Immunity 24, 677–688, June 2006 ª2006 Elsevier Inc. DOI 10.1016/j.immuni.2006.06.002
ReviewTh17: An Effector CD4 T Cell Lineage
with Regulatory T Cell Ties
Casey T. Weaver,1,2,* Laurie E. Harrington,1
Paul R. Mangan,2Maya Gavrieli,3,4
and Kenneth M. Murphy3,4
1Department of Pathology
2Department of Microbiology
University of Alabama at Birmingham
Birmingham, Alabama 35294
3Department of Pathology and Center for Immunology
4Howard Hughes Medical Institute
Washington University School of Medicine
St. Louis, Missouri 63110
The naive CD4 T cell is a multipotential precursor with
defined antigen recognition specificity but substantial
ulatory lineages, contingent upon signals from cells of
the innate immune system. The range of identified ef-
fector CD4 T cell lineages has recently expanded with
description of an IL-17-producing subset, called Th17,
which develops via cytokine signals distinct from,
and antagonized by, products of the Th1 and Th2 line-
ages. Remarkably, Th17 development depends on the
pleiotropic cytokine TGF-b, which is also linked to reg-
ulatory T cell development and function, providing
a unique mechanism for matching CD4 T cell effector
and regulatory lineage specification. Here, we review
Th17 lineage development, emphasizing similarities
and differences with established effector and regula-
esis, and host defense.
The directed development of CD4 effector T cells by
cytokines elicited from pathogen-activated cells of the
innate immune system is a hallmark of adaptive immu-
nity. Until recently, the known universe of adaptive CD4
T cell responses has been encompassed by the Th1-
Th2 paradigm (Mosmann and Coffman, 1989; Murphy
and Reiner, 2002). Development of T helper 1 (Th1) cells,
lar pathogens, is coupled to the sequential actions of in-
terferon-g (IFN-g) and interleukin-12 (IL-12) (Hsieh et al.,
1993; Scharton and Scott, 1993). The development of
asites, is coupled to IL-4 (Min et al., 2004; Shinkai et al.,
2002). The divergence of Th1 and Th2 differentiation is
largely due tocrossregulatory effects ofthese polarizing
cytokines, providing a mechanism whereby first-line in-
nate immune defenses guide appropriate effector T cell
responses that, in turn, orchestrate the host response
to efficiently clear pathogens and establish long-lived
memory for enhanced recall responses. The benefits of
adaptive CD4 T cell responses, however, come at
acost. Inappropriate orpoorly controlledeffector Tcells
can cause host pathology and are particularly deleteri-
ous when directed against self or ubiquitous environ-
mental or commensal floral antigens, which, unlike
most pathogens, cannot be effectively cleared. In this
inflammatory disorders such as autoimmunity and al-
lergy, or atopy. Effector T cell responses are therefore
normally under stringent regulatory control.
Although a key mechanism whereby dysregulated
effector responses are avoided is through intrathymic
tally or physically sequestered from developing thymo-
cytes and because the recombinatorial capacity of anti-
gen-recognition receptors in lymphocytes is so robust.
Hence, evolutionary pressure tomatch the development
regulatory T cell programs has probably been critical to
subsets of regulatory T cells, or Tregs, have been de-
scribed, albeit with incompletely defined lineage rela-
tionships and functions at present (Figure 1). At least
one class of Tregs, so-called natural Tregs (nTregs), is
the product of a developmental lineage distinct from
Th1 and Th2 lineages and therefore represents the
first well-defined expansion of the CD4 T cell functional
represents the only additional effector CD4 T cell arm to
be described since the original discovery of Th1 and
Th2 two decades ago. Th17 cells are characterized by
the production of a distinct profile of effector cytokines,
including IL-17 (or IL-17A), IL-17F, and IL-6, and have
probably evolved to enhance host clearance of a range
of pathogens distinct from those targeted by Th1 and
Th2. Th17 cells develop via a pathway separate from
Th1 and Th2, but with several notable parallels to the
Th1 lineage that have led to some confusion over the
role of the Th1 cells in autoimmunity. The development
of Th17 effectors also shares with some Tregs a require-
ment for TGF-b, establishing an important link between
Th17 and Treg development. As a basis for understand-
ing recent advances in Th17 development and function,
we first highlight features of Th1, Th2, and Treg develop-
ment and function for comparison.
Th1 and Th2 Development: Mechanisms for Lineage
Since the advent of T cell receptor (TCR) transgenic
mouse models, it has been established that naive T cells
functional effectors, e.g., Th1 and Th2, contingent upon
early signals received in concert with antigen (Hsieh
et al., 1992; Seder etal., 1992). There has been extensive
investigation of the factors and signaling pathways that
distinguish differentiation of Th1 and Th2 cells (see Mur-
phy and Reiner, 2002 for review), and although there is
now general consensus on many features that control
these developmental programs, certain details remain
contentious (Berenson et al., 2004). An important facet
with many developmental strategies, is the presence of
reiterative feedback mechanisms that propagate early
lineage decisions once initiated. Th1 differentiation is
initiated by coordinate signaling through the TCR and
STAT1-associated cytokine receptors. Both type I and
receptors (Hibbert et al., 2003; Lucas et al., 2003; Pflanz
et al., 2002), as can the IL-12 family member IL-27
(Hunter, 2005). Receptors for each of these cytokines
are expressed on naive T cell precursors and are acti-
vated by products of pathogen-stimulated cells of the
innate immune system. NK cells are a major source of
IFN-g, whereas plasmacytoid dendritic cells (DCs) are
the primary source of IFN-a. STAT1 signaling down-
to be a ‘‘master regulator’’ of Th1 differentiation (Mullen
tion factor Hlx is selectively expressed by Th1 cells by
virtue of its being a target of T-bet (Mullen et al., 2002)
(Zheng et al., 2004). T-bet potentiates expression of the
Ifng gene and upregulates the inducible chain of the
ated factors. Induction of a competent IL-12 receptor on
developing Th1 cells licenses IL-12 signaling through
STAT4, which further potentiates IFN-g production and
induces expression of IL-18Ra, thereby conferring re-
sponsiveness to IL-18 by mature Th1 cells. The IL-12-
driven component of Th1 development results in mature
dependent or -independent (IL-12 plus IL-18) pathways
(Robinson et al., 1997; Yang et al., 1999). Thus, the later
ture Th1 cells to produce IFN-g in an antigen-indepen-
dent mode, not unlike cells of the innate immune system
such as NK cells.
Although aT-bet-dependent pathway to Th1 develop-
ment has been well described, alternative pathways ap-
pear to exist. For example, the innate phase of the IFN-g
response during Listeria monocytogenes (LM) infection
is unaffected in the absence of T-bet (Way and Wilson,
2004). The adaptive immune response to LM showed
only a modest reduction in the numbers of Th1 cells or
IFN-g secretion by CD4 T cells, suggesting that alterna-
tive pathways can independently contribute to Th1 de-
velopment. Whether these include known pathways,
such as IL-12 and IL-18, or unknown pathways is un-
clear. Nevertheless, there may be greater plasticity in
Th2 differentiation is initiated by TCR signaling in con-
cert with IL-4 receptor signaling via STAT6. Signals that
emanate from the TCR and IL-4 receptors act coopera-
tively to upregulate low expression of GATA-3, a master
regulator of Th2 differentiation (Ouyang et al., 1998,
2000; Zheng and Flavell, 1997). GATA-3 autoactivates
its own expression and drives epigenetic changes in
the Th2 cytokine cluster (Il4, Il5, and Il13 genes) while
suppressing factors critical to the Th1 pathway, such
as STAT4 and the IL-12Rb2 chain. In addition, IL-4 sig-
naling prevents the colocalization of the TCR with IFN-g
receptors at the immunologic synapse of naive T cells
activated by APCs, suggesting another way in which IL-4
may inhibit Th1 development (Maldonado et al., 2004).
Thus, early IL-4 signaling rapidly initiates positive and
negative feedback loops that operate at a number of
levels to reinforce early commitment to Th2 develop-
ment while blocking Th1 development.
Figure 1. Diversification of CD4 T Cell Lineages
Although functional CD4 T cell development has been dominated by the Th1-Th2 paradigm for nearly two decades, the number of defined
lineages has now increased.The cytokines associated with arrows indicate dominant cytokines involved in specification of each of the indicated
lineages. The cytokines listed below each cell type indicate key effector or regulatory cytokines produced by differentiated cells of that lineage
or, in the case of nTreg, a contact-dependent mechanism of suppression. Tn: naive, postthymic CD4 T cell precursors; Tp: thymic precursors.
Dotted lines represent less well-defined lineage relationships.
Importantly, cytokines produced by mature effector
cells themselves can reinforce their own developmental
program through positive andnegative feedback, acting
produced by mature Th1 cells or innate immune cells
induces STAT1 signaling and T-bet expression in anti-
of the IL-12 receptor on developing Th1 cells and sup-
pression of GATA-3. Similarly, IL-4 produced by mature
Th2 cells initiates Th2 development through its upregu-
lation of GATA-3 via STAT6 and suppresses Th1 devel-
opment by blocking IL-12Rb2 expression. The action
of GATA-3 to promote its own transcription through
a cell-intrinsic positive feedback loop represents a po-
tent mechanism for rapidly stabilizing Th2 development.
