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Regulation of the IL-21 Gene by the NF-κB
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J Immunol 2010; 185:2350-2359; Prepublished online 16
July 2010;
doi: 10.4049/jimmunol.1000317
http://www.jimmunol.org/content/185/4/2350
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References
Guobing Chen, Kristine Hardy, Karen Bunting, Stephen
Daley, Lina Ma and M. Frances Shannon
The Journal of Immunology
Regulation of the IL-21 Gene by the NF-kB Transcription
Factor c-Rel
Guobing Chen,* Kristine Hardy,* Karen Bunting,*,1 Stephen Daley,† Lina Ma,* and
M. Frances Shannon*
C
ytokines are a broad class of secreted molecules that play
important regulatory roles during the immune response
and in the development, proliferation, and differentiation
of immune cells. IL-21 is one such cytokine, first described to be
important for the regulation of NK cell and lymphocyte function
(1). It is a four-helix bundle type I cytokine, homologous to
IL-2 and IL-15, and is a ligand for the receptor complex composed
of the unique IL-21R, which is most closely related to the IL-2Rbchain, and the common g-chain (gc), which is shared by the
receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (2, 3). At a molecular level, the IL-21/IL-21R interaction and the resultant signaling
pathways have been well described. IL-21 activates the JAK family protein tyrosine kinases JAK1 and JAK3, which bind to IL-21R
tyrosine 510 (Y510) (4) and gc, respectively (2, 3), and mediate
the activation of STAT1, STAT3, and to a lesser degree STAT5A
and STAT5B (5–7). IL-21 can also transfer signals through the
*Gene Expression and Epigenomics Group, Department of Genome Biology and
†
Immunogenomics Group, Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory,
Australia
1
Current address: Department of Medicine/Hematology-Oncology, Weill Cornell
Medical College, New York, NY.
Received for publication January 29, 2010. Accepted for publication June 17, 2010.
This work was supported by a grant from the National Health and Medical Research
Council of Australia (to M.F.S.). G.C. was supported by the Australian Leadership
Award Scholarship from the Australian Agency for International Development.
Address correspondence and reprint requests to Prof. M. Frances Shannon, Gene
Expression and Epigenomics Group, Department of Genome Biology, John Curtin
School of Medical Research, Australian National University, Canberra, Australian
Capital Territory 2600, Australia. E-mail address: [email protected]
Abbreviations used in this paper: gc, common g-chain; ChIP, chromatin immunoprecipitation; DC, dendritic cell; GC, germinal center; EAE, experimental autoimmune
encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; NS, nonstimulated; P/I,
PMA and ionomycin; SOCS, suppressor of cytokine signaling; TF, transcription factor;
TFH, follicular helper T cell; UBC, ubiquitin-conjugating enzyme.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000317
MAPK and PI3K pathways via the phosphorylation of Shc and
Akt (4).
Although IL-21 is expressed exclusively by activated CD4+
T cells and NKT cells (1, 8–10), it has been shown to have a broad
range of effects on many immune cells, including T and B cells,
NK cells, and dendritic cells (DCs). It can costimulate T cell proliferation together with an anti-CD3 Ab (1), promote proliferation
of CD8+ T cells in a synergistic manner with IL-7 or IL-15 but not
IL-2 (11), and augment antitumor activity of CD8+ T cells (11–15)
and NK cells (16, 17). IL-21 can also costimulate B cell proliferation
(1), induce the differentiation of germinal center (GC) B cells to
plasma cells, and is a crucial regulator of Ab production (18). IL-21
also induces NK cell differentiation and functional maturation, activates the cytotoxic program (16, 19), and has proapoptotic effects
on NK cells in a concentration- and cofactor-dependent manner (20,
21). In myeloid cells, IL-21 induces a proinflammatory response by
augmenting the proliferation and differentiation of DCs (17, 22) and
inducing the expression of the neutrophil chemoattractant CXCL8
in macrophages (23).
More recently, IL-21 has been shown to have an essential role in
the development of Th17 and follicular helper T (TFH) cells (24,
25). IL-21 was found to be highly expressed in the TGF-b– and
IL-6–polarized Th17 subset and was subsequently identified as an
initiator of Th17 development in an IL-6–independent pathway
(24, 25). It could furthermore induce IL-23R expression on activated CD4+ T cells and maintain the expansion of differentiated
Th17 cells, which in turn produced high levels of IL-21. Accordingly, IL-21 has been proposed to have an autocrine function in
the Th17 development process (9, 26, 27). Similarly, IL-21 was
found to be highly expressed in CD4+CXCR5+ICOS+ TFH cells
and is essential for TFH activation and expansion during GC formation (10, 28).
