The Journal of Experimental Medicine
© 2008 Ma et al.
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 7 1551-1557 www.jem.org/cgi/doi/10.1084/jem.20080218
BRIEF DEFINITIVE REPORT
Appropriate immune responses to particular path-
ogens are orchestrated by distinct T cell popula-
tions with relatively stable cytokine expression
profi les that diff erentiate during antigen-specifi c
clonal proliferation. Such diff erentiation of CD4 +
T cells is determined by signals provided by com-
ponents of the innate immune system and is sub-
sequently stabilized by induction and repression
of key transcriptional regulators ( 1 ). Recently,
IL-17 – producing CD4 + T cells (Th17) have
been identifi ed as the key mediators of infl amma-
tion and tissue damage in several animal models
of human diseases (for review see references 1
and 2 ). The commitment of naive murine CD4 +
T cells to the Th17 lineage requires exposure to
TGF- ? and either IL-6 ( 1, 3, 4 ) or IL-21 ( 2, 5,
6 ). These cytokines act in a STAT3-dependent
manner to induce expression of retinoid-related
orphan receptor ? t (ROR ? t), the critical tran-
scription factor necessary for the development of
Th17 cells ( 7 – 10 ), which subsequently induces a
set of eff ector molecules, including IL-17, IL-21,
IL-22, and IL-23R plus several chemokines and
other defense molecules ( 1, 2 ). In contrast to
mice, much less is known about the requirements
for generating human Th17 cells. Indeed, al-
though there is consensus that TGF- ? is not re-
quired for this process, and is actually inhibitory,
controversy exists as to whether IL-6 or IL-23, in
combination with IL-1 ? , is the predominant cue
that mediates commitment of human naive CD4 +
T cells to the Th17 lineage ( 11, 12 ). Given these
diff erences, it is important to dissect further the
molecular requirements for the development of
human Th17 cells.
Although inappropriate Th17 diff erentiation
helps explain autoimmune disease and infl am-
mation, the physiological function of Th17 cells
appears to lie in host protection against specifi c
pathogens ( 1, 2 ). Based on the anatomical distri-
bution of Th17 cells and the capacity of IL-17 to
regulate granulopoiesis, neutrophil chemotaxis,
and production of microbiocidal peptides by
Stuart G. Tangye:
Matthew C. Cook:
S.G. Tangye and M.C. Cook contributed equally to this paper.
Defi ciency of Th17 cells in hyper IgE
syndrome due to mutations in STAT3
Cindy S. Ma , 1 Gary Y.J. Chew , 2,3 Nicholas Simpson , 3 Archana Priyadarshi , 4
Melanie Wong , 6 Bodo Grimbacher , 8 David A. Fulcher , 7 Stuart G. Tangye , 1
and Matthew C. Cook 2,3,5
1 Immunology and Infl ammation Program, Garvan Institute of Medical Research, Darlinghurst 2010, Australia
2 Australian National University Medical School and 3 John Curtin School of Medical Research, Australian National University,
Canberra 2600, Australia
4 Department of Paediatrics and Child Health, and 5 Department of Immunology, The Canberra Hospital, Woden 2606, Australia
6 Department of Immunology, Childrens ’ Hospital at Westmead, and 7 Department of Immunology, Institute of Clinical
Pathology and Medical Research, Westmead Hospital, Westmead 2145, Australia
8 Department of Immunology, Royal Free Hospital & University College London, London WC1E 6BT, England, UK
Hyper – immunoglobulin E syndrome (HIES) is a primary immune defi ciency characterized by
abnormal and devastating susceptibility to a narrow spectrum of infections, most commonly
Staphylococcus aureus and Candida albicans . Recent investigations have identifi ed muta-
tions in STAT3 in the majority of HIES patients studied. Despite the identifi cation of the
genetic cause of HIES, the mechanisms underlying the pathological features of this disease
remain to be elucidated. Here, we demonstrate a failure of CD4 + T cells harboring heterozy-
gous STAT3 mutations to generate interleukin 17 – secreting (i.e., T helper [Th]17) cells in
vivo and in vitro due to a failure to express suffi cient levels of the Th17-specifi c transcrip-
tional regulator retinoid-related orphan receptor ? t. Because Th17 cells are enriched for
cells with specifi cities against fungal antigens, our results may explain the pattern of
infection susceptibility characteristic of patients with HIES. Furthermore, they underscore
the importance of Th17 responses in normal host defense against the common pathogens
S. aureus and C. albicans .
TH17 DEFICIENCY IN HYPER IGE SYNDROME | Ma et al.
