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IMMUNOBIOLOGY
Regulation of Th17 cell differentiation and EAE induction by MAP3K NIK
*Wei Jin,1 *Xiao-Fei Zhou,1 Jiayi Yu,1 Xuhong Cheng,1 and Shao-Cong Sun1
1Department
of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston
Th17 cells play an important role in mediating autoimmune diseases, but the molecular mechanism underlying Th17 differentiation is incompletely understood. We
show here that NF-␬B–inducing kinase
(NIK), which is known to regulate B-cell
maturation and lymphoid organogenesis,
is important for the induction of Th17
cells. NIK-deficient naive CD4 T cells are
attenuated in the differentiation to Th17
cells, although they are competent in
committing to the other effector lineages.
Consistently, NIK knockout mice are resistant to experimental autoimmune encephalomyelitis, a disease model that involves
the function of Th17 cells. This phenotype was also detected in Rag2 knockout
mice reconstituted with NIK-deficient
T cells, confirming a T-cell intrinsic defect. We further show that NIK mediates
synergistic activation of STAT3 by T-cell
receptor and IL-6 receptor signals. NIK
deficiency attenuates activation of STAT3
and induction of STAT3 target genes involved in Th17-commitment program.
These findings establish NIK as an important signaling factor that regulates Th17
differentiation and experimental autoimmune encephalitis induction. (Blood.
2009;113:6603-6610)
Introduction
CD4 T cells play a central role in shaping the immune system for
effective response to microbial infections. Upon activation by an
antigen, naive CD4 T cells differentiate into subsets of effector
T cells, T helper (Th)1, Th2, and Th17 cells, which are characterized by the production of specific cytokines and engagement in
specialized immune functions.1 Th1 cells produce interferon-␥
(IFN-␥) and mediate cellular immune responses against infection
by intracellular pathogens, whereas Th2 cells produce IL-4, IL-5,
and IL-13 and play an important role in antibody responses to
extracellular pathogens.2,3 A signature cytokine produced by the
Th17 cells is IL-17A, which mediates inflammatory responses by
recruiting immune cells and inducing the production of proinflammatory cytokines.4-7 Strong evidence suggests that Th17 cells are
involved in various autoimmune and inflammatory diseases,7,8 such
as experimental autoimmune encephalitis (EAE)5,9 and rheumatoid
arthritis.10-12 Moreover, activated CD4 T cells can also differentiate
into inducible regulatory T cells (iTregs), which suppress the
function of effector T cells, thereby keeping an immune response
under control.
As seen with Th1 and Th2 cells,2,3 the development of Th17
cells is regulated by the specific cytokine microenvironment.13,14
IL-6 and transforming growth factor-␤ (TGF-␤) are critical cytokines that, together with the T-cell receptor (TCR) signal, initiate
the differentiation of Th17 cells from naive CD4 T cells.15-17
Another cytokine, IL-21, is induced by IL-6 in the course of Th17
cell differentiation and may function to sustain the Th17 polarizing
signal in an autocrine manner.18-20 IL-21 also induces the expression of IL-23 receptor (IL-23R), rendering the Th17 cells responsive to IL-23, a cytokine that is involved in the maintenance of the
Th17 population.5,21 In addition, recent studies suggest that Th17
cell differentiation also involves TL1A and its receptor DR3,
members of the TNF and TNF receptor (TNFR) superfamilies,
respectively.22,23 Notably, several other TNF/TNFR family mem-
bers are well-known costimulatory molecules involved in the
activation and differentiation of T cells, although their role in Th17
production is not well characterized.24
The intracellular signaling mechanism mediating Th17 differentiation is still incompletely understood. Nevertheless, the primary
signaling event induced by IL-6 is activation of STAT3, a critical
transcription factor involved in the initiation of the Th17 commitment program.25 IL-6 stimulates the tyrosine phosphorylation of
STAT3, a common mechanism that triggers the dimerization,
nuclear translocation, and DNA binding activity of STAT proteins.26 Activated STAT3 regulates Th17 cell differentiation by
participating in the transcriptional activation of several Th17regulatory genes, including those encoding IL-21, IL-23R, and the
orphan nuclear hormone receptor ROR␥t.20,27,28 Induction of
ROR␥t represents a central step in the Th17-commitment program,
because ROR␥t functions as a Th17 lineage-specific transcription
factor that regulates the expression of the IL-17A gene locus.19,20,27
The transcriptional induction of ROR␥t, as well as the other Th17
regulatory and effector genes, requires not only IL-6 and TGF-␤
but also the TCR signal. However, the signaling molecules
connecting the TCR signal to the Th17-commitment pathway are
poorly defined.
MAP kinase (MAPK) signaling pathways participate in diverse
biological processes, including the differentiation of Th1 and Th2
cells.29 Activation of MAPKs involves a signaling cascade initiated
from activation of a family of upstream kinases, the MAPK kinase
kinases (MAP3Ks).30 In addition to mediating the typical MAP3K
signaling cascades, MAP3Ks also possess other signaling functions. One MAP3K, the NF-␬B–inducing kinase (NIK), has a
central role in mediating a noncanonical NF-␬B signaling pathway.31 This pathway involves processing of an NF-␬B precursor
protein, p100, to a mature NF-␬B subunit, p52, and the nuclear
translocation of p52/RelB NF-␬B complex. The noncanonical
Submitted December 2, 2008; accepted April 24, 2009. Prepublished online as
Blood First Edition paper, May 1, 2009; DOI 10.1182/blood-2008-12-192914.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*W.J. and X.-F.Z. contributed equally to this work.