Remarkably, a further layer of counterregulation may re-
side at the level of physical interactions between chro-
matin domains that contain Th1- or Th2-specific cyto-
kine genes. Direct contacts exist between juxtaposed
regulatory regions in the Th2 cytokine gene cluster on
chromosome 11 and the Ifng promoter on chromosome
10 in the mouse, providing the first demonstration of in-
terchromosomal interactions that could reciprocally
regulate genes involved in divergent lineages (Spiliana-
kis et al., 2005). As a result of such robust counterregu-
latory pathways, Th1 and Th2 development diverge rap-
idly after antigen priming to produce mature effectors
with stable, mutually exclusive expression of IFN-g or
Regulatory T Cells: Strategies for Controlling
Besides effector subsets, CD4 T cells can differentiate
into distinct regulatory subsets characterized by their
of regulatory T cells, nTregs, develops intrathymically,
mounting evidence indicates that other Tregs develop
from naive CD4 T cell precursors in the periphery, so-
called induced, or adaptive, Tregs (aTregs). nTregs ap-
pear to act preferentially in T cell zones of secondary
from naive precursors, thereby terminating effector CD4
T cell development before it begins. Obviously, if com-
pletely dominant, this mechanism would prevent adap-
tive responses to pathogens and risk destruction of
self by preventing the anti-pathogen response in favor
of the anti-self response. aTregs are distinguished by
their differentiation from naive CD4 T cells in peripheral
lymphoid tissues, where they may develop in parallel to
effector T cells and track them to sites of inflammation
to quell effector T cell-driven inflammation as patho-
gen-associated antigens are cleared. At least two types
of aTregs have been described. One, called Tr1, de-
velops under control of IL-10-conditioned DCs and is
marked by high amounts of IL-10 production; as dis-
cussed below, it does not express the transcription fac-
tor Foxp3 (Groux et al., 1997; Wakkach et al., 2003). An-
other is induced from naive precursors under the
influence of TGF-b; these cells are Foxp3+and display
suppressive activities that are indistinguishable from
nTregs, although they develop extrathymically (Chen
et al., 2003; Fantini et al., 2004). Additional types of
terized. There has been generous debate as to whether
these subsets represent truly distinct lineages or over-
lapping and flexible activation states, although recent
evidence that has emerged in parallel with new findings
on the differentiation of Th17 cells (considered below)
may provide some answers.
Following the original functional and phenotypic char-
acterization of Tregs by Sakaguchi and coworkers (re-
viewed in Sakaguchi, 2004), which linked suppressive
activity to a small subset of circulating T cells that stably
express the high-affinity component of the IL-2 receptor
CD25, insights into the developmental lineage of
CD4+CD25+nTregs have come from several studies
showing that the forkhead-winged helix transcription
andisrequiredfor theirdevelopment (Akbarietal.,2003;
Coffer and Burgering, 2004; Fontenot et al., 2003; Gavin
Foxp3 was reported initially as specific to Treg in mice
(Fontenot et al., 2003; Hori et al., 2003; Khattri et al.,
2003) and soon thereafter in humans (Walker et al.,
2003b). Foxp3 appears to bea master regulator of nTreg
development in mice and humans (Fontenot et al., 2003;
Hori et al., 2003; Khattri et al., 2003) insofar as all nTregs
ventional, CD4+CD252T cells induces Treg function and
phenotypic features, including the expression of CD25,
tor-related (GITR) protein, and CTLA4 (Fontenot et al.,
2003; Hori et al., 2003; Khattri et al., 2003; Walker et al.,
Foxp3 develop a lymphoproliferative autoinflammatory
disease caused by an absence of nTreg (Fontenot
et al., 2003; Khattri et al., 2003). A similar syndrome
et al., 2003b).
Subsequent identification of a subset of Foxp3+thy-
mocytes led to the identification of an intrathymic devel-
opmental program for Foxp3+Tregs. Although the pre-
cise mechanisms by which nTregs develop in the
thymus are unknown, results from TCR transgenic sys-
tems generally favor an ‘‘altered selection’’ model in
which development of nTregs requires higher-affinity
TCR interactions during positive selection that are not
so high affinity as to induce negative selection (Aposto-
lou et al., 2002; Bensinger et al., 2001; Jordan et al.,
2001; von Boehmer et al., 2003). Although nTreg devel-
opment was originally thought to require IL-2 and IL-2
signaling (Malek et al., 2002), recent studies indicate
that induction of Foxp3 expression and suppressive
function do not require IL-2 or CD25; rather, both are re-
quired for maintenance of nTreg homeostasis and func-
tion following thymic export (D’Cruz and Klein, 2005;
Fontenot et al., 2005). Thus, autoimmune disease that
is associated with the loss of Tregs in mice deficient for
IL-2, IL-2Ra, or IL-2Rb (Wolf et al., 2001; Almeida et al.,
2002; Malek et al., 2002) is due to poor maintenance of
opment. Notably, an important effect of deficient IL-2
signaling in peripheral Tregs was decreased expression
of TGF-b1 (Fontenot et al., 2005), which appears to be
necessary for maintenance of Foxp3 expression by this
population (Marie et al., 2005). Accordingly, it would
sion by nTregs in the extrathymic environment.
Although programming for Foxp3 expression was ini-
tablish the potential for extrathymic development of
Foxp3+Tregs from naive, CD4+CD252T cell precursors.
Activation of naive murine CD4 T cells in the presence of
TGF-b in vitro induced Foxp3, producing cells with sup-
pressiveactioninvivo (Chenetal., 2003).Similarly, TGF-
b reportedly induced Foxp3 expression in conventional
human CD4 T cells (Fantini et al., 2004). This study also
suggested an autoregulatory loop because TGF-b-in-
duced Foxp3 appeared to inhibit expression of Smad7,
an inhibitor of TGF-b signaling, thus augmenting TGF-
bsignaling. The useofFoxp3 reporter mice,inwhich de-
shown, clearly establish that this isnot due tooutgrowth
of contaminating Foxp3+cells within the CD4+CD252
fraction (Wan and Flavell, 2005). Additional studies
have extended these findings in vivo, particularly in set-
erance induction. Cells functionally and phenotypically
indistinguishable from nTregs developed from RAG-
deficient, Foxp32TCR transgenic precursors after con-
tinuous, low-dose administration of specific peptide
(Apostolou and von Boehmer, 2004), and induction of
Dby-specific transplantation tolerance resulting from in
vivo T cell coreceptor blockade was associated with
the TGF-b-dependent generation of Foxp3-expressing
T cells (Cobbold et al., 2004). In studies of oral tolerance
induction, the development of antigen-specific Foxp3+
tto deLafaille etal., 2004). Finally, Foxp3+cells with Treg
activity also develop from RAG-deficient, monospecific
naive T cell precursors following homeostatic prolif-
eration after transfer into lymphopenic hosts (Curatto
de Lafaille et al., 2004). Thus, although the association
between Foxp3 expression and Treg functional activity
remains valid, it appears that the induction of T cell
expression of Foxp3 may not be limited to the thymus.
In many cases, this appears to be dependent on the ac-
tions of TGF-b, although this has not been rigorously
tested in all settings.
Interestingly, although TGF-b can induce Foxp3 ex-
pression by naive, peripheral CD4 T cells, it does not ap-
pear to be required for intrathymic development of
nTregs. Using transgenic mice that express a domi-
control of the CD4 promoter, CD4+CD25+Tregs develop
and function normally, suggesting that TGF-b signaling
is not required for their intrathymic development (Fahlen
et al., 2005). A caveat here is the timing of ablation of
TGF-b signaling by dnTGF-bRII during thymic develop-
ment. However, normal numbers of Foxp3+nTregs are
found in thymuses of TGF-b1-deficient mice despite re-
duced numbers in the periphery (Marie et al., 2005),
again arguing against a requisite role for TGF-b in nTreg
development. Thus, current data indicate that although
TGF-b is dispensable for intrathymic expression of
Foxp3 and nTreg development, TGF-b is required for
the maintenance of Foxp3 expression and Treg fitness
in the extrathymic environment, and TGF-b can induce
sors. Accordingly, administration of TGF-b expands the
in vivo pool of antigen-specific CD25+Foxp3-express-
ing Treg cells (Peng et al., 2004), and transgenic overex-
in mice (Schramm et al., 2004).
In addition to its role in induction of adaptive Treg de-
velopment, TGF-bisinvolved inatleast some aspectsof
Treg function, although this is an area of considerable
controversy. The contact-dependent suppressor activ-
ity of CD4+CD25+T cells could be abrogated by incuba-
tion with TGF-b1 antibody in both mouse and human
Tregs (Nakamura et al., 2001, 2004). This finding could
be explained by the observation that activation of
CD4+CD25+T cells resulted in the expression of high
amounts of membrane bound TGF-b, mostly in latent
form. Since then, it has been reported that the activation
of CD4+CD25+T cells leads to the upregulation of TGF-
bRII and induces the secretion of TGF-b. Furthermore, it
has been shown that the administration of the TGF-b1-
peptide of TGF-b1 (rLAP) inhibits the suppressive activ-
ity of human and mouse CD4+CD25+T cells (Nakamura
et al., 2004). In a murine model of diabetes mediated
by CD8 T cells, TGF-b1-expressing Foxp3+Treg cells
selectively accumulated in pancreatic lymph nodes
and islets (Green et al., 2003) and suppressed patho-
genic CD8 T cells via a TGF-b-dependent mechanism.