RESULTS AND DISCUSSION
Characteristics of HIES patients and STAT3 mutations
Five unrelated patients with HIES were identifi ed according
to their clinical phenotype ( Table I ). One patient (HIES4)
had a family history of HIES (her mother had succumbed to
infection but was not available for this analysis). All patients
had a history of recurrent infection, including invasive S. aureus
and mucocutaneous candidiasis. In addition, all patients had
features typical of the autosomal-dominant form of HIES, with
abnormalities of the skeleton, teeth, and soft tissue ( Table I ).
Phenotype scores ranged from 26 to 71 according to the pro-
posed HIES score (an age-sensitive scoring with HIES being
most likely in patients with a score > 40) ( 21 ).
Genomic DNA was isolated from each patient, and exons
13 – 21 of STAT3 , which encompass the DNA binding and
SH2 domains, were sequenced. Heterozygous missense mu-
tations were identifi ed in all fi ve patients. Four harbored mu-
tations in the DNA binding domain (R382Q [ n = 2], V432M,
and H437P) and one had a mutation in the SH2 domain
(Q644P; Table I ). The R382 residue is a common site of
mutation, accounting for ? 30% (18/58) of reported HIES
genotypes, with R382Q in 8 of those 18 ( 19, 20 ). Most
of the mutations we identifi ed have previously been dem-
onstrated to impair the ability of STAT3 to translocate to
the nucleus and activate transcription of STAT3 target genes
( 19, 20, 22 ).
Heterozygous STAT3 mutations abrogate the generation
of Th17 cells in vivo
To test the hypothesis that missense mutations in STAT3
would confer a defect in human Th17 cell diff erentiation,
CD4 + T cells from HIES patients were examined and com-
pared with those from matched normal controls. Flow cyto-
metric analysis revealed normal development of CD4 + T cells,
epithelium in mice, it is likely that Th17 cells confer immunity
to pathogens on mucosal and possibly other epithelial surfaces
( 8, 13 ). For instance, Th17 cells appear to have a predominant
role in antifungal immune responses in both humans and
mice ( 14 – 16 ).
Hyper-IgE (HIES), or Job ’ s, syndrome (OMIM 147060)
is a primary immune defi ciency characterized by abnormal
susceptibility to a narrow spectrum of infections, most com-
monly Staphylococcus aureus and Candida albicans ( 17, 18 ). Pa-
tients with HIES often suff er from mucocutaneous candidiasis,
whereas local and invasive S. aureus infections lead to persis-
tent eczematoid eruptions, recurrent skin and joint abscesses,
and lung damage. In addition to recurrent tissue-destructive
infections, the HIES phenotype includes coarse facies, osteo-
penia resulting in fractures after relatively minor trauma, sco-
liosis and craniosynostosis, delayed shedding of the primary
dentition, and short stature ( 18 ). Patients also exhibit dra-
matic increases in serum IgE ( > 10 times normal) ( 17, 18 ).
Recently, heterozygous missense mutations and short dele-
tions in STAT3 have been identifi ed in the majority of HIES
patients studied ( 19, 20 ). Despite the identifi cation of the
genetic cause of HIES, the mechanisms underlying the path-
ological features of this disease remain to be elucidated. Based
on the observations that (a) defects in immunity to both
Candida and S. aureus are prominent features of HIES, espe-
cially at epithelial surfaces ( 18 ), (b) human Th17 cells have
specifi cities for fungal antigens, including C. albicans ( 14 ), (c)
heterozygous STAT3 mutations are responsible for HIES
in the majority of cases ( 19, 20 ), and (d) the development of
murine Th17 cells is compromised in mice lacking expression
of STAT3 in their CD4 + T cells ( 7, 9, 10 ), we hypothe-
sized that STAT3 mutations conferred specifi c susceptibility
in humans with HIES to infection via an inability to generate
Table I. Characteristics of HIES patients
HIES 1 HIES 2 HIES 3 HIES 4 HIES 5
S. aureus pneumonia
S. aureus abscesses
Other serious infection
Parenchymal lung damage
Retained primary teeth
Phenotype score ( 21 )
Affected STAT3 domain
DNABD, DNA binding domain; SH2, src homology 2 domain.