IL-21 appears to have both positive and negative regulatory effects in immune responses and has additionally been recognized for
its antitumor effects in a number of preclinical tumor models, including models of melanoma and lymphoma (24, 25, 29). It is also
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IL-21 is a member of the common g-chain–dependent cytokine family and is a key modulator of lymphocyte development,
proliferation, and differentiation. IL-21 is highly expressed in activated CD4+ T cells and plays a critical role in the expansion
and differentiation of the Th cell subsets, Th17 and follicular helper T (TFH) cells. Because of its potent activity in both myeloid
and lymphoid cell immune responses, it has been implicated in a number of autoimmune diseases and has also been used as
a therapeutic agent in the treatment of some cancers. In this study, we demonstrate that c-Rel, a member of the NF-kB family of
transcription factors, is required for IL-21 gene expression in T lymphocytes. IL-21 mRNA and protein levels are reduced in the
CD4+ cells of rel2/2 mice when compared with rel+/+ mice in both in vitro and in vivo models. A c-Rel binding site identified in the
proximal promoter of il21 is confirmed to bind c-Rel in vitro and in vivo and to regulate expression from the il21 promoter in
T cells. Downstream of IL-21 expression, Th17, TFH, and germinal center B cell development are also impaired in rel2/2 mice. The
administration of IL-21 protein rescued the development of TFH cells but not germinal center B cells. Taken together, c-Rel plays
an important role in the expression of IL-21 in T cells and subsequently in IL-21-dependent TFH cell development. The Journal
of Immunology, 2010, 185: 2350–2359.
The Journal of Immunology
Materials and Methods
Animals and Ag immunization
All animals were maintained in a specific pathogen-free facility. c-Rel knockout (rel2/2) mice were obtained from Prof. S. Gerondakis (Burnet Institute
South Australia, Australia) and fully backcrossed 10 generations onto
C57BL/6. Wild-type C57BL/6 mice (rel+/+) were purchased from the Australian Phenomics Facility, Australian National University (Canberra, Australian Capital Territory, Australia). Eight- to 12-wk-old female mice were
used in all experiments. Myelin oligodendrocyte glycoprotein (MOG) peptide (aa 35–55; MEVGWYRSPFSRVVHLYRNGK, MOG35–55) was synthesized by the Biomolecular Resource Facility, Australian National
University. The MOG35–55 peptide was dissolved in PBS at 2 mg/ml and
emulsified in an equal volume of CFA consisting of IFA (Difco, Ann Arbor,
MI) plus 4 mg/ml heat-inactivated Mycobacterium tuberculosis (strain H37
RA; Difco). One hundred-microliter emulsions were injected s.c. into each
mouse. Spleens were isolated from immunized mice after 14 d.
EAE induction and clinical scoring
EAE induction was performed using MOG35–55 immunization as indicated
above plus 100 ml 1.5 mg/ml p-toxin tail vein injections, immediately
before and 2 d after MOG35–55 immunization. The clinical assessment of
EAE was performed every day until day 35 after immunization and scored
with a standard grading system. Grades 0–5 represented no overt signs of
disease, limp tail, limp tail plus hind limb weakness, hind limb paralysis,
forelimbs paralysis, and moribund state/death by EAE/sacrifice for humane
reasons, respectively.
EAE development in bone marrow-reconstituted mice
The CD45.1 congenic wild-type C57BL/6 (rel+/+) mice were irradiated
with two doses of 450 rad spaced 4 h apart, followed by i.v. injection of
107 bone marrow cells from CD45.2 rel+/+, rel2/2 or a mixture of CD45.1
rel+/+ and CD45.2 rel2/2 at a 1:1 ratio. The chimeric mice were immunized with MOG35–55 on week 4 after the reconstruction of immune system. The splenocytes were isolated for flow cytometry of TFH and GC
B cells at day 14 after immunization.