Thus, adoptive transfers of CD4+CD25+Treg cells did
not suppress naive or activated islet-reactive CD8 T
cells bearing dnTGF-bRII. Similarly, the inhibition of
CD45RBhiCD4 T cells by Treg in an adoptive model of
colitis required TGF-b signaling (Fahlen et al., 2005). In-
terestingly, however, neither intact TGF-b signaling nor
TGF-b production was required by Treg for their ability
to suppress the CD45RBhipopulation, suggesting that,
at least in this model, Treg may act instead to induce
or process TGF-b production by other cells.
Given the dominance of Tregs in suppressing naive
and effector T cell functions in many experimental set-
tings, how is the effector T cell response recruited to
enhance pathogen clearance, or, how are the Tregs
themselves suppressed to allow protective effector
responses? Pasare and Medzhitov found that Treg
suppression was reversed in the setting of TLR-induced
activation of DCs, thereby linking pathogen-associated
molecular patterns, or PAMPs, to abrogation of Treg
dominance (Pasare and Medzhitov, 2003). Furthermore,
reversal of Treg suppression required TLR-induced,
MyD88-dependent production of IL-6 and an unidenti-
fied cofactor by DCs. This finding provided a vital link
between pathogen-induced activation of innate immune
cells and the release of Treg dominance to permit initia-
tionofaneffectorresponse byadaptiveimmunecells. In
an extension of these studies, we found that IL-1 syner-
gized with IL-6 to subvert Treg suppression through
siveness to IL-2 (Kubo et al., 2004). In these and other
studies (Yamazaki et al., 2003), TLR activation of DCs
also reversed the anergic state of Tregs, permitting their
robust proliferation while retaining their suppressive
function once proinflammatory cytokine production
subsided. Additional studies (Klein et al., 2003; Walker
et al., 2003a) showed that Tregs can proliferate in vivo
similarly to naive T cells after immunization without
losing suppressive function either in vivo or in vitro.
Thus, Treg suppression is reversed in the context of
pathogen-elicited activation of DCs through TLR-stimu-
lated production of IL-6 and IL-1, permitting linked re-
versal of Treg dominance to pathogen-induced activa-
tion of innate immune cells. Furthermore, the active
recruitment of Treg proliferation in the same setting pro-
vides a mechanism whereby the effector T cell response
is accompanied by an expanded pool of Tregs, which
are poised to reassert dominant suppression once the
inciting pathogen is cleared and ongoing development
of effector T cells ceases.
Th17: Effector T Cells with Immunopathogenic
The breakthrough leading to discovery of the Th17 line-
age came from murine models of autoimmunity. Experi-
mental autoimmune encephalitis (EAE) and collagen-
induced arthritis (CIA), two prototypical autoimmune
mouse models, have historically been associated with
unchecked Th1 responses, based largely on studies in
which disease development was ablated by treatment
with neutralizing antibodies specific for IL-12p40 or
gene-targeted mice deficient in the p40 subunit of IL-12
(Constantinescu et al., 1998; Leonard et al., 1995; Segal
came intoquestion withthediscoverybyOppmannetal.
that a new IL-12 family member, IL-23, shares with IL-12
dimer is composed of IL-12p40 and IL-12p35, whereas
theIL-23 heterodimeriscomposedoftheIL-12p40 chain
paired with IL-23p19. Given that key experimental data
linking EAE and CIA to Th1 autoimmunity were based
on protection associated with manipulations that tar-
geted the IL-12p40 subunit, it became unclear whether
protective effects were truly due to inhibition of IL-12 or
might involve IL-23. Indeed, data from a number of stud-
ies were inconsistent with a simple Th1 or IL-12–IFN-g
naling (Bettelli et al., 2004; Ferber et al., 1996; Kageyama
et al., 1998; Matthys et al., 1998, 1999; Willenborg et al.,
In an elegant series of studies, Cua and coworkers
resolved this paradox when they revisited the immuno-
pathologic basis for EAE and CIA using mice deficient
in IL-12, IL-23, or both (Cua et al., 2003; Murphy et al.,
2003). Strikingly, it was found that disease development
was ablated in mice deficient in IL-23, but not IL-12.
Thus, mice deficient in the IL-23p19 subunit (lacking
IL-23 only) or the IL-12p40 subunit (lacking both IL-23
and IL-12) were resistant to EAE and CIA, whereas
IL-12p35-deficient mice (lacking IL-12 only) remained
study that found that mice lacking the IL-12 receptor
complex also succumbed to EAE (Zhang et al., 2003).
Thus, it appears that IL-23, not IL-12, is critically linked
to autoimmunity in these models.
Clues to the pathogenic role for IL-23 came from anal-
yses of the cytokine phenotypes of effector CD4 T cells
primedwith typeIIcollageninthe CIA studiesbyMurphy
found that IL-23 elicited production of the proinflamma-
tory cytokine IL-17 from CD4 T cells of the effector and
memory phenotype (Aggarwal et al., 2003). IL-17, or IL-
17A, is the founding member of a six-member family of
cytokines (IL-17A–F; reviewed by Kolls and Linde ´n,
2004) that was initially described as a product of human
CD4 T cells and has been linked to a number of T cell-
driven inflammatory conditions. Notably, diminished fre-
from the draining lymph nodes of collagen-immunized,
IL-23p19-deficient mice that were protected from arthri-
tis development; a reciprocal pattern of IFN-g+and
veloped exacerbated disease compared to wild-type
controls. Thus, a positive correlation was established
between the availability of IL-23 and IL-17-producing
correlation was established between IL-12 and IFN-g-
with these findings, impaired joint inflammation was
reported in IL-17-deficient mice after type II collagen im-
also decreased disease severity (Koenders et al., 2005a;
of IL-17 in the joints exacerbated disease (Koenders
et al., 2005b; Lubberts et al., 2001). Collectively, these
data strongly implicated a role for the IL-23-induced
development of IL-17-producing effector T cells in auto-
immune inflammation; the production of classical Th1
cells alone did not induce disease.
More direct proof of a link between IL-17-producing T
cell effectors and immune pathogenesis came with data
showing that proteolipid protein peptide (PLP) primed
CD4 T cells enriched for production of IL-17 by culture
in IL-23 induced severe EAE in recipient mice after pas-
sive transfers, whereas Th1 cells enriched by culture in
fined functional and phenotypic differences between so-
that gene-expression profiles of these two subsets were
quite distinct. Thus, although IL-12-polarized cells, i.e.,
prototypic Th1 cells, preferentially expressed genes as-
sociated with cytotoxicity (IFN-g, FasL, and granzymes),
IL-23-polarized cells expressed genes associated with
chronic inflammation (IL-17, IL-17F, IL-6, TNF-a, and
proinflammatory chemokines). These results therefore
confirmed a new role for Th17 cells in immunopatho-
genesis and strongly suggested that Th1 and Th17 cells
represent distinct effector subsets that develop under
differential IL-12 or IL-23 conditioning.
Th17 Differentiation: A Distinct Developmental
Pathway and New Role for TGF-b in Adaptive
as a unique effector T cell subset were defined in the
foregoing studies, the pathway leading to Th17 differen-
tiation has begun to emerge only more recently and has
held some interesting surprises. Two distinct models of
Th17 differentiation were proposed after establishment
of the central role for Th17 cells in EAE and CIA (Bettelli
it was proposed that the early differentiation of Th1 and
Th17 from naive CD4 T cell precursors was shared, and
thus Th1 and Th17 diverged contingent upon selective
availability of IL-12 and IL-23 acting on a common
‘‘Th1 precursor’’ or ‘‘pre-Th1’’ intermediate that coex-
pressed IL-12 and IL-23 receptors. In the second model,
it was proposed that Th1 and Th17 differentiation were
nonoverlapping and represented distinct lineages. In
view of the fact that intact T-bet and STAT4 are strictly
required for disease development in EAE and CIA,
whereas IL-12, IFN-g, and STAT1 are not, a corollary of
the latter model predicts that T-bet and STAT4 contrib-
ute to disease development through Th17-independent
In a pair of reports (Harrington et al., 2005; Park et al.,
2005), direct support for a Th1-independent pathway of
Th17 differentiation was established (Figure 2). In our
studies, we found that IL-23 failed to induce IL-17 pro-
duction from Th1-polarized cells, indicating that Th1
cells are not IL-23 responsive. Furthermore, both type
II and type I interferons, which activate STAT1-induced
expression of T-bet and Th1 commitment, strongly in-
hibited Th17 development. Together, these findings in-
dicated that not only was the Th1 pathway nonpermis-
sive to Th17 development, but the Th1 product IFN-g
Parallel findings were made for the Th2 lineage: Th2-po-
larized cells were unresponsive to IL-23, and IL-4 po-
tently inhibited Th17 development. Indeed, neutraliza-
tion of IFN-g and IL-4—whether by blocking antibodies
orgenetic deficiency—was required toinduce apprecia-
ble IL-17-producing effectors under the conditions ex-
amined. Accordingly, we found that key signaling com-
ponents of Th1 and Th2 differentiation—STAT1, T-bet,
STAT4, and STAT6—were each dispensable for Th17
development. Park et al. extended these studies
in vivo, showing that immunization of mice deficient in
IFN-g or T-bet led to unimpaired Th17 development
(Park et al., 2005). In sum, these data established that
IL-17-producing effectors develop via a lineage that is
distinct from, and antagonized by, the Th1 and Th2 line-
ages. These findings provide potential explanations for
a number of the paradoxical effects observed in the de-
velopment of EAE and CIA in mice with Th1-lineage de-
fects. In view of the pathogenic potential of Th17 cells
and the potent suppressive effects of IFN-g on Th17 de-
velopment, it is now not surprising that mice with tar-
geted deficiencies of IFN-g, IFN-gR, IL-12, and STAT1
to the observed disease resistance of T-bet-deficient
mice, it is now apparent that this is probably not due
to a defect in Th17 development, implicating T cell-inde-
pendent mechanisms by which T-bet contributes to
Although IL-23 appears to be required for Th17-medi-
ated immunopathology, three new reports indicate that
IL-23 is not required for Th17 commitment (Bettelli
et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006).