JEM VOL. 205, July 7, 2008
BRIEF DEFINITIVE REPORT
cells could be detected after stimulation of CD4 + T cells from
two HIES patients ( Fig. 1, B and C ). These fi ndings were
corroborated by examination of IL-17 secretion by stimu-
lated CD4 + T cells. Control CD4 + T cells secreted abundant
quantities of IL-17, whereas IL-17 was undetectable in cul-
tures of CD4 + T cells from four of fi ve HIES patients ( Fig.
1 D ). In the fi fth patient, the concentration of IL-17 was
> 20-fold lower than the average amount produced by con-
trol CD4 + T cells ( Fig. 1 D ).
To exclude a diff erence in the rate of IL-17 secretion by
HIES CD4 + T cells, production of IL-17 by activated CD4 +
T cells from healthy donors and HIES patients was measured
over an 11-d time course. In cultures of healthy CD4 + T
cells, IL-17 was fi rst detected after 4 d, peaked on days 5 – 6,
and remained stable or declined at later times ( Fig. 1 E ). In
contrast, no IL-17 was detected in the supernatants of HIES
CD4 + T cells at any of the time points examined ( Fig. 1 E ).
We also assessed expression of IL-22, a Th17-derived cyto-
kine thought to be important in epithelial immunity ( 23, 24 ).
IL-22 mRNA ( Fig. 1 F ) was signifi cantly reduced in acti-
vated CD4 + T cells from HIES patients compared with
healthy controls (P < 0.01). Furthermore, there was a com-
plete absence of IL-22 secretion in cultures of activated CD4 +
T cells ( Fig. 1 G ). This fi nding demonstrates that production
of both IL-17 and IL-22 by in vivo – generated Th17 cells is
dependent on functional STAT3 protein and confi rm that
IL-22 is a signature Th17 cytokine.
HIES CD4 + T cells fail to up-regulate expression of ROR ? t,
the Th17 lineage – specifi c transcriptional regulator
Diff erentiation of Th17 cells is determined by ROR ? t ( 8 ).
Because induction of this key transcription factor in mice is
regulated by STAT3 ( 7, 9, 10 ), we tested for a possible defect
in the expression of ROR ? t mRNA in HIES CD4 + T cells.
We found a fourfold reduction of ROR ? t message in stimu-
lated HIES CD4 + T cells compared with control CD4 + T
cells ( Fig. 2 A ).
Given that STAT3 monomers must undergo homodimer-
ization to be functional ( 25 ), a fourfold reduction in expression
of ROR ? t is consistent with the prediction that a single mutant
allele of STAT3 would reduce functional STAT3 homodimers
with comparable proportions of naive (CD45RA + CD27 + ),
central memory (CD45RA ? CD27 + ) and eff ector memory
(CD45RA ? CD27 ? ) cells in HIES patients and healthy con-
trols (not depicted).
To investigate whether Th17 cells were present within
the circulating T cell population of HIES patients in vivo,
total CD4 + T cells were stimulated in vitro with anti-CD3
mAb, anti-CD28 mAb, and IL-2 for 4 – 5 d. The proportion
of Th17 cells within the population was assessed by intracel-
lular staining for IL-17 after restimulation with PMA and
ionomycin. A small but consistent population of IL-17 – ex-
pressing cells was detected in the CD4 + T cells examined for
all healthy donors (1.2%; n = 5; Fig. 1, A and C ). In contrast,
there was a signifi cant reduction in IL-17 + cells from the
cultures of stimulated STAT3 mutant HIES CD4 + T cells
(0.44%; n = 5; P < 0.05; Fig. 1, B and C ). Indeed, no IL-17 +
Figure 1. HIES patients have a defect in the generation of IL-17 –
producing CD4 + T cells in vivo. Intracellular expression of IL-17A and
IL-2 in purifi ed CD4 + T cells from a healthy donor (A) and a HIES patient
(B) activated for 5 d (see Materials and methods). (C) The frequency of IL-
17A + cells, determined by intracellular cytokine expression, and (D) IL-17A
secretion by CD4 + T cells from fi ve healthy donors and fi ve HIES patients
that had been stimulated with anti-CD3 mAb, anti-CD28 mAb, and IL-2
for 4 – 5 d, as determined by ELISA. Each symbol represents the value from
an individual donor or patient, and the line represents the mean. (E) Time
course of IL-17A secretion by CD4 + T cells isolated from a normal donor
and HIES patient. Similar results were obtained when CD4 + T cells from all
HIES patients were examined over a culture period that continued for up
to 11 d. (F and G) Expression of IL-22 mRNA (relative to housekeeping
genes; F) and secreted protein (G) by normal and HIES CD4 + T cells was
determined after 2 and 5 d, respectively, of stimulation with CD3/CD28/
IL-2. *, P < 0.05; **, P < 0.01.