Administration of rIL-21
Recombinant murine IL-21 protein (0.5 mg/injection) (594-ML; R&D
Systems, Minneapolis, MN) or PBS as a control was used for i.v. injection
at days 0, 4, 7, 10, and 13 after MOG35–55 immunization. The splenocytes
were isolated for flow cytometry of TFH and GC B cells at day 14 after
immunization.
CD4+ T cell purification and stimulation
CD4+ T cells were purified from spleens of rel+/+ and rel2/2 mice as
described previously (52). CD4+ T cells (1 3 106 cells/ml) were stimulated
for the indicated times with the appropriate activating Ab, cytokines or
chemical. The stimuli used were anti-CD3 Ab (553058; BD Pharmingen,
San Diego, CA) at 1/100, anti-CD28 Ab (553295; BD Pharmingen) at 1/
200, PMA (Sigma-Aldrich, St. Louis, MO) at 10 ng/ml, calcium ionomycin A23187 (Sigma-Aldrich) at 1 mM, TGF-b (240-B; R&D Systems) at
1 ng/ml, IL-6 (406-ML; R&D Systems) at 10 ng/ml, IL-21 (594-ML; R&D
Systems) at 50 ng/ml, and ICOS (313511; BioLegend, San Diego, CA) at
10 ng/ml. For proliferation assays, the CD4+ T cells were first labeled with
CFSE (53) and then stimulated with the appropriate stimuli.
Cell culture
EL-4.IL-2 (EL-4) murine thymoma cells were maintained in RPMI 1640
medium supplemented with 10% FCS, 10 mM HEPES, 2 mM Lglutamine, and antibiotics. EL-4 cells were transfected by electroporation
with the indicated plasmids as previously described (52) and stimulated at
1 3 106 cells/ml with 10 ng/ml PMA and 1 mM calcium ionomycin
A23187 (P/I).
Plasmids preparation
The mouse IL-21 luciferase reporter, IL-21/pXPG, was constructed by inserting the 2260 to +33 bp promoter region of the mouse IL-21 gene into the
pXPG luciferase reporter (54) using a BglI/SalI PCR product amplified
from a BAC plasmid (RP23-98I15) containing the mouse IL-21 gene
(Children’s Hospital Oakland Research Institute, Oakland, CA). Mutation
of the c-Rel binding site in the IL-21 promoter luciferase reporter (mutIL-21)/
pXPG was performed using the QuickChange II Site-Directed Mutagenesis
Kit (Stratagene). The 2239 to 2242 bp sequence of the murine IL-21 promoter was mutated from TTCC to GGTA. The human c-Rel cDNA in
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associated with autoimmunity and autoimmune diseases, such as
systemic lupus erythematosus (30), NOD (31, 32), experimental
allergic encephalomyelitis (EAE) (33), rheumatoid arthritis (34,
35), and in a model of autoimmunity in the sanroque mouse model
(36). The administration, reduction, or neutralization of IL-21 may
therefore represent a potential therapy for immunopathologies in
which IL-21 expression is dysregulated. This warrants further investigation of the regulation of the il21 gene in lymphocytes and
in IL-21–dependent immune responses.
IL-21 is reported to be mainly expressed in activated CD4+
T cells, especially in TFH cells, Th17 cells and NKT cells (1, 8–
10). The transcription factors (TFs) that have been implicated in
IL-21 gene expression to date are NFATc2 and T-bet, which activate and repress IL-21, respectively (37, 38). However, IL-21 is still
expressed in NFATc2-deficient mice, implying that additional TFs
might be involved in the regulatory events of il21 gene expression
(37). Because we and others have shown that members of the
NF-kB family of TFs, including c-Rel and RelA, play important
roles in the regulation of other inducible cytokines, such as IL-2, in
activated CD4+ T cells (39, 40), we predicted that these factors
might also play a role in the regulation of IL-21 in these cells.
The NF-kB family of TFs is composed of five family members:
p50 (NF-kB1), p52 (NF-kB2), p65 (RelA), RelB, and c-Rel, which
function as homo- or heterodimers to regulate genes important for
inducible immune and inflammatory responses (reviewed in Ref.