In each of these studies, the development of IL-17-pro-
ducing effectors in a primary response in vitro was rela-
tively undiminished under conditions of IL-23 deficiency,
and Th17 development was not enhanced by addition of
that induced Th1 and Th17 differentiation, respectively
(Harrington et al., 2005). Thus, in striking parallel to Th1
development, Th17 development is initiated indepen-
dently of a requirement for signaling by an IL-12 family
of the variable component of the IL-12 receptor, IL-
12Rb2, the variable component of the IL-23 receptor,
ness. IL-23 signaling is therefore not required for Th17
commitment and early IL-17 production but instead ap-
pears to be important for amplifying and/or stabilizing
the Th17 phenotype. This is in agreement with early re-
ports that found that IL-23 was shown to augment IL-17
production from the memory pool of CD4 T cells, but
notfromnaivecells(Aggarwal etal., 2003).Inthisregard,
it is notable that IL-12 actions in Th1 development are
linked to additional differentiation events, and it is not
unlikely that this is paralleled for Th17. Specifically, IL-12
induces expression of the IL-18Ra chain, resulting in an
alternative pathway for IFN-g expression that is TCR
independent. Assuming that a similar pathway exists
downstream of IL-23 signaling in developing Th17 cells,
this could provide an important mechanism by which
Th17 effector function is augmented. In this regard, pre-
liminary studies from our lab indicate that both IL-1,
whose receptor shares signaling components with the
IL-18R, and IL-18 may act in concert with IL-23 to acti-
vate a TCR-independent pathway of IL-17 expression
(Y. Lee and C.T.W., unpublished data). Thus, in another
parallel with the Th1 lineage, Th17 cells appear to have
TCR-dependent and -independent mechanisms for ex-
pression of effector cytokines.
If IL-23 is not required for Th17 commitment, what is?
Remarkably, three independent groups that addressed
this question from different initial premises arrived at
the same conclusion: TGF-b is necessary for initiation
of Th17 differentiation (Bettelli et al., 2006; Mangan
tion that LPS-activated dendritic cells stimulate naive
T cell proliferation by subverting the suppressive activity
ofTregs(Kuboetal., 2004;Pasare and Medzhitov, 2003),
Veldhoen et al. showed that naive T cells activated in the
presence of CD4+CD25+Tregs exhibited suppressed
amounts of IFN-g and IL-2 production but expressed
high amounts of IL-17. Antibody blockade of TGF-b in
cultures of LPS-activated DCs, Tregs, and naive CD4
opment.Importantly,inaddition toTGF-b, differentiation
of IL-17-producing effector cells required soluble den-
driticcell factorselicitedbyTLR- andMyD88-dependent
signaling. In addition to TLR4, TLR3 and TLR9 signals
(elicited by LPS, polyI:C, and CpG, respectively) were ef-
fective at inducing DC factors that could act in concert
with TGF-b to differentiate Th17cells.Strikingly, analysis
of DC-derived factors that acted in concert with TGF-b
led to the identification of IL-6 as a critical cofactor.
Thus, the same inflammatory cytokine previously identi-
fiedas asignalfor reversalofTregsuppressionwasnow
Using an APC-free culture system, it was shown that
Th17 development could be reconstituted by TGF-b and
IL-6 alone, indicating that other DC-drive factors (e.g.,
costimulators) were not required. IL-1b and TNF-a were
found to amplify the Th17 response induced by TGF-b and
Finally, an analysis of transcription-factor expression
by IL-17-producing T cells showed that Th17 effectors
polarized cells and GATA-3 compared to Th2-polarized
cells, supporting and extending previous findings that
distinct from Th1 and Th2 (Harrington et al., 2005; Park
et al., 2005).
We independently identified TGF-b as a critical factor
for Th17 commitment (Mangan et al., 2006). Following
the identification of IFN-g and IL-4 as potent inhibitors
mote Th17 development through its effects to inhibitTh1
and Th2 development and the cytokines driving Th1 and
Th2 specification. The addition of exogenous TGF-b1 to
primary cultures of naive CD4 T cells induced a modest
but appreciable population of Th17 effectors, which
was markedly enhanced in the presence of neutralizing
antibodies to IL-4 and IFN-g. Using IFN-g-deficient
APCs andT cells, or IFN-gR-deficientT cells, exogenous
TGF-b induced even greater Th17 development, inde-
pendently of IL-23. Importantly, TGF-b upregulated the
IL-23R component of the IL-23 receptor, in contrast to
the effects of IFN-g, which upregulated the IL-12Rb2
component oftheIL-12 receptor but notIL-23R.Accord-
ingly, the antagonistic effects of TGF-b versus IFN-g sig-
naling early in the activation of naive T cells deviates
lineage development toward Th17 or Th1, with concom-
itant upregulation of the inducible components of the
IL-23 or IL-12 receptor, respectively.
Because TGF-b had been previously linked to the de-
velopment of Foxp3+Tregs, we examined the pheno-
types of T cell populations generated by exogenous
TGF-b addition; we found that an appreciable fraction
of Foxp3+T cells were induced, albeit minor in compar-
ison to the numbers of IL-17-positive cells. Notably, ex-
pression of IL-17 or Foxp3 was restricted to separate
subsets; thus, TGF-b-driven Th17 and Treg develop-
ment from naive precursors were mutually exclusive.
Importantly, we found that under conditions where IL-6
was supplemented, development of Foxp3+cells was
eliminated. Conversely, blockade of IL-6 permitted en-
hanced development of the Foxp3+subpopulation, sug-
gesting that IL-6 blocked Treg development while en-
hancing Th17 development.
To examine the requirement for TGF-b in Th17 devel-
opment in vivo, we explored TGF-b1-deficient mice.
Mice homozygous for TGF-b1 deficiency were essen-
tially devoid of Th17 cells, which are normally enriched
in the lamina propria of the intestine and in mesenteric
lymph nodes (unpublished data; Stark et al., 2005).
Mice hemizygous for TGF-b1 deficiency showed an
intermediate phenotype compared to controls, and
circulating amounts of IL-17 correlated with these
phenotypes. Thus, although not directly establishing
an in vivo link between TGF-b and Th17 development,
these studies support a critical role for TGF-b1 in the
development of Th17 cells.
In a final, independent set of studies, Kuchroo and co-
pendent and also showed that the addition of IL-6 con-
trolled the relative frequency of Th17 versus Tregs that
developed in the presence of TGF-b (Bettelli et al.,
2006). Using Foxp3-EGFP knockin reporter mice to track
cells were induced to express Foxp3 upon activation in
the presence of TGF-b. In a survey that examined the ef-
fects of proinflammatory cytokines on the development
of Foxp3+T cells, only IL-6 potently suppressed the fre-
quency of cells expressing Foxp3; cocultures of TGF-b
with IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-18, and
TNF-a did not appreciably suppress the frequency of
Foxp3+T cells that developed from naive precursors.
When the phenotype of the cells cultured with TGF-b plus
IL-6 was examined, a majority were IL-17+. Thus, in agree-
ment with our own studies, IL-6 appeared to divert the
development of Foxp3+regulatory cells towards the
Th17 lineage, an effect that was independent of IL-23.
The addition of IL-6 therefore suppressed the TGF-b-
induced generation Foxp3+Tregs, while reciprocally pro-
were supplemented by the finding that resistance of EAE
development in IL-6-deficient mice was correlated with
a deficiency of IL-17+cells in EAE infiltrates.