The online version of this article contains a data supplement.
BLOOD, 25 JUNE 2009 䡠 VOLUME 113, NUMBER 26
© 2009 by The American Society of Hematology
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JIN et al
NF-␬B pathway is stimulated by a subset of TNFR family members
and plays a critical role in B-cell maturation and lymphoid
organogenesis.31 In addition to regulating noncanonical NF-␬B,
NIK appears to possess other signaling functions. At least in cell
line models, NIK phosphorylates MEK1, thereby causing activation of the downstream MAPK, ERK.32 Moreover, overexpressed
NIK induces the phosphorylation of STAT3 in a prostate cancer cell
line.33 Notably, the catalytic activity of NIK is rapidly stimulated
upon ligation of TCR and the costimulatory molecule CD28,34
suggesting a role for NIK in regulating the signaling events
involved in T-cell activation or differentiation.
In the present study, we investigated the function of NIK in
T-cell differentiation using NIK knockout mice. We show that NIK
has a critical role in Th17 cell differentiation and EAE induction,
although this MAP3K is dispensable for the differentiation of Th1,
Th2, and iTreg cells. We provide evidence that NIK is required for
synergistic activation of STAT3 by the TCR and IL-6 signals. Thus,
our data establish NIK as a critical regulator of Th17 differentiation
and shed light on a new function of NIK in mediating TCR
signaling.
Methods
Mice
NIK knockout mice, from a 129Sv/Ev background,35 were provided by
Amgen (Thousand Oaks, CA) and maintained in the specific pathogen-free
facility of M. D. Anderson Cancer Center. NIK⫹/– heterozygous mice were
bred to generate age- and sex-matched NIK⫹/⫹ and NIK–/– mice that were
used in the experiments. Rag2 knockout (Rag2–/–) mice, from a 129Sv/Ev
background, were obtained from Taconic (Germantown, NY). All animal
experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas M. D.
Anderson Cancer Center.
Antibodies, reagents, and plasmids
Functional grade anti–mouse (m) CD3⑀ (145-2C11) and anti-mCD28
(37.51) antibodies as well as the blocking antibodies for mIFN-␥ (XMG1.2)
and mIL-4 (11B11) were from eBioscience (San Diego, CA). Fluorescencelabeled antibodies for mCD4-Pacific blue (L3T4), mCD25-PE-Cy7 (PC61.5),
mCD62L-APC (MEL-14), mCD44-APC-Cy7 (IM7), mIL-17A-PE
(eBio17B7), mIFN-␥-FITC (XMG1.2), mIL-4-PE (11B11), and mFoxp3FITC (FJK-16s) were also purchased from eBioscience. PE-conjugated
anti–phospho-STAT3 (pY705) was from BD Biosciences (San Jose, CA).
The recombinant mIL-6 and hTGF-␤ were purchased from PeproTech
(Rocky Hill, NJ), and recombinant LIGHT (lymphotoxin-like, exhibits
inducible expression, and competes with HSV glycoprotein D for HVEM, a
receptor expressed by T lymphocytes) was from R&D Systems (Minneapolis, MN). Phorbol 12-myristate 13-acetate (PMA) and ionomycin were
from Sigma (St Louis, MO), and monensin was from eBioScience. The
retroviral vector pCLXSN(GFP) was a modified version of pCLXSN,36
in which the neomycin gene was replaced with a GFP gene. pCLXSN(GFP)-NIK was created by inserting the human NIK cDNA into the
pCLXSN(GFP) vector.
10 ␮g/mL anti–IFN-␥), or iTreg (10 units/mL IL-2, 5 ng/mL TGF-␤)
conditions. When indicated, recombinant LIGHT (500 ng/mL) was added
to the culture as a costimulatory molecule. After the indicated times, the
cells were subjected to intracellular cytokine staining (ICS) and real-time
RT-PCR analyses.
For proliferation assays, naive CD4 T cells were labeled with carboxyl
fluorescent succinimidyl ester (CFSE, 1.25 ␮g/mL in PBS) for 5 minutes at
room temperature. After 2 washes with RPMI medium or PBS (supplemented with 5% FCS), the cells were stimulated with anti-CD3/CD28
under Th0 or Th17 conditions, as described above. After 72 hours, CFSE
intensity was determined by flow cytometry.
Retroviral infection of CD4 T cells
Naive CD4 T cells were activated with plate-bound anti-CD3 (4 ␮g/mL)
plus anti-CD28 (4 ␮g/mL) in 24-well plates for 36 hours and then infected
with pCLXSN(GFP) or pCLXSN(GFP)-NIK retroviruses. After 36 hours of
infection, infected cells were enriched by cell sorting based on GFP
expression and subjected to Th17 differentiation assays.
ICS
T cells isolated from spleen and central nervous system (CNS; brain and
spinal cord) of immunized mice or from in vitro cultures were stimulated
for 4 hours with PMA (50 ng/mL) and Ionomycin (500 ng/mL) in the
presence of monensin (10 ␮g/mL). After the stimulation period, cells were
fixed in 2% paraformaldehyde and permeablized in 0.5% saponin before
staining for relevant cytokines. The stained cells were analyzed by flow
cytometry.