41). Compared with the other NF-kB proteins, c-Rel expression is
primarily restricted to mature cells of the myeloid and lymphoid
lineages (42, 43). Although dispensable for hematopoietic progenitor cell differentiation, c-Rel is critical for the normal function of
B cells, T cells, macrophages, and DCs and for the expression of
genes encoding a number of cytokines and TFs during immune cell
activation (44). Specific examples of the requirement for c-Rel in
B cells include the maintenance of B cell viability and promotion
of cell cycle progression through the regulation of the antiapoptotic
genes Bcl-2 and Bcl-XL (45–47) and activation of the cell cycle
genes E2F3a and cyclin E (47), respectively. Decreased numbers
of GC B cells in c-Rel knockout (rel2/2) mice as well as in p502/2 or
p652/2 fetal liver-reconstituted mice (48, 49) also point to an important role for c-Rel and other NFkB family members in B cell
activation and Ig affinity maturation during normal B cell development. Although the early stages of T cell development are not impaired in rel2/2 mice, even though peripheral CD4+ and CD8+ cell
number are slightly reduced (39, 50), Th1 polarization and the expression of associated cytokine genes are significantly impaired in
rel2/2 mice under specific Ag challenge (39, 51). The role of c-Rel
in specific T cell subsets, including the now well-characterized
Th17 and TFH cell populations, has not been fully investigated;
however, c-Rel regulates a number of important activation markers
and differentiation factors in CD4+ T cells, including IL-2, IL-3,
GM-CSF, IFN-g, CD25 (IL-2Ra), and c-myc (41, 44).
In this study, we investigated a role for c-Rel in the inducible
expression of IL-21 in activated CD4+ T cells. In rel2/2 mice,
we show that induction of IL-21 expression is significantly impaired in activated CD4+ T cells as well as in Th17 and TFH cells
from these mice. This appears to be a direct regulatory effect
because c-Rel protein can bind to the promoter of il21 and can
regulate activity of the il21 promoter. We also show that the development of Th17 cells, TFH cells, and GC B cells are impaired in
rel2/2 mice and that the Tfh defect, but not the GC B cell defect,
can be rescued by the administration of rIL-21. Taken together,
these findings point to a novel and critical role for c-Rel in the
control of IL-21–mediated lymphocyte development and in activated T cell-dependent immunity.
2351
2352
a pRc/CMV expression vector (Invitrogen Life Technologies, Carlsbad, CA)
was reported previously (52). Sequence analysis was used to confirm the
integrity of all constructs.
RNA preparation and quantitative PCR
Total RNA was extracted from CD4+ T cells isolated from rel+/+ mice and
rel2/2 mice and from EL-4 cells and reverse transcribed as described previously (40). SYBR Green real-time PCR was performed using an ABI
PRISM 7700 sequence detection system (PerkinElmer, Wellesley, MA) as
described previously (52). To normalize for inefficiencies in cDNA synthesis and RNA input, PCRs for the ubiquitin-conjugating enzyme (UBC)
E2D2 were conducted in parallel. The primer pairs used were IL-21 forward, 59-TCA GCT CCA CAA GAT GTA AAG GG-39, and IL-21 reverse,
59-GGG CCA CGA GGT CAA TGA T-39; and UBC forward, 59-AAG AGA
ATC CAC AAG GAA TTG AAT G-39, and UBC reverse, 59-CAA CAG
GAC CTG CTG AAC ACT G-39.
DNA binding assay
Transfection of T cell lines and luciferase reporter assays
fluorochromes (eBioscience, San Diego, CA), CXCR5 and GL7 (BD
Pharmingen), biotinylated IL-21 (R&D systems), goat anti-murine IL-21
and anti-goat IgG (Santa Cruz Biotechnology), and streptavidin fluorochrome conjugates (eBioscience). For flow cytometric analysis of cytokines, CD4+ cells from rel+/+ or rel2/2 mice were simulated with
P/I plus GolgiStop for 4–6 h, followed by cell surface marker staining,
then were fixed and permeated before staining with the anti-cytokine Abs.
For the absolute cell number counting, cells were stained with 7aminoactinomycin D to exclude the dead cells and mixed with calibrite
beads (BD Biosciences, San Jose, CA) immediately before flow cytometric
analysis. Data were acquired using a FACSCalibur or LSR II flow cytometer and analyzed with FlowJo software. The absolute cell number was
calculated using a ratio based on the number of beads: the cell number =
beads added/beads counted 3 cell counted. The proliferation profiles were
analyzed using FlowJo software.
Computational promoter analysis
A region of DNA sequence corresponding to 1000 bp upstream and 500 bp
downstream of transcription start site of the IL-21 gene was analyzed by the
rVista tools (http://rvista.dcode.org/) and the TRANSFAC database (56).