Figure 2. Model of Branching Th17 and
Adaptive Treg Lineage Development
This model emphasizes distinct pathways
leading to mature Th17 effector cells or
Foxp3+adaptive Tregs (aTreg), induced by
tial effects of IL-6 and IL-23. Naive CD4 T cells
(Tn) activated by antigen presented on imma-
ture DCs that do not produce IL-6 production
are induced by TGF-b to express Foxp3 and
develop into aTregs (top panel). Tns activated
6 are induced by TGF-b to upregulate IL-23R
and become competent for IL-17 production
sponsiveness to IL-18 and IL-1, which can act
synergistically with IL-23 to induce Th17 cyto-
tion. Alternatively, TCR stimulation by antigen
can induce Th17 cytokine production directly,
without a requirement for IL-23, IL-1, or IL-18.
Dotted lines indicate possible positive feed-
back loops by which cytokine products of
Th17 (IL-6) or aTreg cells (TGF-b1) may rein-
force lineage development.
The foregoing studies identified a critical role for TGF-
b, and IL-6, in Th17 development and suggested that IL-
23 likely functioned subsequent to Th17 commitment,
perhaps to expand committed Th17 effectors or main-
tain and extend their function. In view of the in vitro find-
ings that IL-17 effectors could develop independently of
IL-23, we reexamined the requirements for IL-23 in vivo
using an infectious model based on sublethal challenge
tium, for which we had found an intact IL-23–IL-17 axis
essential for host protection (Mangan et al., 2006). In
wild-type mice, challenge with C. rodentium stimulated
a rapid, robust CD4 T cell effector response in the distal
colon and draining lymph nodes that was dominated by
Th17 cells. Notably, upon inoculation of IL-23p19-defi-
cient mice with this pathogen, comparable, robust
Th17 responses were identified despite the absence of
IL-23. However, unlike the wild-type controls, which
cleared the infection and resolved the colonic inflamma-
tion, IL-23-deficient animals had a markedly impaired
inflammatory response in the colonic tissues, failed to
clear the pathogen, and rapidly succumbed to infection,
indicating that induction of IL-17-producing effectors
alone was inadequate for host protection. Thus, while
IL-23 is dispensable for the differentiation of IL-17-com-
petent T cells in vitro and in vivo, it is indispensable for
a fully protective Th17 response. This could be due to
limitations of a positive feedback loop that upregulates
proinflammatory cytokines induced by Th17 cells (Fig-
ure 2), especially IL-6, IL-1, and TNF-a.
The remarkable balancing act of adaptive immunity—
how to facilitate the targeted destruction of pathogens
withoutexcessive collateral damage toself—isnowhere
better exemplified than in the shared use of TGF-b in
controlling the newly described Th17 effector lineage
and adaptive Treg development. It is perhaps fitting
that TGF-b should be central to this yin/yang interplay,
given its complex and often apparently inconsistent bi-
ology (Wahl, 1994).Wenow haveaglimpseofanelegant
and self-correcting or homeostatic mechanism, wherein
the same factor driving one response drives a compen-
satory response that controls or terminates the initial re-
sponse. Thus, in a setting of pathogen-driven inflamma-
tion, naive T cells that recognize foreign antigen may
receive signals from regulatory T cells themselves
(TGF-b) to initiate Th17 development, while the patho-
gen-induced signal that acts in concert with TGF-b to
switch the response to a protective Th17 response (IL-
6) also subverts Treg dominance and drives expansion
of Tregs that could be poised to terminate further T ef-
tiating the inflammatory response is cleared. Alterna-
tively, the production of TGF-b, contributed perhaps
by the turnover of effete, apoptotic host cells under nor-
mal, nonpathogenic states (Chen et al., 2001), may initi-
ate the development of Tregs that could maintain dom-
inant suppressive function in the absence of pathogenic
challenges but contribute to Th17 effector development
when necessary. The unique role of TGF-b is made all
the more intriguing by the fact that it is also required
for the maintenance of Foxp3+Tregs in the periphery,
induced through the actions of IL-2 (Fontenot et al.,
2005; Marie et al., 2005).
Although the complexities of TFG-b biology remain
a formidable challenge, the newly identified links be-
tween TGF-b and Th17 development promise the solu-
tion of many conundrums that have precluded a com-
plete understanding of autoimmune pathogenesis.
Theyalso raisemanyintriguing questions. First,elucida-
tion of this new effector lineage and the description of
cytokine factors driving its development beg the ques-
tion of which transcription factor (or factors) controls
Th17 lineage specification. For Th1, Th2, and Treg line-
ages, key transcription factors have been identified
that specify the genotypic and phenotypic characteris-
tics of these lineages. Thus, T-bet specifies Th1,
GATA-3 specifies Th2, and Foxp3 specifies Treg devel-
opment. Is there an analogous factor that links TGF-
b and IL-6 signaling to Th17 development? Related to
this, how do TGF-b and IL-6 signaling cooperate to
specify Th17 commitment, whereas the absence of IL-
6induces Foxp3,therebyspecifying Tregdevelopment?
Given the abundance of existing data on signaling path-
ways that TGF-b and IL-6 recruit and the target-gene re-
to be answered soon. Our current understanding of
competitive inhibition of the TGF-b and IFN-g signaling
cascades offers good predictions as to how IFN-g acts
on the naive T cell to inhibit Th17 development (Ulloa
et al., 1999) and vice versa (Lin et al., 2005). It is likely
that similar considerations of intersections of the
STAT3-mediated IL-6 signaling pathway with the
pathway will contribute additional new insights.
Although discovery of the Th17 lineage originated in
revisionist studies of autoimmune mechanisms, its true
origins no doubt lie in the evolutionary pressures to pro-
tect vertebrates from certain classes of microbial patho-
gens (McKenzie et al., 2006). In retrospect, it is apparent
that the Th1-Th2 paradigm was insufficient to encom-
pass the entire spectrum of pathogenic challenges. As
the classes of pathogens for which Th1 and Th2 are pro-
tective became better defined—primarily those adapted
for intracellular survival in phagocytes for at least a por-
tion of their life cycle, or parasites such as helminths, re-
spectively—it became increasingly apparent that Th1
and Th2 cells offer protection against only a portion of
the pathogen landscape, implying the existence of addi-
tional effector lineages. Almost certainly, therefore, the
Th17 lineage evolved to control certain classes of path-
ogens not covered by Th1 and Th2. Given the growing
association of IL-23 and/or IL-17 to host protection in
a number of bacterial infection models (e.g., Klebsiella
pneumoniae, Borrelia burgdorferi, Bordetella pertussis,
and Citrobacter rodentium), it is likely that Th17 cells
evolved to cope with a range of extracellular bacterial
pathogens (Fedele et al., 2005; Happel et al., 2005; In-
fante-Duarte et al., 2000; Mangan et al., 2006) as well
tissues such as the gut, which are colonized by abun-
dant commensal bacterial species. Th17 cells may
also play an important role in the clearance of fungi, al-
though more studies will be needed to define the range
of pathogens linked to this lineage. In this regard, our
own recent studies have found that mice deficient in
IL-6 fail to develop a protective Th17 response upon
challenge with C. rodentium, establishing a critical role
for this cytokine in Th17 development induced by infec-
tion (D. O’Quinn, P.R.M., and C.T.W., unpublished data).
The links between IL-6, Th17 development, and host
protection offer new possibilities for improved vaccine
development that were previously unappreciated.
If TGF-b and IL-6 can induce IL-17-producing effec-
tors independently of IL-23, what is the precise role of
IL-23 in amplifying or maintaining Th17 responses in
vivo, and where does it act? The studies of Cua and co-
workers clearly identify a requirement for IL-23 in Th17-
dependent autoimmune pathogenesis, and there are
ment in at least some bacterial infections. Hence, Th17
lineage commitment is independent of IL-23, although
host protection and autoimmunity linked to Th17 devel-
opment are not. This indicates that IL-23 signaling in
developing Th17 cells is necessary in vivo for critical
downstream actions on Th17 precursors; whether this
is related to Th17 survival, cell number amplification,
or enhanced functional properties is yet to be defined.
Direct effects on non-T cells are also possible. In this re-
gard,itwill beimportant todefineatwhatanatomicsites
IL-23 actions on Th17 precursors occur. While it has
been assumed that IL-23 actions are exerted at sites
of T effector cell induction—i.e., T cell zones of second-
ary lymphoid tissues—in fact, we have very little infor-
mation regarding the temporal and spatial effects of
IL-12 family-member actions in situ. The principal site
with an important role for IL-23 in homeostasis to the in-
testinal flora but also raising the possibility that IL-23
acts primarily at effector sites, rather than inductive
sites, whether to recruit Th17 cells, enhance their func-
tion, or prevent their death. Given the finding that IL-1
and IL-18 can synergize with IL-23 to induce potent
IL-17 production from Th17-polarized effectors inde-
lished data), a potent positive feedback loop with IL-23
at its epicenter exists at sites of inflammation.