Figure 2. Defi ciency of ROR ? t mRNA in HIES patients . (A) ROR ? t
mRNA expression in CD4 + cells activated for 2 d with CD3/CD28/IL-2 in
fi ve HIES patients and fi ve normal controls. (B) ROR ? t mRNA expression
after 2 d of culture is proportional to the number of Th17 cells as deter-
mined by intracellular cytokine expression after 4 – 5 d.
TH17 DEFICIENCY IN HYPER IGE SYNDROME | Ma et al.
by 75%. In our cohort of patients, the quantity of ROR ? t
mRNA correlated with the number of Th17 cells induced after
stimulation in vitro , which would be expected if ROR ? t spec-
ifi es Th17 diff erentiation ( Fig. 2 B ).
HIES CD4 + T cells can proliferate, produce Th1-
and Th2-type cytokines, and undergo in vivo
differentiation to T regulatory (T reg) and T follicular
helper (T FH ) cell phenotypes
The defect in IL-17 secretion and Th17 cell diff erentiation by
HIES CD4 + T cells did not appear to result from a general defect
in lymphocyte activation. HIES CD4 + T cells proliferated to a
similar extent as control counterparts ( Fig. 3 A ). Furthermore,
there was no signifi cant defect in production of IFN- ? ( Fig. 3,
B – E ), TNF- ? ( Fig. 3 F ), IL-4, or IL-13 ( Fig. 3, G and I ). IFN- ?
secretion by activated HIES CD4 + T cells was reduced com-
pared with controls ( Fig. 3 E ), but this reduction did not reach
statistical signifi cance, and there was no defi ciency of IFN- ? –
producing cells when assessed by fl ow cytometry after restimula-
tion with PMA/ionomycin. Interestingly, there was a consistent
and signifi cant reduction ( ? 3.5-fold; P < 0.01) in IL-10 pro-
duction by CD4 + T cells from all HIES patients examined rela-
tive to production by control CD4 + T cells ( Fig. 3 H ). This
probably refl ects a specifi c intrinsic requirement for STAT3 in
IL-10 production by activated CD4 + T cells, as recently demon-
strated in mice that specifi cally lack STAT3 in their CD4 + T
cells ( 26 ). Despite reductions in secretion of IFN- ? and IL-10,
measurable amounts of these cytokines were clearly present
in the cultures of activated CD4 + T cells from HIES patients
( Fig. 3 I ), which contrasts the complete defi cit in IL-17 produc-
tion by these cells ( Fig. 1, D and E ). Interestingly, HIES CD4 +
T cells consistently gave rise to an increased frequency of cells
expressing IL-2 compared with normal controls ( Fig. 3, B – D ).
Furthermore, T reg cells (defi ned as CD4 + CD25 hi CD127 lo cells)
( 27 ) and T FH cells (defi ned as CD4 + CXCR5 + cells) ( 28 ) were
found to be present in normal numbers ex vivo in HIES patients
( Fig. 3, J and K ).