Real-time quantitative RT-PCR
Total RNA was isolated from T cells using TRI reagent (Molecular
Research Center, Cincinnati, OH) and subjected to cDNA synthesis using
MMLV reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo (dT)
primers. Real-time quantitative PCR was performed using iCycler Sequence Detection System (Bio-Rad, Hercules, CA) and iQ SYBR Green
Supermix (Bio-Rad). The expression of individual genes was calculated by
a standard curve method and normalized to the expression of GAPDH. The
gene-specific primer sets (all for murine genes) were: IL-17A, 5⬘CTCAGACTACCTCAACCGTTC-3⬘, and 5⬘-TGAGCTTCCCAGATCACAGAG-3⬘; IL-17F, 5⬘-CCCATGGGATTACAACATCACTC-3⬘, and 5⬘CACTGGGCCTCAGCGATC-3⬘; ROR␥t, 5⬘-CAAGTCATCTGGGATCCACTAC-3⬘, and 5⬘-TGCAGGAGTAGGCCACATTACA-3⬘; IL-21, 5⬘ATCCTGAACTTCTATCAGCTCCAC-3⬘, and 5⬘-GCATTTAGCTATGTGCTTCTGTTTC-3⬘; IL-22, 5⬘-TCCGAGGAGTCAGTGCTAAA-3⬘, and
5⬘-AGAACGTCTTCCAGGGTGAA-3⬘; IL-23R, 5⬘-GCCAAGAAGAC
CATTCCCGA-3⬘, and 5⬘-TCAGTGCTACAATCTTCTTCAGAGGACA3⬘; and GAPDH, 5⬘-CTC ATG ACC ACA GTC CAT GCC ATC-3⬘, and
5⬘-CTG CTT CAC CAC CTT CTT GAT GTC-3⬘.
Adoptive transfer of T and B cells
B220⫹ B cells and CD90.2⫹ T cells were isolated from the splenocytes of
NIK⫹/⫹ and NIK–/– mice using magnetic beads (Miltenyi Biotec). The
purity of the isolated cells was more than 95%, as determined by flow
cytometry. NIK⫹/⫹ B cells (5 ⫻ 106) were mixed with either NIK⫹/⫹ or
NIK–/– T cells (10 ⫻ 106) and then injected into the tail vein of nonirradiated Rag2–/– mice. Recipient mice were subjected to EAE studies 16 hours
after the adoptive transfer.
CD4 T-cell differentiation and proliferation assays
CD4 T cells were isolated from splenocytes using a CD4 T-cell Isolation Kit
(Miltenyi Biotec, Auburn, CA) and then subjected to flow cytometric cell
sorting (FACSAria, BD Biosciences) to purify naive CD4 T cells
(CD4⫹CD25⫺CD44loCD62Lhi). Purified naive CD4 T cells were stimulated
with the indicated amounts of plate-bound anti-CD3 and anti-CD28 under
Th0 (10 ␮g/mL anti–IL-4, 10 ␮g/mL anti–IFN-␥), Th1 (10 ng/mL IFN-␥,
10 ng/mL IL-12, 10 ␮g/mL anti–IL-4), Th2 (10 ng/mL IL-4, 10 ␮g/mL
anti–IFN-␥), Th17 (20 ng/mL IL-6, 5 ng/mL TGF-␤, 10 ␮g/mL anti–IL-4,
Induction and evaluation of EAE
The encephalitogenic peptide (residues 35-55, Met-Glu-Val-Gly-Trp-TyrArg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) of myelin oligodendrocyte glycoprotein (MOG) was purchased from Genemed
Synthesis Inc. (San Francisco, CA, 95% purity). To induce acute EAE, mice
were injected s.c. (in the back region) with 300 ␮g of the MOG35-55 peptide
in CFA containing 5 mg/mL heat-killed Mycobacterium tuberculosis (H37Ra
strain; BD Diagnostics, Franklin Lakes, NJ). On the day of immunization
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BLOOD, 25 JUNE 2009 䡠 VOLUME 113, NUMBER 26
and 48 hours later, the mice were also injected i.v. with Pertussis toxin
(200 ng/mouse; List Biological Laboratories, Campbell, CA) in PBS. This
immunization procedure was repeated once on day 8 after the initial
immunization. Mice were examined daily for EAE disease symptoms,
which were scored using a standard method: 0, no clinical signs; 1, limp
tail; 2, paraparesis (weakness, incomplete paralysis of 1 or 2 hind limbs); 3,
paraplegia (complete paralysis of 2 hind limbs); 4, paraplegia with fore limb
weakness or paralysis; and 5, moribund state or death.
Analysis of in vivo T-cell differentiation and CNS infiltration
At the indicated times after MOG35-55 immunization, the mice were killed
for splenocyte preparation and CNS infiltration analysis. CD90.2⫹ T cells
were isolated from the splenocytes using magnetic beads (Miltenyi Biotec)
and subjected to ICS as described above. For the preparation of CNS
lymphocytes, brains and spinal cords were excised and dissociated for
1 hour at 37°C by digestion with collagenase IV (0.5 mg/mL; Invitrogen)
and DNase I (10 ␮g /mL; Roche, Indianapolis, IN) in RPMI medium.