Finally, what are the implications for the other arm of
the adaptive response, the B cell? The connection be-
tween Th17 and host protection against extracellular
bacteria has important implications for immunoglobulin
isotype switching. Because opsonizing antibodies rep-
resent a fundamental component of the adaptive re-
sponse for enhancing phagocytic clearance of extracel-
immunoglobulin class switching that favors targeting to
activating Fc receptors on phagocytic cells. In this re-
gard, it is notable that TGF-b has long been recognized
as a cytokine favoring class switching of IgA and
IgG2b, Ig subclasses important in mucosal barrier func-
tion and antibacterial protection. This is contrasted with
Th1 cytokine IFN-g and IgG1 and IgE class switching by
the Th2 cytokine IL-4 (Stavnezer, 1996). IL-6 isalso a po-
tent B cell growth and differentiation factor. Thus, TGF-
b and IL-6, the cytokines critical for Th17 development,
have symmetrical links with B cell maturation and
class-switching functions aimed at blocking mucosal
colonization by commensal bacteria and clearing inva-
in IL-23- and IL-17A-deficient mice are defective (Ghi-
lardi et al., 2004; Nakae et al., 2002), although detailed
pathogen challenge studies have not yet been reported.
covery of this unique pathway and its intimate relation-
ship to T regulatory cell and B cell pathways, the foun-
dation has been laid for a new era in understanding
adaptive immune regulation, with its attendant opportu-
nities for improved therapies for host defense and
ful comments and suggestions. We also thank Noelle LeLievre for
editorial assistance. We offer our apologies to colleagues whose
work could not be adequately cited or discussed due to space lim-
itations. This work was supported by the NIH (grants AI35783,
AI57956, and DK64400 to C.T.W.), Sankyo Co. Ltd. (C.T.W.), and
the Howard Hughes Medical Institute (K.M.M.).
Aggarwal, S., Ghilardi, N., Xie, M.H., de Sauvage, F.J., and Gurney,
A.L. (2003). Interleukin-23 promotes a distinct CD4 T cell activation
state characterized by the production of interleukin-17. J. Biol.
Chem. 278, 1910–1914.
Akbari, O., Stock, P., DeKruyff, R.H., and Umetsu, D.T. (2003). Role
of regulatory T cells in allergy and asthma. Curr. Opin. Immunol.
Almeida, A.R., Legrand, N., Papiernik, M., and Freitas, A.A. (2002).
Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape
a population of regulatory cells that controls CD4+ T cell numbers.
J. Immunol. 169, 4850–4860.
Apostolou, I., and von Boehmer, H. (2004). In vivo instruction of sup-
pressor commitment in naive T cells. J. Exp. Med. 199, 1401–1408.
Apostolou, I., Sarukhan, A., Klein, L., and von Boehmer, H. (2002).
Origin of regulatory T cells with known specificity for antigen. Nat.
Immunol. 3, 756–763.
Becker, C., Wirtz, S., Blessing, M., Pirhonen, J., Strand, D., Becht-
hold, O., Frick, J., Galle, P.R., Autenrieth, I., and Neurath, M.F.
(2003). Constitutive p40 promoter activation and IL-23 production
in the terminal ileum mediated by dendritic cells. J. Clin. Invest.
Bensinger, S.J., Bandeira, A., Jordan, M.S., Caton, A.J., and Laufer,
T.M. (2001). Major histocompatibility complex class II-positive corti-
cal epithelium mediates the selection of CD4(+)25(+) immunoregula-
tory T cells. J. Exp. Med. 194, 427–438.
Berenson, L.S., Ota, N., and Murphy, K.M. (2004). Issues in T-helper
1 development–resolved and unresolved. Immunol. Rev. 202, 157–
Bettelli, E., and Kuchroo, V.K. (2005). IL-12- and IL-23-induced
T helper cell subsets: birds of the same feather flock together.
J. Exp. Med. 201, 169–171.
Bettelli, E., Sullivan, B., Szabo, S.J., Sobel, R.A., Glimcher, L.H., and
Kuchroo, V.K. (2004). Loss of T-bet, but not STAT1, prevents the de-
velopment of experimental autoimmune encephalomyelitis. J. Exp.
Med. 200, 79–87.
Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M.,
Weiner, H.L., and Kuchroo, V.K. (2006). Reciprocal developmental
pathways for the generation of pathogenic effector TH17 and regu-
latory T cells. Nature 441, 235–238.
Chen, W., Frank, M.E., Jin, W., and Wahl, S.M. (2001). TGF-beta re-
leased by apoptotic T cells contributes to an immunosuppressive
milieu. Immunity 14, 715–725.
Chen, W., Jin, W., Hardegen, N., Lei, K.J., Li, L., Marinos, N.,
McGrady, G., and Wahl, S.M. (2003). Conversion of peripheral
CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by
TGF-beta induction of transcription factor Foxp3. J. Exp. Med. 198,
Cobbold, S.P., Castejon, R., Adams, E., Zelenika, D., Graca, L.,
Humm, S., and Waldmann, H. (2004). Induction of foxP3+ regulatory
T cells in the periphery of T cell receptor transgenic mice tolerized to
transplants. J. Immunol. 172, 6003–6010.
Coffer, P.J., and Burgering, B.M. (2004). Forkhead-box transcription
factors and their role in the immune system. Nat. Rev. Immunol. 4,
Constantinescu, C.S., Wysocka, M., Hilliard, B., Ventura, E.S., Lavi,
E., Trinchieri, G., and Rostami, A. (1998). Antibodies against IL-12
prevent superantigen-induced and spontaneous relapses of exper-
imental autoimmune encephalomyelitis. J. Immunol. 161, 5097–
Cua, D.J., Sherlock, J., Chen, Y., Murphy, C.A., Joyce, B., Seymour,
B., Lucian, L., To, W., Kwan, S., Churakova, T., et al. (2003). Inter-
leukin-23 rather than interleukin-12 is the critical cytokine for auto-
immune inflammation of the brain. Nature 421, 744–748.
Curatto de Lafaille, M.A., Lino, A.C., Kutchukhidze, N., and Lafaille,
J.J. (2004). CD25- T cells generate CD25+Foxp3+ regulatory T cells
by peripheral expansion. J. Immunol. 173, 7258–7268.
D’Cruz, L.M., and Klein, L. (2005). Development and function of
agonist-induced CD25+Foxp3+ regulatory T cells in the absence
of interleukin 2 signaling. Nat. Immunol. 6, 1152–1159.
Fahlen, L., Read, S., Gorelik, L., Hurst, S.D., Coffman, R.L., Flavell,
R.A., and Powrie, F. (2005). T cells that cannot respond to TGF-
beta escape control by CD4(+)CD25(+) regulatory T cells. J. Exp.
Med. 201, 737–746.
Fantini, M.C., Becker, C., Monteleone, G., Pallone, F., Galle, P.R.,
and Neurath, M.F. (2004). Cutting edge: TGF-beta induces a regula-
tory phenotype in CD4+CD25- T cells through Foxp3 induction and
down-regulation of Smad7. J. Immunol. 172, 5149–5153.
Fedele, G., Stefanelli, P., Spensieri, F., Fazio, C., Mastrantonio, P.,
and Ausiello, C.M. (2005). Bordetella pertussis-infected human
monocyte-derived dendritic cells undergo maturation and induce
Th1 polarization and interleukin-23 expression. Infect. Immun. 73,
Ferber, I.A., Brocke, S., Taylor-Edwards, C., Ridgway, W., Dinisco,
C., Steinman, L., Dalton, D., and Fathman, C.G. (1996). Mice with
a disrupted IFN-gamma gene are susceptible to the induction of ex-
perimental autoimmune encephalomyelitis (EAE). J. Immunol. 156,
Fontenot, J.D., Gavin, M.A., and Rudensky, A.Y. (2003). Foxp3 pro-
grams the development and function of CD4+CD25+ regulatory
T cells. Nat. Immunol. 4, 330–336.
Fontenot, J.D., Rasmussen, J.P., Gavin, M.A., and Rudensky, A.Y.
(2005). A function for interleukin 2 in Foxp3-expressing regulatory
T cells. Nat. Immunol. 6, 1142–1151.
Gavin, M., and Rudensky, A. (2003). Control of immune homeostasis
bynaturally arisingregulatory CD4+ T cells. Curr. Opin. Immunol. 15,
Ghilardi, N., Kljavin, N., Chen, Q., Lucas, S., Gurney, A.L., and De
Sauvage, F.J. (2004). Compromised humoral and delayed-type hy-
persensitivity responses in IL-23-deficient mice. J. Immunol. 172,
Green, E.A., Gorelik, L., McGregor, C.M., Tran, E.H., and Flavell, R.A.
(2003). CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells
through TGF-beta-TGF-beta receptor interactions in type 1 diabe-
tes. Proc. Natl. Acad. Sci. USA 100, 10878–10883.
Groux, H., O’Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de
Vries,J.E., andRoncarolo, M.G.(1997).ACD4+T-cell subsetinhibits
antigen-specific responses and prevents colitis. Nature 389, 737–
Happel, K.I., Dubin, P.J., Zheng, M., Ghilardi, N., Lockhart, C., Quin-
ton, L.J., Odden, A.R., Shellito, J.E., Bagby, G.J., Nelson, S., and
Kolls, J.K. (2005). Divergent roles of IL-23 and IL-12 in host defense
against Klebsiella pneumoniae. J. Exp. Med. 202, 761–769.
Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy,
T.L., Murphy, K.M., and Weaver, C.T. (2005). Interleukin 17-produc-
ing CD4+ effector T cells develop via a lineage distinct from the
T helper type 1 and 2 lineages. Nat. Immunol. 6, 1123–1132.
Hibbert, L., Pflanz, S., De Waal Malefyt, R., and Kastelein, R.A.