It has been recently reported that Th17 cells express the
chemokine receptor CCR6 ( 14, 29 ). Because IL-17 – produc-
ing cells were absent from the CD4 + T cell compartment of
HIES patients, we enumerated CD4 + CCR6 + T cells in pe-
ripheral blood. In healthy donors, ? 20% of CD4 + T cells ex-
pressed CCR6 ( Fig. 3 L ). In contrast, < 10% of CD4 + T cells
expressed CCR6 in HIES patients ( Fig. 3 L ), consistent with
the suggestion that CCR6 identifi es Th17 cells. Further-
more, because human CCR6 + CD4 + T cells have been
shown to secrete both IL-17 and IFN- ? under some circum-
stances ( 29 ), defi ciency of this subset might account for the
reduction in IFN- ? sometimes observed in HIES. It is im-
portant to emphasize that previous assessments of IFN- ? pro-
duction in HIES are inconsistent, which could also be due to
heterogeneity of HIES cohorts before genotyping for STAT3
Figure 3. CD4 + T cells from HIES patients undergo normal prolif-
eration and differentiation into Th1-, Th2-, T reg – , and T FH -type
effector cells. (A) CFSE profi les of normal (outline black histogram) and
HIES (solid red histogram) CD4 + T cells after stimulation with CD3/CD28
mAb and IL-2 for 5 d. (B) Intracellular expression of IFN- ? and IL-2 in
CD4 + T cells purifi ed from normal donors and HIES patients after activa-
tion for 4 – 5 d with CD3/CD28/IL-2. The FACS plots in B are representative
of one normal control and one HIES patient. (C and D) Frequency of CD4 +
T cells from normal controls and HIES patients that expressed IL-2 (C) or
IFN- ? (D). (E – I) Secretion of IFN- ? (E), TNF- ? (F), IL-4 (G), IL-10 (H), and
IL-13 (I) by normal and HIES CD4 + T cells stimulated with anti-CD3/CD28
mAb and IL-2 for 5 d. (J and K) CD4 + T cells were labeled with either (J)
CD25 and CD127 to identify T reg cells (CD25 hi CD127 lo ) or (K) CXCR5 to
identify T FH cells, as indicated by the gated populations. The values repre-
sent the frequency of gated cells and are from one healthy donor and one
HIES patient and are representative of at least four patient and control
samples. (L) PBMCs were labeled with anti-CD4 and CCR6 mAb, and the
frequency of CD4 + CCR6 + cells in healthy donors and HIES patients was
enumerated. For all graphs (E – I and L), each symbol represents the value
from an individual donor or patient, and the line represents the mean.
*, P < 0.05; **, P < 0.01; ns, not signifi cant.
JEM VOL. 205, July 7, 2008
BRIEF DEFINITIVE REPORT
and IL-23 ( 31 ) to both activate STAT3. Furthermore, al-
though IL-23 can also activate STAT-1, -4, and -5 ( 31 ), it
would appear that IL-23 – induced activation of STAT3 plays
a predominant role in amplifying the generation of Th17 cells
from human naive CD4 + T cells induced by IL-6 ( Fig. 2 ).
Our fi ndings identify a fundamental defect in Th17 diff er-
entiation in HIES. Thus, enumeration of IL-17 production by
activated CD4 + T cells, coupled with measuring the frequency
of CCR6 + CD4 + T cells, emerges as a relatively quick and sim-
ple procedure that could be used to screen patients suspected of
having HIES due to inactivating mutations in STAT3 . In ad-
dition, our fi ndings also point to a crucial role for Th17 cells in
the normal host response. Infections with Candida spp. and
S. aureus , the most prevalent etiological agents in the infections
encountered in patients with HIES, are also important patho-
gens in other clinical settings. Mechanisms for defense against
Candida and other fungal infections have been contentious. Al-
though human Th17 cells respond selectively to Candida ( 14 ),
other evidence suggests that production of IL-17 and IL-23 in-
creases susceptibility to fungal infection in vivo ( 32 ). Further-
more, a role for T reg cells has been proposed, whereby
down-regulation of Th17-mediated infl ammation is necessary
to prevent fungal-induced pathology ( 33 ). However, we did not
detect a defi ciency of T reg cells, at least by cell surface pheno-
type, in HIES. Our observations using CD4 + T cells from
HIES patients supports the data of Acosta-Rodriguez et al. ( 14 )
that Th17 cells play a crucial role in coordinating the host
defense against Candida . This most likely refl ects a direct func-
tion of IL-17 produced by Th17 cells, as demonstrated by the
requirement of the IL-17R in protective immune responses in
mice to infection with C . albicans ( 15 ). Recent evidence from
Milner et al. ( 30 ) demonstrates that CD4 + T cells from HIES
patients fail to diff erentiate in vitro into IL-17 – producing
became available. Indeed, recent evidence indicates that some
HIES-like patients with normal STAT3 exhibit defi ciencies
of IFN- ? production, whereas STAT3-defi cient patients
have no IFN- ? defect ( 30 ).