The results were present in the University of California Santa Cruz genome
browser.
Statistical analysis
The CD4+ cell proliferation profiles were analyzed using CFSE timeseries cell number data (57). Briefly, the cell number in each division at
each time point was determined using the FlowJo software. The precursor
cohort numbers were generated by dividing the cell numbers in the ith
division by 2(i+0.5), where i is the division number, and used for fitting the
normal distributions for cells in each division. All data were analyzed
using a Student t test, with statistical significance represented by the following p values and symbols: pp , 0.05; ppp , 0.01; and pppp , 0.001.
Results
IL-21 expression is regulated by c-Rel in T cells
Chromatin immunoprecipitation (ChIP) analysis of c-Rel binding was performed as previously described (55) with some minor modifications. Briefly,
CD4+ T cells from rel+/+ or rel2/2 mice stimulated with P/I for 6 h were
fixed with 1% formaldehyde for 15 min, and the chromatin sonicated to
200- to 1000-bp fragments. c-Rel immunoprecipitation was performed on
precleared cell lysates for 1 h at 4˚C with 3 mg anti–c-Rel Ab (sc-71; Santa
Cruz Biotechnology). Quantitative PCR was performed on purified DNA
from the anti–c-Rel or no Ab (control) immunoprecipitations using the
forward primer 59-GGC AGG GAT GGA TAG AGT CC-39 and reverse
primer 59-CAC CTT GGT GAA TGC TGA AA-39 corresponding to the
upstream regions of the IL-21 promoter flanking the c-Rel binding site.
Gene expression profiling using mRNA isolated from rel+/+ and
rel2/2 splenic CD4+ T cells stimulated with PMA and ionomycin
for 6 h indicted that the gene encoding the cytokine IL-21 was
regulated by c-Rel (S. Lee, G. Chen, and M.F. Shannon, unpublished data). The role of c-Rel in il21 gene expression was confirmed using quantitative real-time PCR to detect il21 mRNA
expression levels in rel+/+ and rel2/2CD4+ T cells stimulated with
P/I or Abs to CD3 and CD28 (anti-CD3/CD28) for the times indicated. In rel+/+CD4+ T cells, il21 mRNA was induced in a timedependent manner in response to both P/I and anti-CD3/CD28
stimulation (Fig. 1A, 1B). In rel2/2 cells, il21 mRNA expression
was greatly reduced under both stimulation conditions, especially
at later time points (Fig. 1A, 1B). il21 was expressed at higher
levels in rel+/+CD4+ T cells that were prestimulated with antiCD3/CD28 and then reactivated with other stimuli (Fig. 1C). Loss
of c-Rel also affected the expression of il21 in anti–CD3/CD28prestimulated cells that were restimulated with P/I, PMA, or ionomycin alone, anti-CD3/CD28, CD3 alone, and anti-CD3/ICOS for
6 h (Fig. 1C) or for 24 h (Fig. 1D). Interestingly, IL-6 restimulation
induced higher levels of il21 in the absence of c-Rel (Fig. 1C, 1D).
Measurement of IL-21 protein following stimulation with either
P/I or anti-CD3/CD28 by ELISA (Fig. 2A) revealed that IL-21
protein was reduced in activated CD4+ cells from rel2/2 mice. In
an intracellular FACS assay using splenocytes stimulated with P/I,
the percentage of CD4+IL-21+ cells in rel+/+ was higher than in the
rel2/2 mouse (Fig. 2B, 2C). The percentage of IL-21+ cells within
the CD4+ population was also decreased in rel2/2 when compared
with rel+/+ mice (Fig. 2D, 2E).
From these data, c-Rel appears to be important for inducible il21
mRNA and protein expression in activated CD4+ T cells.
Flow cytometry
c-Rel binds to and activates the promoter region of the IL-21 gene
The following Abs were used in flow cytometric analysis of murine cell
markers: CD4, CD95, B220, ICOS, IL-17a, and IFN-g with conjugated
To determine whether IL-21 was a direct target of the TF, c-Rel,
the il21 promoter sequence, was analyzed for potential c-Rel or
EL-4 (5 3 106) cells were electroporated with a Bio-Rad Gene Pulser at
270 V and a capacitance of 975 microfarads. For luciferase assays, EL-4
T cells were transfected in duplicate with 10 mg empty pXPG plasmid or
IL-21/pXPG or muIL-21/pXPG reporter plasmids described above either
alone or in combination with 10 mg empty pRc/CMV or a c-Rel expression
plasmid. Transfected cells were recovered overnight and left unstimulated
or stimulated for the indicated times with P/I, and cell lysates were harvested and analyzed as described previously (52). Thirty micrograms of
total protein was analyzed for luciferase activity using a Turner BioSystems microplate luminometer.
