiagen).
PCR amplification and sequencing. For each sample, three replicate 25 ml PCR
amplifications were performed, each containing 5ng of purified DNA, 0.2mM
dNTPs,1.5mMMgCl2,1.25UPlatinumTaqDNApolymerase,2.5mlof103PCR
buffer, and 0.2mM each of broad-range bacterial forward and reverse primers as
described previously34, flanking the V1–V3 variable region. The primers were
modified to include adaptor sequences required for 454 sequencing, with the
addition of a unique 6–8 base barcode in the reverse primer. The forward primer
(59-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGAGTTTGATCCTGGCTC
AG-39) consisted of the 454 Lib-L primer B (underlined) and the broad-range
universal bacterial primer 8F (italics); the reverse primer (59-CCATCTCATCCC
TGCGTGTCTCCGACTCAGNNNNNNNATTACCGCGGCTGCTGG-39) con-
sisted of the 454 Lib-L primer A, barcode (NNNNNNN), and the broad-range
primer 534R (italics). Thecycling conditions were: 94uC for3min, then 25 cycles
of 94uC for 30s, 56uC for 30s, and 72uC for 1 min. The three replicate PCR
products were pooled and subsequently purified using the Qiaquick PCR
Purification Kit (Qiagen). The purified PCR products were sequenced unidirec-
tionallyona 454GSFLXTitaniumpyrosequencing platformfollowingthe Roche
454 recommended procedures.
Sequenceprocessingandanalysis.SequenceswereconvertedtostandardFASTA
format using Vendor 454 software. Sequences shorter than 200base pairs (bp),
containing undetermined bases or homopolymer stretches longer than 8bp, or
failing to align with the V1–V3 region were excluded from the analysis. Using the
454 base quality scores, which range from 0 to 40 (0 being an ambiguous base),
sequencesweretrimmedusingasliding-windowtechnique,suchthattheminimum
averagequalityscoreoverawindowof50basesneverdroppedbelow35.Sequences
weretrimmedfromthe39-enduntilthiscriterionwasmet.Sequenceswerealigned
to the V1–V3 region of the 16S gene, using as template the SILVA reference
alignment35and the Needleman-Wunsch algorithm with default scoring options.
Potentially chimaeric sequences were removed using the chimaera uchime pro-
gram36. Sequences were grouped into operational taxonomic units (OTUs) using
the average neighbour algorithm. Sequences with distance-based similarity of97%
or greater were assigned to the same OTU. For each fecal sample, OTU-based
microbial diversity was estimated by calculating the Shannon diversity index37.
Phylogenetic classification to genus level was performed for each sequence, using
the Bayesian classifier algorithm described by Wang and colleagues, using a
database of known 16S sequences generated by the Ribosomal Database Project
(RDP)38. For each experiment, data were analysed on each taxon level individu-
ally.ThecountdatawasrescaledusingDESeqRpackage39.Bacteriawithlessthan
10 mean count in both conditions were removed from further analysis and bac-
teriawithstatisticallysignificantdifferencesbetweentwoconditions(forexample,
WT and KO), were determined using binomial test (from DESeq package).
Bacteria with fold-change greater than two and FDR50.05 were declared
significant.
31. Stromnes, I. M. & Goverman, J. M. Active induction of experimental allergic
encephalomyelitis. Nature Protocols 1, 1810–1819 (2006).
32. Burich, A. et al. Helicobacter-induced inflammatory bowel disease in IL-10- and T
cell-deficient mice. Am. J. Physiol. Gastrointest. Liver Physiol. 281, G764–G778
(2001).
33. Ubeda, C. et al. Vancomycin-resistant Enterococcus domination of intestinal
microbiota is enabled by antibiotic treatment in mice and precedes bloodstream
invasion in humans. J. Clin. Invest. 120, 4332–4341 (2010).
34. Nossa,C.W.etal.Designof16SrRNAgeneprimersfor454pyrosequencingofthe
human foregut microbiome. World J. Gastroenterol. 16, 4135–4144 (2010).
35. Schloss, P. D. et al. Introducing mothur: open-source, platform-independent,
community-supported software for describing and comparing microbial
communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009).
36. Edgar, R. C. et al. UCHIME improves sensitivity and speed of chimera detection.
Bioinformatics 27, 2194–2200 (2011).
37. Magurran, A. E. Measuring Biological Diversity (Blackwell, 2004).
38. Wang, Q. et al. Naive Bayesian classifier for rapid assignment of rRNA sequences
into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007).
39. Anders, S. & Huber, W. Differential expression analysis for sequence count data.
Genome Biol. 11, R106 (2010).
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©2012