Dispersed cells were passed through a 40-␮m nylon mesh and collected by
centrifugation. The cells were then resuspended in RPMI medium, layered
onto a Percoll density gradient (Biochrom, Berlin, Germany), and centrifuged for 30 minutes (625 g, 22°C). CNS lymphocytes were isolated by
collection of the interphase fraction between 30% and 70% Percoll. After
intensive washing in Hanks balanced-salt solution, cells were analyzed by
flow cytometry.
Statistical analysis
Two-tailed unpaired t test statistical analysis was performed using the Prism
software. P values less than .05 and less than .01 mean significant and very
significant, respectively.
Results
NIK deficiency in mice attenuates in vivo generation of Th17
cells and induction of EAE
To assess the in vivo role of NIK in regulating T-cell differentiation
and immune responses, we analyzed the effect of NIK deficiency
on the induction of a T-cell-mediated autoimmune disease, EAE.5,9
Because the NIK knockout mice are from a genetic background
(129Sv) known to be less susceptible to EAE, we used a protocol
that involved 2 rounds of immunization with MOG35-55 and
Pertussis toxin. After 2 weeks of the initial immunization, the
NIK⫹/⫹ mice started to display EAE disease symptoms (Figure
1A, and Figure S1, available on the Blood website; see the
Supplemental Materials link at the top of the online article). By
day 25, all of these control mice developed EAE (Figure S1).
The average disease score initially peaked on day 17 and lasted
until at least day 35 (Figure 1A). In contrast to the NIK⫹/⫹ mice,
none of the NIK–/– mice had EAE symptoms at least until day 35
(Figures 1A, S1).
To examine MOG35-55-induced T-cell differentiation in vivo, we
analyzed the different subsets of CD4 effector T cells during the
early and late phases of the EAE disease. We performed ICS to
detect the in vivo frequency of Th17 and Th1 cells in the spleen of
NIK⫹/⫹ and NIK–/– mice. During the early phase of EAE (day 14), a
significant level of IL-17A-producing Th17 cells (around 1%) and
IFN␥-producing Th1 cells (around 1%) was detected in the spleen
of NIK⫹/⫹ mice (Figure 1B,C). The production of Th1 cells was not
inhibited, but rather enhanced, in the NIK–/– mice (Figure 1B,C). In
contrast, these mutant animals displayed a severe reduction in the
population of Th17 cells (Figure 1B,C). Compared with the early
phase of EAE, the late phase (day 35) produced a higher level of
Th17 cells in the NIK⫹/⫹ spleen (Figure 1D,E). Importantly, the
REGULATION OF Th17 CELL DIFFERENTIATION BY NIK
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frequency of Th17 cells in the NIK–/– spleen was still low at this
late time point (Figure 1D,E).
In addition to IL-17A, Th17 cells produce several other
cytokines, including the IL-17A homologue, IL-17F, and IL-22. To
further confirm the critical role of NIK in Th17 cell production in
vivo, we analyzed the expression of these Th17-expressing genes
by real-time PCR using splenic T cells derived from MOGimmunized (day 35) NIK⫹/⫹ and NIK–/– mice. Consistent with the
ICS results, the expression of IL-17A, but not of IFN-␥, was
severely attenuated in the NIK–/– T cells (Figure 1F). Moreover, the
NIK–/– T cells also failed to express IL-17F and IL-22 (Figure 1F).
Together, these results suggest that NIK has a critical role in
mediating MOG35-55-induced Th17 cell production and EAE
pathogenesis.
NIK expression in T cells is required for EAE induction
NIK has an important signaling role in lymphoid stromal cells,
which is required for the development of lymph nodes and Peyer
patches.31 However, previous studies suggest that these lymphoid
organs are not required for immune responses involved in the
induction of EAE or antigen-induced arthritis.37,38 Nevertheless, it
is important to determine whether the EAE-resistant phenotype of
the NIK–/– mice was due to their lymphoid organ abnormalities or
the T-cell intrinsic defect in Th17 differentiation. To address this
question, we performed lymphocyte transfer studies using Rag2–/–
mice as recipients. Because the Rag2–/– mice lack lymphocytes,
they are completely resistant to EAE induction. We transferred
NIK⫹/⫹ or NIK–/– T cells, together with NIK⫹/⫹ B cells, into the
Rag2–/– recipients. As expected, transfer of NIK⫹/⫹ T cells into
Rag2–/– mice rendered these mice susceptible to EAE induction
(Figure 2A). Similar to the wild-type mice, the Rag2–/– mice that
had been transferred with NIK⫹/⫹ T cells began to develop EAE
symptoms around 2 weeks of immunization and all of them became
sick on day 22 (Figure 2A). In contrast, none of the NIK–/– T-cell
recipients developed EAE (Figure 2A). Furthermore, the disease
score of the NIK⫹/⫹ T-cell recipients was steadily increasing until
day 25, but no disease symptom was observed in the NIK–/–
T-cell recipients throughout of the 26-day experimental period
(Figure 2B). Thus, NIK expression in T cells is critical for the
induction of EAE.
To examine whether NIK has a T-cell intrinsic role in the
differentiation of Th17 cells in vivo, we analyzed the frequency of
Th17 cells in the spleen of immunized mice at 2 different time
points. The NIK–/– T-cell recipients produced a significantly lower
level of Th17 cells compared with the NIK⫹/⫹ T-cell recipients
(Figure 2C). This defect was particularly striking on day 24, thus
suggesting the requirement of NIK in Th17 cell production in vivo.