IL-21 sandwich ELISA
Anti–IL-21 Ab (sc-17651; Santa Cruz Biotechnology) was coated onto
a 96-well ELISA plate overnight. The sera from unimmunized rel+/+
and rel2/2, or cultured medium from CD4+ T cells from rel+/+ or rel2/2
mice stimulated with PMA plus ionomycin or anti-CD3 plus anti-CD28
for 6 h, were incubated in the coated plates, and bound protein was
detected with a biotinylated anti–IL-21 Ab (BAF594; R&D Systems)
and a streptavidin/HRP-conjugated Ab (Bio-Rad, Hercules, CA). A tetramethylbenzidine substrate (BD Pharmingen) was added, the reaction stopped with 1 M H2SO4, and color development was detected on a microplate
reader (Molecular Devices) at 450 nm.
Chromatin immunoprecipitation
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Recombinant c-Rel homodimer binding to predicted c-Rel binding sites was
measured using a modified enzyme-linked immunoassay (52). Briefly,
biotinylated, double-stranded, sense and antisense oligonucleotides (25mer) corresponding to consensus, mutant, and gene-specific NF-kB/Rel
binding sequences were generated. NeutrAvidin-coated 96-well strip plates
(Pierce Chemical Co., Rockford, IL) were incubated with 50 nM oligonucleotides followed by blocking with 3% skim milk in binding buffer (10
mM Tris [pH 7.5], 10 mM MgCl2, 5 mM EDTA, 10 mM DTT, 0.2%
Nonidet P-40, 1% glycerol, 0.4% sucrose, and 0.5 mg/ml BSA). Purified
recombinant human c-Rel protein produced in Escherichia coli was serially diluted (0.4–56 nM) in binding buffer containing 3% skim milk and
5 mg/ml poly(deoxyinosinic-deoxycytidylic) (GE Healthcare, Piscataway,
NJ) and incubated for 1 h. c-Rel binding was detected with an anti–c-Rel
Ab (sc-71; Santa Cruz Biotechnology, Santa Cruz, CA) and a secondary
HRP-conjugated Ab. A tetramethylbenzidine substrate (BD Pharmingen)
was added, the reaction was stopped with 1 M H2SO4, and color development was detected on a microplate reader (Molecular Devices, Sunnyvale,
CA) at 450 nm.
c-Rel REGULATES IL-21 EXPRESSION
The Journal of Immunology
2353
FIGURE 1. IL-21 gene expression was reduced in
rel2/2CD4+ T cells. A–D, IL-21 mRNA levels were
measured by quantitative RT-PCR in splenic CD4+
T cells isolated from rel+/+ (n) or rel2/2 (N) mice. Cells
were stimulated with P/I (A), anti-CD3/CD28 (B), or
stimulated with anti-CD3/CD28 for 2 d, then restimulated with the stimuli indicated and assayed 6 h (C) or
24 h (D) later. Average IL-21 mRNA levels are expressed relative to UBC mRNA levels from triplicate experiments. NS, nonstimulated.
ELISA-based DNA binding assay to test the binding ability of this
predicted c-Rel site, recombinant c-Rel bound to the il21 promoter
site in a sequence-specific manner (Fig. 3B). To determine
whether this site bound c-Rel in vivo, ChIP assays were performed with murine splenic CD4+ T cells that were either left
FIGURE 2. IL-21 protein was reduced in rel2/2
CD4+ T cells. A, IL-21 protein levels were measured
by ELISA in culture medium from rel+/+ (n) or rel2/2
(N) CD4+ T cells stimulated with P/I or anti-CD3/CD28
for 6 h. B–E, IL-21 protein detected by intracellular Ab
staining in splenocytes stimulated with P/I for 6 h from
rel+/+ (n) or rel2/2 (N) mice. B, Representative flow
cytometry plots showing CD4 versus IL-21 expression.