(2003). IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce
T-Bet and IL-12Rbeta2 in naive T cells. J. Interferon Cytokine Res.
Hori, S., Nomura, T., and Sakaguchi, S. (2003). Control of regulatory
T cell development by the transcription factor Foxp3. Science 299,
Hsieh, C.S., Heimberger, A.B., Gold, J.S., O’Garra, A., and Murphy,
K.M. (1992). Differential regulation of T helper phenotype develop-
ment by interleukins 4 and 10 in an alpha beta T-cell-receptor trans-
genic system. Proc. Natl. Acad. Sci. USA 89, 6065–6069.
Hsieh, C.S., Macatonia, S.E., Tripp, C.S., Wolf, S.F., O’Garra, A., and
produced by Listeria-induced macrophages. Science 260, 547–549.
Hunter, C.A. (2005). New IL-12-family members: IL-23 and IL-27,
cytokines with divergent functions. Nat. Rev. Immunol. 5, 521–531.
Microbial lipopeptides induce the production of IL-17 in Th cells.
J. Immunol. 165, 6107–6115.
Jordan, M.S., Boesteanu, A., Reed, A.J., Petrone, A.L., Holenbeck,
A.E., Lerman, M.A., Naji, A., and Caton, A.J. (2001). Thymic selection
of CD4+CD25+ regulatory T cells induced by an agonist self-pep-
tide. Nat. Immunol. 2, 301–306.
Kageyama, Y., Koide, Y., Yoshida, A., Uchijima, M., Arai, T., Miya-
moto, S.,Ozeki, T., Hiyoshi, M., Kushida, K.,and Inoue, T. (1998). Re-
duced susceptibility to collagen-induced arthritis in mice deficient in
IFN-gamma receptor. J. Immunol. 161, 1542–1548.
Klein, L., Khazaie, K., and von Boehmer, H. (2003). In vivo dynamics
of antigen-specific regulatory T cells not predicted from behavior
in vitro. Proc. Natl. Acad. Sci. USA 100, 8886–8891.
Koenders, M.I., Lubberts, E., Oppers-Walgreen, B., van den Bersse-
laar, L., Helsen, M.M., Di Padova, F.E., Boots, A.M., Gram, H., Joos-
ten, L.A., and van den Berg, W.B. (2005a). Blocking of interleukin-17
during reactivation of experimental arthritis prevents joint inflamma-
tion and bone erosion by decreasing RANKL and interleukin-1. Am.
J. Pathol. 167, 141–149.
Koenders, M.I., Lubberts, E., Oppers-Walgreen, B., van den Bersse-
laar, L., Helsen, M.M., Kolls, J.K., Joosten, L.A., and van den Berg,
W.B. (2005b). Induction of cartilage damage by overexpression of
T cell interleukin-17A in experimental arthritis in mice deficient in
interleukin-1. Arthritis Rheum. 52, 975–983.
Kolls, J.K., and Linde ´n, A. (2004). Interleukin-17 family members and
inflammation. Immunity 21, 467–476.
Kubo, T., Hatton, R.D., Oliver, J., Liu, X., Elson, C.O., and Weaver,
C.T. (2004). Regulatory T cell suppression and anergy are differen-
tially regulated by proinflammatory cytokines produced by TLR-
activated dendritic cells. J. Immunol. 173, 7249–7258.
Langrish, C.L., Chen, Y., Blumenschein, W.M., Mattson, J., Basham,
B., Sedgwick, J.D., McClanahan, T., Kastelein, R.A., and Cua, D.J.
(2005). IL-23 drives a pathogenic T cell population that induces
autoimmune inflammation. J. Exp. Med. 201, 233–240.
Leonard, J.P., Waldburger, K.E., and Goldman, S.J. (1995). Preven-
tion of experimental autoimmune encephalomyelitis by antibodies
against interleukin 12. J. Exp. Med. 181, 381–386.
Lin, J.T., Martin, S.L., Xia, L., and Gorham, J.D. (2005). TGF-beta 1
uses distinct mechanisms to inhibit IFN-gamma expression in
CD4+ T cells at priming and at recall: differential involvement of
Stat4 and T-bet. J. Immunol. 174, 5950–5958.
Lubberts, E., Joosten, L.A., Oppers, B., van den Bersselaar, L., Coe-
nen-de Roo, C.J., Kolls, J.K., Schwarzenberger, P., van de Loo, F.A.,
and van den Berg, W.B. (2001). IL-1-independent role of IL-17 in sy-
novial inflammation and joint destruction during collagen-induced
arthritis. J. Immunol. 167, 1004–1013.
Lubberts, E., Koenders, M.I., Oppers-Walgreen, B., van den Bersse-
laar, L., Coenen-de Roo, C.J., Joosten, L.A., and van den Berg, W.B.
(2004). Treatment with a neutralizing anti-murine interleukin-17 anti-
body after the onset of collagen-induced arthritis reduces joint in-
flammation, cartilage destruction, and bone erosion. Arthritis
Rheum. 50, 650–659.
Lucas, S., Ghilardi, N., Li, J., and de Sauvage, F.J. (2003). IL-27 reg-
ulates IL-12 responsiveness of naive CD4+ T cells through Stat1-
dependent and -independent mechanisms. Proc. Natl. Acad. Sci.
USA 100, 15047–15052.
Maldonado, R.A., Irvine, D.J., Schreiber, R., and Glimcher, L.H.
(2004). A role for the immunological synapse in lineage commitment
of CD4 lymphocytes. Nature 431, 527–532.
Malek, T.R., Yu, A., Vincek, V., Scibelli, P., and Kong, L. (2002). CD4
regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient
mice. Implications for the nonredundant function of IL-2. Immunity
Mangan, P.R., Harrington, L.E., O’Quinn, D.B., Helms, W.S., Bullard,
D.C., Elson, C.O., Hatton, R.D., Wahl, S.M., Schoeb, T.R., and
Weaver, C.T. (2006). Transforming growth factor-beta induces
development of the T(H)17 lineage. Nature 441, 231–234.
beta1 maintains suppressor function and Foxp3 expression in
CD4+CD25+ regulatory T cells. J. Exp. Med. 201, 1061–1067.
Matthys, P., Vermeire, K., Mitera, T., Heremans, H., Huang, S., and
Billiau, A. (1998). Anti-IL-12 antibody prevents the development
and progression of collagen-induced arthritis in IFN-gamma recep-
tor-deficient mice. Eur. J. Immunol. 28, 2143–2151.
Matthys, P., Vermeire, K., Mitera, T., Heremans, H., Huang, S.,
Schols, D., De Wolf-Peeters, C., and Billiau, A. (1999). Enhanced
autoimmune arthritis in IFN-gamma receptor-deficient mice is
conditioned by mycobacteria in Freund’s adjuvant and by increased
expansion of Mac-1+ myeloid cells. J. Immunol. 163, 3503–3510.
the IL-23-IL-17 immune pathway. Trends Immunol. 27, 17–23.
Min, B., Prout, M., Hu-Li, J., Zhu, J., Jankovic, D., Morgan, E.S., Ur-
ban, J.F., Jr., Dvorak, A.M., Finkelman, F.D., LeGros, G., and Paul,
W.E. (2004). Basophils produce IL-4 and accumulate in tissues after
infection with a Th2-inducing parasite. J. Exp. Med. 200, 507–517.
Mosmann, T.R., and Coffman, R.L. (1989). Th1 and Th2 cells: Differ-
ent patterns of lymphokine secretion leads to different functional
properties. Annu. Rev. Immunol. 7, 145–173.
Mullen, A.C., High, F.A.,Hutchins, A.S., Lee,H.W., Villarino, A.V., Liv-
ingston, D.M., Kung, A.L., Cereb, N., Yao, T.P., Yang, S.Y., and
Reiner, S.L. (2001). Role of T-bet in commitment of TH1 cells before
IL-12-dependent selection. Science 292, 1907–1910.
Mullen, A.C., Hutchins, A.S., High, F.A., Lee, H.W., Sykes, K.J., Cho-
dosh, L.A., and Reiner, S.L. (2002). Hlx is induced by and genetically
interacts with T-bet to promote heritable T(H)1 gene induction. Nat.
Immunol. 3, 652–658.
Murphy, C.A., Langrish, C.L., Chen, Y., Blumenschein, W., McClana-
han, T., Kastelein, R.A., Sedgwick, J.D., and Cua, D.J. (2003). Diver-
gent pro- and antiinflammatory roles for IL-23 and IL-12 in joint au-
toimmune inflammation. J. Exp. Med. 198, 1951–1957.
Murphy, K.M., and Reiner, S.L. (2002). The lineage decisions of
helper T cells. Nat. Rev. Immunol. 2, 933–944.
Sekikawa, K., Asano, M., and Iwakura, Y. (2002). Antigen-specific
T cell sensitization is impaired in IL-17-deficient mice, causing sup-
pression of allergic cellular and humoral responses. Immunity 17,
Nakae, S., Nambu, A., Sudo, K., and Iwakura, Y. (2003). Suppression
of immune induction of collagen-induced arthritis in IL-17-deficient
mice. J. Immunol. 171, 6173–6177.