Naive CD4 + T cells fail to differentiate into Th17 cells
Human naive CD4 + T cells do not produce IL-17 when stimu-
lated through CD3 and CD28 ( 11, 12 ). Thus, the detection of
IL-17 in the cultures of total CD4 + T cells activated with anti-
CD3/CD28 mAb refl ects production by memory CD4 + T cells
that have diff erentiated to the Th17 lineage in vivo. The re-
quirement of STAT3 in CD4 + T cell diff erentiation in vivo ap-
pears to be specifi c to the Th17 lineage because CD4 + T cells
from HIES patients could give rise to eff ector cells with features
of Th1-, Th2-, T reg – , and T FH -type cells in vivo. Because
STAT3 is ubiquitously expressed and functions downstream of
numerous cytokine receptors ( 25 ), it was possible that the in-
ability to generate Th17 cells in vivo was a consequence of a re-
quirement for STAT3 function in non-CD4 + T cells, for
instance antigen-presenting cells, that instruct the diff erentiation
of CD4 + T cells. It was therefore important to demonstrate an
intrinsic requirement of STAT3 in naive CD4 + T cells for their
commitment to the Th17 lineage. Thus, naive CD4 + T cells
were sort-purifi ed and then cultured with beads coated with anti-
CD2, anti-CD3, and anti-CD28 together with various com-
binations of IL-1 ? , IL-6, and IL-23. Secretion of IL-17A by
normal CD4 + T cells was not detected until the cells had been
subjected to three rounds of stimulation in vitro with anti-CD2,
anti-CD3 plus anti-CD28 mAb-coated beads, and either IL-1 ?
plus IL-6 or IL-23, with each stimulation period lasting 4 – 5 d
( Fig. 4 A ). The combination of IL-1 ? plus IL-6 was approx-
imately fi vefold more eff ective at inducing production of IL-
17A by activated naive CD4 + T cells than IL-23, and there was
a further two- to threefold increase in the presence of all three
cytokines ( Fig. 4 A ). In complete contrast, when naive HIES
CD4 + T cells were cultured under the same optimized condi-
tions they failed to produce IL-17A ( Fig. 4 A ). Although these
culture conditions induced IL-17A, we were unable to detect
IL-17A – expressing cells by intracellular staining (not depicted).
Because only ? 1% of total activated CD4 + T cells expressed IL-
17A, yet secreted up to 10,000 pg/ml IL-17A (see Fig. 1, A – E ),
our inability to detect IL-17A + cells in cultures of naive CD4 + T
cells most likely refl ects the fact that these cells secreted 10 – 20-
fold less IL-17A than memory CD4 + T cells.
Consistent with the defect in diff erentiation of naive HIES
CD4 + T cells into Th17 cells in the presence of IL-1 ? , IL-6,
and IL-23, we demonstrated a defect in induction of ROR ? t
in T cells in response to each of these stimuli. The induction
of ROR ? t mRNA was > 10-fold greater in IL-23 – containing
culture conditions in normal CD4 + T cells compared with
those from HIES patients ( Fig. 4 B ). Interestingly, induction
of ROR ? t expression appeared to be absolutely dependent
on IL-23, whereas there was a lesser eff ect of IL-6 ( Fig. 4 B ).
These fi ndings recapitulate the in vivo Th17 diff erentiation
defect in HIES and are consistent with the ability of IL-6 ( 25 )
Figure 4. Naive CD4 + T cells from HIES patients fail to differenti-
ate to the Th17 lineage in vitro in response to the STAT3-activating
cytokines IL-6 and IL-23. Naive CD4 + T cells isolated from a normal
donor ( ? , ? ) and a HIES patient ( ? , ? ) were cultured with anti-CD2,
anti-CD3, and anti-CD28 mAb-coated beads either alone (CD3) or in the
presence of IL-1 ? plus IL-6 (IL-1 ? +IL-6), IL-23 (IL-23), or all three cyto-
kines (IL-1 ? +IL-6+IL-23). The cells were harvested after 5 d and then
recultured under the same conditions for another two rounds of stimula-
tion. (A) IL-17A secretion was determined after either 3 d ( ? , ? ; total
time of culture, 12 d) or 6 d of the third round of restimulation ( ? , ? ;
total time of culture, 15 d). (B) Expression of ROR ? t mRNA in cells stimu-
lated for 5 d ( ? , ? ) or 15 d ( ? , ? ) was determined by quantitative PCR.
TH17 DEFICIENCY IN HYPER IGE SYNDROME | Ma et al.
TCTGTCCAACCTACCCTTCG; exon 20, GGACAGAGTGTGCACA-
GATGTAA and CACTGGAGCAAGCAAAACAA; and exon 21, GCTTA-
AGTCTTTTCCCCTTCG and GCATTTGCCTATCTATCCTCCA.