We next analyzed the infiltration of T cells into the CNS during
the initial and a later stage of EAE. Consistent with their
development of EAE, the NIK⫹/⫹ T-cell recipients displayed
infiltration of CD4⫹ T cells into the CNS on both day 14 and
day 24 (Figure 2D). On the other hand, a significantly lower level
of CNS-infiltrating T cells were detected in the NIK–/– T-cell
recipients, particularly during the early stage (day 14; Figure 2D).
To assess the existence of Th17 and Th1 effector T cells within the
CNS of the immunized Rag2–/– recipient mice, we performed
real-time PCR assays to detect the expression of IL-17A and IFN-␥
genes in CNS tissue. As expected, a high level of IL-17A and IFN-␥
expression was detected within the CNS of NIK⫹/⫹ T-cell recipients (Figure 2E). In sharp contrast, the CNS of the NIK–/– T-cell
recipients had a considerably lower level of IL-17A expression
(Figure 2E). Interestingly, these mutant recipients also expressed a
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BLOOD, 25 JUNE 2009 䡠 VOLUME 113, NUMBER 26
Figure 1. NIK knockout mice are resistant to EAE induction and defective in Th17 differentiation. (A) Age- and sex-matched NIK–/– and NIK⫹/⫹ mice (7 ⫹/⫹ and 5 –/–)
were immunized with MOG35-55 peptide on day 0 and day 8 and monitored daily for EAE disease symptoms. Data are representative of 3 experiments. (B,C) MOG35-55immunized mice were killed on day 14. Splenic T cells were stimulated for 4 hours with PMA plus ionomycin and subjected to ICS and flow cytometry to determine the
frequency of Th1 and Th17 cells among CD4⫹CD44⫹ cells based on their production of IFN-␥ and IL-17A, respectively. Data are presented as a representative flow cytometry
graph (B) and mean value of multiple mice (each circle or square in this and all the subsequent figures represents an individual mouse). (D,E) MOG35-55-immunized mice
(7 NIK⫹/⫹ and 5 NIK–/–) were killed on day 35. The frequency of Th1 and Th17 cells in the spleen was analyzed as described in panels B and C. (F) Splenic T cells from day 35
MOG35-55-immunized mice were stimulated for 4 hours with PMA plus ionomycin. RNA was prepared from the cells and subjected to real-time PCR assays to detect the relative
expression of the indicated genes (fold relative to one of the NIK⫹/⫹ samples). Data are representative of 2 experiments and are presented as mean value of multiple (7 NIK⫹/⫹
and 5 NIK–/–) mice.
substantially lower level of the Th1 cytokine, IFN-␥, in the CNS
(Figure 2E). These results suggest that in addition to regulating
Th17 cell differentiation, NIK may play a role in the CNS
infiltration or activation of Th1 cells. Because both Th17 and Th1
cells contribute to the pathogenesis of EAE,39,40 these findings also
explain the drastic EAE-resistance phenotype of the NIK–/– T-cell
recipient mice.
NIK is required for Th17 cell differentiation in vitro
To further examine the T-cell intrinsic role of NIK in regulating
Th17 cell differentiation, we used an in vitro T-cell differentiation
model. The NIK⫹/⫹ and NIK–/– naive CD4 T cells were stimulated
by anti-CD3/anti-CD28 under Th1, Th2, Th17, and iTreg polarizing conditions. Loss of NIK did not affect the differentiation of
naive CD4 T cells to Th1 or Th2 cells (Figure 3A). Previous studies
suggest that NIK mutant (aly) and NIK–/– mice have reduced
numbers of nTregs, probably due to the NF-␬B signaling defect in
thymic stromal cells.41,42 Interestingly, the expression of NIK in
naive T cells appeared to be less important for the differentiation of
iTregs, as the NIK–/– T cells only displayed a moderate reduction in
the iTreg induction (Figure 3B). Overall, these results suggest that
NIK is largely dispensable for naive T-cell differentiation to Th1,
Th2, and iTreg lineages. In contrast, a parallel experiment revealed
that the production of Th17 cells was severely attenuated in the
NIK–/– T cells (Figure 3B bottom panels). These findings suggest
that NIK has a specific role in regulating naive CD4 T-cell
differentiation to the Th17 lineage.