Nakamura,K.,Kitani, A., andStrober, W.(2001). Cell contact-depen-
dent immunosuppression by CD4(+)CD25(+) regulatory T cells is
mediated by cell surface-bound transforming growth factor beta.
J. Exp. Med. 194, 629–644.
Nakamura, K., Kitani, A., Fuss, I., Pedersen, A., Harada, N., Nawata,
H., and Strober, W. (2004). TGF-beta 1 plays an important role in the
mechanism of CD4+CD25+ regulatory T cell activity in both humans
and mice. J. Immunol. 172, 834–842.
Oppmann, B., Lesley, R., Blom, B., Timans, J.C., Xu, Y., Hunte, B.,
Vega, F., Yu, N., Wang, J., Singh, K., et al. (2000). Novel p19 protein
engages IL-12p40 to form a cytokine, IL-23, with biological activities
similar as well as distinct from IL-12. Immunity 13, 715–725.
Ouyang, W., Ranganath, S.H., Weindel, K., Bhattacharya, D., Mur-
phy, T.L., Sha, W.C., and Murphy, K.M. (1998). Inhibition of Th1
development mediated by GATA-3 through an IL-4-independent
mechanism. Immunity 9, 745–755.
Ouyang, W., Lohning, M., Gao, Z., Assenmacher, M., Ranganath, S.,
Radbruch, A., and Murphy, K.M. (2000). Stat6-independent GATA-3
autoactivation directs IL-4-independent Th2 development and com-
mitment. Immunity 12, 27–37.
Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y.H.,
Wang, Y., Hood, L., Zhu, Z., Tian, Q., and Dong, C. (2005). A distinct
lineage of CD4 T cells regulates tissue inflammation by producing
interleukin 17. Nat. Immunol. 6, 1133–1141.
Pasare, C., and Medzhitov, R. (2003). Toll pathway-dependent
blockade of CD4+CD25+ T cell-mediated suppression by dendritic
cells. Science 299, 1033–1036.
Peng, Y., Laouar, Y., Li, M.O., Green, E.A., and Flavell, R.A. (2004).
TGF-betaregulatesin vivo expansion
CD4+CD25+ regulatory T cells responsible for protection against
diabetes. Proc. Natl. Acad. Sci. USA 101, 4572–4577.
Pflanz, S., Timans, J.C., Cheung, J., Rosales, R., Kanzler, H., Gilbert,
J., Hibbert, L., Churakova, T., Travis, M., Vaisberg, E., et al. (2002).
IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein,
induces proliferation of naive CD4(+) T cells. Immunity 16, 779–790.
Robinson, D., Shibuya, K., Mui, A., Zonin, F., Murphy, E., Sana, T.,
Hartley, S.B., Menon, S., Kastelein, R., Bazan, F., and O’Garra, A.
(1997). IGIF does not drive Th1 development but synergizes with
IL-12 for interferon-gamma production and activates IRAK and
NFkappaB. Immunity 7, 571–581.
Sakaguchi, S. (2000). Regulatory T cells: Key controllers of immuno-
logic self-tolerance. Cell 101, 455–458.
Sakaguchi, S. (2004). Naturally arising CD4+ regulatory T cells for
immunologic self-tolerance and negative control of immune
responses. Annu. Rev. Immunol. 22, 531–562.
Scharton, T.M., and Scott, P. (1993). Natural killer cells are a source
of interferon gamma that drives differentiation of CD4+ T cell sub-
sets and induces early resistance to Leishmania major in mice.
J. Exp. Med. 178, 567–577.
Schramm, C., Huber, S., Protschka, M., Czochra, P., Burg, J.,
Schmitt, E., Lohse, A.W., Galle, P.R., and Blessing, M. (2004).
TGFbeta regulates the CD4+CD25+ T-cell pool and the expression
of Foxp3 in vivo. Int. Immunol. 16, 1241–1249.
Seder, R.A., Paul, W.E., Davis, M.M., and Fazekas de St Groth, B.
(1992). The presence of interleukin 4 during in vitro priming deter-
mines the lymphokine-producing potential of CD4+ T cells from
T cell receptor transgenic mice. J. Exp. Med. 176, 1091–1098.
Segal, B.M., Dwyer, B.K., and Shevach, E.M. (1998). An interleukin
(IL)-10/IL-12 immunoregulatory circuit controls susceptibility to
autoimmune disease. J. Exp. Med. 187, 537–546.
Shinkai, K., Mohrs, M., and Locksley, R.M. (2002). Helper T cells reg-
ulate type-2 innate immunity in vivo. Nature 420, 825–829.
Spilianakis, C.G., Lalioti, M.D., Town, T., Lee, G.R., and Flavell, R.A.
(2005). Interchromosomal associations between alternatively ex-
pressed loci. Nature 435, 637–645.
Stark, M.A., Huo, Y., Burcin, T.L., Morris, M.A., Olson, T.S., and Ley,
K. (2005). Phagocytosis of apoptotic neutrophils regulates granulo-
poiesis via IL-23 and IL-17. Immunity 22, 285–294.
Stavnezer, J. (1996). Immunoglobulin class switching. Curr. Opin.
Immunol. 8, 199–205.
Szabo, S.J., Kim, S.T., Costa, G.L., Zhang, X., Fathman, C.G., and
Glimcher, L.H. (2000). A novel transcription factor, T-bet, directs
Th1 lineage commitment. Cell 100, 655–669.
Ulloa,L.,Doody, J., andMassague,J.(1999).Inhibitionof transform-
ing growth factor-beta/SMAD signalling by the interferon-gamma/
STAT pathway. Nature 397, 710–713.
Veldhoen,M.,Hocking, R.J.,Atkins,C.J.,Locksley, R.M.,andStock-
inger, B. (2006). TGFbeta in the context of an inflammatory cytokine
milieu supports de novo differentiation of IL-17-producing T cells.
Immunity 24, 179–189.
von Boehmer, H., Aifantis, I., Gounari, F., Azogui, O., Haughn, L.,
Apostolou, I., Jaeckel, E., Grassi, F., and Klein, L. (2003). Thymic
selection revisited: how essential is it? Immunol. Rev. 191, 62–78.
Wahl, S.M. (1994). Transforming growth factor beta: the good, the
bad, and the ugly. J. Exp. Med. 180, 1587–1590.
Wakkach, A., Fournier, N., Brun, V., Breittmayer, J.-P., Cottrez, F.,
and Groux, H. (2003). Characterization of denritic cells that induce
tolerance and T regulatory 1 cell differentiation in vivo. Immunity
Walker, L.S., Chodos, A., Eggena, M., Dooms, H., and Abbas, A.K.
(2003a). Antigen-dependent proliferation of CD4+ CD25+ regulatory
T cells in vivo. J. Exp. Med. 198, 249–258.
Walker, M.R., Kasprowicz, D.J., Gersuk, V.H., Benard, A., Van Land-
eghen, M., Buckner, J.H., and Ziegler, S.F. (2003b). Induction of
FoxP3 and acquisition of T regulatory activity by stimulated human
CD4+CD25- T cells. J. Clin. Invest. 112, 1437–1443.
Wan, Y.Y., and Flavell, R.A. (2005). Identifying Foxp3-expressing
suppressor T cells with a bicistronic reporter. Proc. Natl. Acad.
Sci. USA 102, 5126–5131.
Way, S.S., and Wilson, C.B. (2004). Cutting edge: immunity and IFN-
gamma production during Listeria monocytogenes infection in the
absence of T-bet. J. Immunol. 173, 5918–5922.
Willenborg, D.O., Fordham, S., Bernard, C.C., Cowden, W.B., and
Ramshaw, I.A. (1996). IFN-gamma plays a critical down-regulatory
role in the induction and effector phase of myelin oligodendrocyte
glycoprotein-induced autoimmune encephalomyelitis. J. Immunol.
Wolf, M., Schimpl, A., and Hunig, T. (2001). Control of T cell hyperac-
tivation in IL-2-deficient mice by CD4(+)CD25(-) and CD4(+)CD25(+)
T cells: evidence for two distinct regulatory mechanisms. Eur. J. Im-
munol. 31, 1637–1645.
Yamazaki, S., Iyoda, T., Tarbell, K., Olson, K., Velinzon, K., Inaba, K.,
and Steinman, R.M. (2003). Direct expansion of functional CD25+
CD4+ regulatory T cells by antigen-processing dendritic cells.
J. Exp. Med. 198, 235–247.
Yang, J., Murphy, T.L., Ouyang, W., and Murphy, K.M. (1999). Induc-
tion of interferon-gamma production in Th1 CD4+ T cells: evidence
for two distinct pathways for promoter activation. Eur. J. Immunol.
and Rostami, A. (2003). Induction of experimental autoimmune
encephalomyelitis in IL-12 receptor-beta 2-deficient mice: IL-12
responsiveness is not required in the pathogenesis of inflammatory
demyelination in the central nervous system. J. Immunol. 170, 2153–
Zheng, W., and Flavell, R.A. (1997). The transcription factor GATA-3
is necessary and sufficient for Th2 cytokine gene expression in CD4
T cells. Cell 89, 587–596.
IFN-gamma expression. J. Immunol. 172, 114–122.