CD4 + T cell isolation, phenotyping, and cell culture. Whole blood was
collected from normal donors and HIES patients, and PBMCs were prepared
by centrifugation using Ficoll-Paque. CD4 + T cells were then isolated by
positive selection using the CD4-DYNA beads. Purifi ed CD4 + T cells were
cultured in 24-well plates (2 × 10 5 cells/ml) with immobilized 5 μ g/ml anti-
CD3 mAb, 750 ng/ml anti-CD28 mAb, and 20 U/ml rIL-2 ( 37 ). CD4 + T
cells were also labeled with CFSE ( 37 ), and their proliferation was determined
after 5 d of in vitro culture. For in vitro diff erentiation to the Th17 lineage,
naive CD4 + T cells were isolated by sorting CD45RA + CD27 + cells ( 37 ) and
then culturing them with anti-CD2 – , anti-CD3 – , and anti-CD28 – coated
beads (one bead per fi ve cells), and 20 U/ml rIL-2 alone or in the presence of
20 ng/ml IL-23 only, 10 ng/ml IL-1 ? plus 50 ng/ml IL-6, or IL-1 ? , IL-6,
and IL-23. The cells were harvested every 4 – 5 d before being washed and re-
cultured under the same conditions at 2 × 10 5 cells/ml ( 11, 12 ).
The frequencies of CD4 + T cells that were of a naive, conventional
memory, or eff ector memory phenotype, were determined by enumerating
CD4 + T cells that were either CD45RA + CD27 + , CD45RA ? CD27 + , or
CD45RA ? CD27 ? , respectively. T reg and T FH cells were quantitated by
determining the frequency of CD4 + T cells that had a CD25 hi CD127 lo ( 27 )
or CXCR5 + ( 28 ) phenotype, respectively.
Intracellular cytokine staining and ELISA. After 5 or 6 d of activation
with the conditions mentioned above, CD4 + T cells were harvested and re-
stimulated for 6 h with 100 ng/ml PMA and 750 ng/ml ionomycin, with 10
μ g/ml brefeldin A added after 2 h. After this time, cells were fi xed in 2%
formaldehyde (Sigma-Aldrich) and stained for expression of intracellular cy-
tokines (IL-2, IL-17A, and IFN- ? ). All antibody staining and wash steps
were performed in 0.5% saponin solution ( 37 ). Data were acquired using a
FACSCanto or LSRII (BD Biosciences) and analyzed using FlowJo software
(Tree Star). IL-17A in culture supernatants was measured using the human
IL-17A ELISA Ready-SET-Go kit according to the manufacturer ’ s instruc-
tions (eBioscience). IL-22 secretion was measured using the Human IL-22
ELISA Development kit from PeproTech. Secretion of IL-4, IL-10, IL-13,
IFN- ? , and TNF- ? was determined by ELISA using purifi ed and biotinyl-
ated cytokine-specifi c mAb (IL-4: 8D4-8 and MP4-25D2; IL-10: JES3-
12G8 and JES3-9D7; IL-13: PVM13-1 and polyclonal; IFN- ? : NIB42 and
4S.B3; TNF- ? : MAb1 and MAb11; from BD Biosciences and eBioscience)
as the capture and detection mAbs, respectively, and visualization with strep-
tavidin – horseradish peroxidase and TMB substrate.
mRNA quantitation. After 2 d of culture, CD4 + T cells were harvested and
washed with PBS. Total RNA was isolated using the QIAGEN RNeasy kit
according to the manufacturer ’ s instructions. For quantitative RT-PCR, total
RNA was reverse transcribed using oligo-dT according to the Invitrogen pro-
tocol. cDNA expression was determined using the ABI Prism 7900 sequence
detection system and Syber-green reagents (Applied Biosystems). The fol-
lowing primers were used: ROR ? t , TCAGTCATGAGAA CACAAATTGA
and GCACCCCTCACAGGTGATAA; UBE2D2 , TGAAGAGAATCCA-
CAAGGAATTGA and CAACAGGACCTG CTGAACACTG; and IL22 ,
AGGCTCAGCAACAGGCTAAG and TCCTTCAGCTTTTGCACATT.
Fluorescence signals were measured over 40 PCR cycles, and the cycle (Ct)
at which signals crossed a threshold set within the logarithmic phase was re-
corded. The Ct for the target gene was subtracted from the Ct for UBE2D2
( ? Ct). The relative amount of mRNA was calculated as 2 Δ Ct .
We thank Drs. Carola Vinuesa and Elissa Deenick for helpful discussions, Gill Tangye
for establishing the cytokine ELISAs, and Karen Fenimore for providing reagents.