Because NIK can be activated by both the TCR34 and certain
TNFRs involved in T-cell costimulation,31,43 we examined whether
specific TNFR members are involved in Th17 cell differentiation
and whether NIK is required for such costimulation. Stimulation of
the wild-type T cells under Th17 conditions either did not affect or
even down-regulated the expression of several TNFRs (Figure
S2A). Interestingly, however, one TNFR member, herpes virus
entry mediator (HVEM), was strongly induced when naive T cells
were stimulated under Th17 conditions compared with the Th0 or
Th1 conditions (Figure S2A,B). Furthermore, the HVEM ligand,
LIGHT, greatly promoted Th17 cell differentiation, and this
costimulatory effect was largely blocked in the NIK–/– T cells
(Figure S2C). Interestingly, however, NIK became less important
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BLOOD, 25 JUNE 2009 䡠 VOLUME 113, NUMBER 26
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Figure 2. NIK-deficient T cells are defective in mediating EAE induction when adoptively transferred to Rag2–/– mice. (A,B) T cells isolated from NIK⫹/⫹ and NIK–/– mice
were mixed with B cells derived from NIK⫹/⫹ mice and adoptively transferred into Rag2–/– mice. One day after the cell transfer, recipient mice were immunized with MOG35-55
peptide as described in Figure 1A. EAE incidence (A) and disease scores (B) were monitored daily. Mice transferred with NIK⫹/⫹ and NIK–/– donor T cells are indicated as filled
circles and squares, respectively. (C) Rag2–/– mice were transferred with the indicated donor T cells together with wild-type B cells. MOG35-55-immunized mice were killed on
day 14 or day 24. Splenic T cells were stimulated for 4 hours with PMA plus ionomycin and subjected to ICS and flow cytometry to determine the frequency of Th17 cells among
CD4⫹CD44⫹ cells based on IL-17A production. Data are mean value of the indicated number of mice (each circle or square represents 1 individual mouse). (D) Rag2–/– mice
were transferred with the indicated donor T cells together with wild-type B cells. After 14 or 24 days of MOG immunization, flow cytometry was performed to determine the
frequency of CD4⫹CD45⫹ cells infiltrating to the brain and spinal cord. Data are mean value of the indicated number of mice. (E) MOG-immunized Rag2–/– recipients were killed
on day 24 for isolating RNA from total CNS tissue. Real-time PCR was performed to determine the relative expression of the indicated genes as described in Figure 1F.
for Th17 induction when the T cells were stimulated with a high
dose of TCR stimuli either in the presence or absence of LIGHT
(Figure S2D). Thus, it appears that NIK regulates the strength of
the TCR and costimulatory signals involved in Th17 differentiation.
NIK is dispensable for T-cell proliferation but is required for the
induction of genes involved in Th17 commitment and effector
functions
To further characterize the molecular mechanism by which NIK
regulates Th17 cell differentiation, we examined the effect of NIK
deficiency on the induction of T-cell proliferation and expression of
genes involved in Th17 lineage commitment and effector functions. When stimulated under Th0 conditions, NIK⫹/⫹ and NIK–/–
naive CD4 T cells displayed comparable proliferation efficiency
(Figure 4A). Similarly, NIK⫹/⫹ and NIK–/– T cells did not show
significant difference in proliferation upon stimulation under Th17
conditions (Figure 4A). Thus, the NIK deficiency has no significant
effect on the proliferation potential of naive CD4 T cells, a finding
that is consistent with the dispensable role of NIK in the induction
of Th1 and Th2 cells (Figure 3A).
We next examined the role of NIK in regulating the gene
expression program involved in the induction of Th17 differentiation. As previously demonstrated,7 stimulation of wild-type naive
CD4 T cells under Th17 conditions resulted in the induction of
genes encoding IL-21, IL-23R, and the Th17 lineage specific
transcription factor ROR␥t, as well as the Th17 effector cytokines
IL-17A, IL-17F, and IL-22 (Figure 4B). Consistent with the ICS
results (Figure 3B), the induction of IL-17A gene expression was
largely blocked in the NIK-deficient T cells (Figure 4B). Similarly,
the mutant T cells had a defect in the induction of IL-17F and IL-22
genes. Interestingly, the NIK deficiency also attenuated the induction of the major Th17-regulatory genes, IL-21, IL-23R, and
ROR␥t (Figure 4B). Thus, NIK is not simply required for IL-17A
gene induction but is rather involved in the signal transduction that
mediates the Th17-specific gene expression program.
To further confirm that the loss of NIK was responsible for the
defect of the NIK–/– T cells in Th17 differentiation, we reconstituted these mutant T cells in vitro with NIK by retroviral transduction. To assure efficient selection of the transduced cells, we used a
retroviral vector expressing green fluorescence protein (GFP) and
sorted the GFP-positive cells for the Th17 differentiation experiment. As expected, parallel experiment using uninfected cells
revealed a severe defect of NIK–/– T cells in Th17 differentiation, as
indicated by the defect in IL-17A induction (Figure 4C left panel).
Furthermore, reconstitution of the NIK–/– T cells with NIK, but not
GFP, rescued the induction of IL-17A (Figure 4C right panel).
These results further emphasize the T-cell intrinsic role of NIK in
the regulation of Th17 cell differentiation.
NIK mediates the synergistic induction of STAT3 tyrosine
phosphorylation by TCR and IL-6R signals
In B cells, NIK is known to induce the processing of NF-␬B2
precursor protein p100,44 a central step in noncanonical NF-␬B
activation.31 However, stimulation of T cells through the TCR
signaling pathway does not lead to appreciable p100 processing.45
Because NIK activation by the TCR signal occurs rapidly,34 we
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JIN et al
dimerization and nuclear translocation.26 STAT3 phosphorylation
can be stimulated by IL-6,7 although early studies also suggested
the induction of this event by the TCR signal.46 To examine the role
of NIK in regulating STAT3 phosphorylation, we used a flow
cytometry approach that allows detection of STAT3 phosphorylation on a single-cell basis. Stimulation of naive CD4 T cells with
IL-6 for 25 minutes led to the induction of STAT3 tyrosine
phosphorylation (Figure 5, top left panel). This signaling event
appeared to be independent of NIK, as it similarly occurred in
NIK-deficient T cells. Furthermore, the IL-6 stimulated STAT3
phosphorylation was transient, which was almost reversed to the
background level after 50 minutes (Figure 5 top right panel). The
TCR signal also induced STAT3 tyrosine phosphorylation, albeit
with a lower magnitude and more transient kinetics (Figure 5
middle panels). Remarkably, however, the TCR signal potently
synergized with IL-6, leading to much stronger and prolonged
induction of STAT3 tyrosine phosphorylation (Figure 5 bottom
panels). Moreover, the synergistic induction of STAT3 phosphorylation by the TCR and IL-6 signals was substantially attenuated in
the NIK–/– T cells. These results suggest that optimal activation of
STAT3 in naive CD4 T cells requires both the IL-6R and TCR
signals and that NIK plays a critical role in mediating this
functional interplay.