This work was supported by the National Health and Medical Research Council
(NHMRC) of Australia. C.S. Ma, G.Y.J. Chew, and S.G. Tangye are the recipients of
Research Fellowships awarded by the NHMRC.
The authors have no confl icting fi nancial interests.
eff ector cells in response to stimulation with C. albicans and
S. aureus . Defi ciency of IL-17 is not necessarily the only mech-
anism of immunodefi ciency in HIES because IL-22 has been
shown to contribute to protection against Klebsiella pneumoniae
in mice ( 23 ), and we show that IL-22 production is also re-
duced signifi cantly in HIES.
Although our fi ndings help to explain the spectrum of infec-
tions in HIES, other aspects of the complex phenotype require
elucidation. A similar cell-intrinsic eff ect of STAT3 acting within
chondrocytes or osteoblasts may explain some of the skeletal
defects as evidenced in mice with a conditional hematopoietic
cell-specifi c disruption of the STAT3 gene ( 34 ). Craniosynosto-
sis and midline abnormalities, for example, can result from de-
fects in the transcription factor TWIST, which is induced by
STAT3 ( 35, 36 ). The mechanism of the HIES component of
the phenotype is also likely to be complex because STAT3 op-
erates intrinsically in B cells and is activated in response to many
regulatory cytokines, including IL-6, IL-10, and IL-21, and, as
shown here, T helper signals are also disrupted.
Finally, illumination of the aspects of the HIES phenotype
that arise as a consequence of Th17 defi ciency may sound a
note of caution when considering IL-17 antagonism in auto-
immune and infl ammatory diseases ( 9, 10 ). On the other hand,
administration of IL-17 or IL-22 may represent a therapeutic
approach to controlling specifi c pathogen infections in HIES
patients, as well as in other patient groups who exhibit suscep-
tibility to Candida .
MATERIALS AND METHODS
Reagents. Allophycocyanin-conjugated anti – IFN- ? mAb was purchased
from Invitrogen, and FITC – anti – IL-17A was purchased from eBioscience.
Alexa 647 – anti-CXCR5, allophycocyanin – anti-CD25, FITC – anti-CD45RA,
PE-conjugated anti – IL-2 and anti-CD27, PE-Cy7 – anti-CD4, and PE – anti-
CCR6 were purchased from BD Biosciences. ECD-conjugated anti-CD4 and
PE – anti-CD127 were from Beckman Coulter. Recombinant human IL-2
(rIL-2) was purchased from Endogen, recombinant human IL-1 ? and IL-6
were from PeproTech, and recombinant human IL-23 was purchased from
eBioscience. Anti-CD28 mAb was purchased from BD Biosciences, anti-CD3
mAb (Spv-T3b) was provided by H. Spits (NKI, Amsterdam, Netherlands),
and anti-CD2 – , anti-CD3 – , and anti-CD28 – coated beads were purchased
from Miltenyi Biotec (T cell activation/expansion kit). PMA, calcium iono-
phore (ionomycin), brefeldin A, and saponin were all purchased from Sigma-
Aldrich. CFSE was purchased from Invitrogen.
Patients. Five normal donors and fi ve patients with the clinical diagnosis of
HIES were recruited from Immunology Clinics in Canberra and Sydney,
Australia. The cited HIES score ( 21 ) is age sensitive; therefore, the low score
in the 7-yr-old patient did not argue against testing her for mutations in
STAT3 . All studies were approved by the institutional human research ethics
committees (Canberra and Westmead hospitals).
Identifi cation of STAT3 mutations in HIES patients. Genomic
DNA was isolated from saliva samples, and exons 13 – 21 encompass-
ing the DNA binding and SH2 domains were amplifi ed by PCR and se-
quenced (ABI Prism 7700). The following primers were used: exons 12 – 14,
TAGTTTAAAGAAATGCCCAGGAGCACAGAG and TTGGCCTA-
AGTGACTTTTTGGAATAACTACAGC; exon 15, TGCTGTGCT-
GCTTAGACTGG and CCCCTGTACGTAGCCTCTCCA; exon 16,
GAGATGCGGGTGAAGAGATT and CTTGTTTAGATGAGGGATG-
GTG; exon 17, AGTGCCCCCTCCTTTTAGTT and CCCAAATGAA-
CAGCCCTATG; exons 18 and 19, TGCACACACACAACAGTGC and
JEM VOL. 205, July 7, 2008 Download full-text
BRIEF DEFINITIVE REPORT
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Submitted: 1 February 2008
Accepted: 17 April 2008
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