Figure 3. NIK is required for differentiation of Th17 cells but is dispensable for
the differentiation of other CD4 effector cells. (A) Naive CD4 T cells isolated from
NIK⫹/⫹ and NIK–/– mice were stimulated for 72 hours with plate-bound anti-CD3 and
anti-CD28 (1 ␮g/mL) under Th0, Th1, or Th2 conditions followed by flow cytometry to
measure the frequency of IFN-␥ producing Th1 cells and IL-4 producing Th2 cells.
(B) Naive CD4 T cells were stimulated for 72 hours with plate-bound anti-CD3 and
anti-CD28 (1 ␮g/mL) under Th0, Th17, or iTreg conditions and then subjected to flow
cytometry to determine the frequency of Foxp3 producing iTregs and IL-17A
producing Th17 cells. Data in both panels A and B are representative of 3 independent experiments.
reasoned that NIK might be involved in the regulation of the early
signaling events involved in Th17 lineage commitment. In this
regard, the transcription factor STAT3 is a key molecule that
initiates the Th17 commitment program.7 STAT3 activation involves its phoshorylation at tyrosine 705, which triggers its
Discussion
The results presented in this paper demonstrate a novel and
unexpected function of NIK in the regulation of T-cell mediated
immune functions. Using T-cell transfer and in vitro differentiation
approaches, we have shown that NIK has a T-cell intrinsic function
in the regulation of Th17 cell differentiation. Consistent with these
findings, NIK deficiency renders mice resistant to the induction of
EAE, an autoimmune disease that involves Th17 cells. Like the
role of NIK in T-cell differentiation, the function of NIK in
mediating EAE is T-cell intrinsic, as revealed by T-cell transfer into
Rag2–/– mice. Our findings thus establish NIK as a critical signaling
molecule that regulates Th17 differentiation and EAE induction.
Figure 4. NIK is dispensable for naive CD4 T-cell
proliferation but is required for the induction of
Th17-specific genes. (A) Naive CD4 T cells from NIK⫹/⫹
and NIK–/– mice were labeled with CFSE and stimulated
for 72 hours with plate-bound anti-CD3 and anti-CD28
under Th0 or Th17 conditions. Cell proliferation was
measured by flow cytometry and determined based on
the dilution of CFSE during cell division. The intensity of
CFSE is reduced to one-half after each cell division
(indicated by numbers). Data are representative of 3 independent experiments. (B) Naive NIK⫹/⫹ and NIK–/– CD4
T cells were stimulated with plate-bound anti-CD3 and
anti-CD28 (1 ␮g/mL) under Th0 or Th17 conditions for
18 hours. Real-time quantitative RT-PCR assays were
performed to determine the relative expression of the
indicated genes (fold to the NIK⫹/⫹ Th0 sample). Data are
representative of 3 independent experiments. (C) Naive
NIK⫹/⫹ and NIK–/– CD4 T cells were either directly subjected to Th17 differentiation assays as described in
panel B (left) or preinfected with pCLXSN(GFP) or pCLXSN(GFP)-NIK retroviral vectors (right). The infected cells
were enriched by cell sorting based on GFP expression
and then subjected to differentiation assays. Relative
expression of IL-17A was determined by real-time PCR
and presented as fold relative to NIK⫹/⫹ Th0 sample (left
panel) or to the Th0 GFP sample (right panel).
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BLOOD, 25 JUNE 2009 䡠 VOLUME 113, NUMBER 26
Figure 5. NIK regulates the synergistic induction of STAT3 tyrosine phosphorylation by the TCR and IL-6R signals. NIK⫹/⫹ and NIK–/– naive CD4 T cells were
stimulated for the indicated times with IL-6 (20 ng/mL), anti-CD3/CD28 (2 ␮g/mL), or
IL-6 plus anti-CD3/CD28. Site-specific tyrosine phosphorylation of STAT3 (Tyrosine 705) was analyzed by flow cytometry. Data are representative of 3 experiments.
NIK is known as a central player in the noncanonical NF-␬B
signaling pathway, which involves the processing of NF-␬B2
precursor protein p100 to p52 and nuclear translocation of the
p52/RelB heterodimer.31 This NIK-specific pathway is stimulated
primarily in B cells and lymphoid stromal cells by a subset of
TNFR family members.31 On the other hand, activation of T cells
by T-cell mitogens does not lead to appreciable processing of
p100.45 We also failed to detect significant p100 processing in CD4
T cells stimulated with anti-CD3/CD28 under Th17 conditions
(data not shown). Consistently, NIK deficiency does not lead to a
global defect in naive CD4 T-cell activation or differentiation. Both
the proliferation and Th1/Th2 differentiation are competent in the
NIK–/– T cells. However, the fact that NIK is activated by the TCR
signal34 suggests a signaling role for this kinase in T cells. Notably,
NIK activation by the TCR signal occurs rapidly,34 which is in
sharp contrast to the slow induction of the noncanonical NF-␬B
signaling by TNFRs.31 These previous findings, together with our
observation that NIK is required for the induction of Th17regulatory genes, suggest the involvement of NIK in early signaling events of the Th17 commitment program. Indeed, our current
study demonstrates that NIK is required for the synergistic
activation of STAT3 by the TCR and IL-6R signals. In naive
T cells, the IL-6 stimulated STAT3 phosphorylation occurs weakly
and transiently, but this signaling event is greatly potentiated and
prolonged by TCR ligation. Moreover, the NIK deficiency severely
attenuated the synergistic activation of STAT3 by the TCR and
IL-6R signals. Given the critical role of STAT3 in the induction of
Th17 commitment, the current finding suggests that regulation of
STAT3 activation is an important mechanism by which NIK
participates in the induction of Th17 differentiation. On the other
hand, our study does not exclude the involvement of p100
processing or degradation in the Th17 induction. To address this
possibility, mice expressing a processing-defective p100 would be
important.
A recent study suggests that NIK is involved in LIGHTstimulated STAT3 phosphorylation in a tumor cell line.34 Interestingly, our current study suggests that LIGHT has an important
costimulatory role in the induction of Th17 cell differentiation.
Although LIGHT stimulates both the lymphotoxin beta receptor
and HVEM, only the latter is expressed in T cells.47 We have shown
that the expression of HVEM is greatly promoted by the Th17-
REGULATION OF Th17 CELL DIFFERENTIATION BY NIK
6609
polarizing conditions and that, accordingly, LIGHT promotes the
induction of Th17 cells in vitro. Our findings are consistent with the
important function of the LIGHT/HVEM signaling pair in mediating inflammatory responses in vivo.48,49 A recent study demonstrates that HVEM expression in T cells is partially required for
T-cell mediated induction of colitis, although HVEM has an
opposite function when it is expressed in non-T cells.49 It is
currently unclear whether T-cell-specific knockout of HVEM
affects Th17 development in vivo. We have further shown that NIK
is required for the induction of Th17 cell differentiation by the TCR
and LIGHT signals. This function of NIK is particularly prominent
when T cells are stimulated with low doses of anti-CD3/CD28 but
becomes less striking in the presence of high doses of the TCR
stimuli. It is important to note, though, that the role of NIK in Th17
cell differentiation is not limited to LIGHT costimualtion, as loss of
NIK also affects the production of Th17 cells induced in the
absence of LIGHT. It is thus unclear whether NIK mediates
LIGHT-stimulated signaling in T cells or promotes the T-cell
costimulation by acting on the TCR signal. On the other hand, our
data clearly demonstrate a role for NIK in regulating the TCR
signal, particularly in the activation of STAT3.
The effect of NIK deficiency on EAE induction is striking. Both
regular NIK–/– mice and NIK–/– T cell-transferred Rag2–/– mice are
completely resistant to EAE induction by MOG immunization.
This phenotype cannot be solely attributed to the deficiency in
IL-17 production, because IL-17A knockout mice show reduced
severity, but not blocked incidence, of EAE disease9 and IL-17Fdeficient mice only have a minor phenotype in EAE induction.44
However, because Th17 cells produce additional cytokines and
possibly other factors, it is likely that the combinational function of
the Th17-derived inflammatory factors is more significant than any
of the single cytokine in mediating autoimmunity. Indeed, mice
lacking the transcription factors ROR␥t and ROR␣, in which Th17
cell development is impaired, are completely resistant to EAE
induction.50 Importantly, our data suggest that NIK is critical for
Th17 cell development, rather than simply controls the expression
of IL-17A and IL-17F. This finding at least partially explains the
critical role of NIK in mediating EAE induction. However, it is also
possible that NIK regulates additional functions of T cells involved
in the pathogenesis of EAE. In this regard, we have shown that NIK
deficiency in T cells reduces the expression of the Th1 cytokine,
IFN-␥, during the effector phase of EAE. Because NIK is
dispensable for Th1 cell production both in vivo (in spleen) and in
vitro, this finding indicates that NIK may regulate the CNS
migration or activation of Th1 cells. Notwithstanding, our current
study clearly demonstrates a role for NIK in regulating the
signaling program mediating Th17 cell differentiation, which may
contribute to the resistance of NIK–/– mice to EAE induction.
Acknowledgments
We thank Amgen for providing the NIK knockout mice.
This study was supported by grants from the National Institutes
of Health (NIH, Bethesda, MD; AI064639, GM084459, and
AI057555).
Authorship
Contribution: W.J. and X.-F.Z. performed research, analyzed data,
and prepared figures; J.Y. contributed to experimental work; X.C.
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6610
BLOOD, 25 JUNE 2009 䡠 VOLUME 113, NUMBER 26
JIN et al
provided technical assistance; and S.-C.S. supervised the overall
research and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Shao-Cong Sun, Department of Immunology,
The University of Texas M. D. Anderson Cancer Center, 7455
Fannin St, Box 902, Houston, TX 77030; e-mail: ssun@
mdanderson.org.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2009 113: 6603-6610
doi:10.1182/blood-2008-12-192914 originally published
online May 1, 2009
Regulation of Th17 cell differentiation and EAE induction by MAP3K NIK
Wei Jin, Xiao-Fei Zhou, Jiayi Yu, Xuhong Cheng and Shao-Cong Sun
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