D, Representative flow cytometry histogram showing
the percentage of IL-21+ cells in CD4+ population.
Solid line, P/I stimulated; dashed line, unstimulated;
black line, rel2/2; and gray line, rel2/2. The number
on the right or left represented IL-21+ and IL-212 cells,
respectively. Summaries of the data in B and D are
shown in C and E, respectively. All experiments were
performed in triplicate. NS, nonstimulated.
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NF-kB binding sites. A binding site matching the consensus site
for c-Rel (58) was found at position 2234 to 2243 bp
(GTGAATTCCA) upstream of the transcription start site of the
murine il21 gene (Fig. 3A). This site was conserved in human,
mouse, rat, dog, opossum, and chicken (Fig. 3A). Using an in vitro
2354
c-Rel REGULATES IL-21 EXPRESSION
that c-Rel also bound to the proximal promoter of il2, which has
previously been shown to be bound and regulated by c-Rel in
these cells (Fig. 3C) (40).
To test the specific function of the c-Rel binding site in the il21
promoter, a luciferase reporter construct containing 294 bp of the
il21 promoter, encompassing the c-Rel binding site identified
above, was transfected into EL-4 T cells and luciferase activity
measured over time. The il21 reporter construct showed almost no
activity in nonstimulated cells but was activated in a time-dependent
manner with P/I stimulation (Fig. 3D, 3E). When c-Rel was cotransfected into these cells, the il21 reporter construct showed higher
levels of activity in response to c-Rel over-expression, particularly
at later time points (Fig. 3D). Mutation of the c-Rel binding site
(muIL-21) abolished the ability of the il21 reporter to respond to
P/I stimulation (Fig. 3E). In addition, overexpression of c-Rel led to
an increase in promoter activity for the wild-type but not the mutated
construct (Fig. 3E), suggesting that c-Rel is at least partially responsible for the activity of the il21 promoter. These results show that the
il21 gene is a direct target of c-Rel.
FIGURE 3. c-Rel binds to the promoter of the IL-21 gene and regulates
IL-21 gene activity. A, Schematic of the IL-21 promoter (21000 to +500 bp
of transcription start site) in the University of California Santa Cruz
browser, showing the location of the c-Rel binding site and conservation
across species. B, Recombinant c-Rel binding to oligonucleotides corresponding to a c-Rel consensus site (black bar), mutated c-Rel consensus
(white dotted bar), IL-21 promoter potential c-Rel binding site (gray bar),
and an IL-2 promoter c-Rel binding site (open bar). C, ChIP analysis of cRel binding to the IL-2 and IL-21 proximal promoter regions in rel+/+CD4+
T cells. Cells were left unstimulated (open bar) or were stimulated with P/I
for 6 h (filled bar). D and E, A luciferase reporter plasmid containing the
IL-21 proximal promoter sequence (IL-21) was cotransfected into EL4 cells
with and without a c-Rel expression plasmid, in the presence or absence of
P/I stimulation, and analyzed for luciferase activity. D, Luciferase activity
measured in transfected cells activated with P/I stimulation over time. E,
Comparison of c-Rel overexpression on the activity of the wild-type il21
reporter (IL-21) or the il21 reporter containing a mutant c-Rel binding site
(muIL-21) in nonstimulated cells (open bar) or cells stimulated with P/I for
12 h (filled bar). pXPG, empty luciferase reporter plasmid; CMV, empty
expression plasmid. All experiments were performed three times.
unstimulated or were stimulated with P/I for 6 h. c-Rel binding
was detected at the il21 proximal promoter only in P/I-activated
cells (Fig. 3C), As a positive control for the assay, we showed
Th17 and TFH cells are known sources of IL-21 in vivo (9, 10). To
determine the specific CD4+ Th cell subsets in which c-Rel plays
a role in the regulation of IL-21, we first generated Th17 cells
in vitro by treatment of CD4+ T cells from the spleens of rel+/+ or
rel2/2 mice with anti-CD3, TGF-b, and IL-6 and measured the
expression of IL-21 in the percentage of Th17 subset distinguished
with the markers CD4+IFN-g2IL-17+. Compared with rel+/+derived Th17 cells (CD4+IFN-g2IL-17+), the levels of IL-21
mRNA and protein were reduced by .50% in Th17 cells derived
from rel2/2CD4+ T cells (Fig. 4A, 4B), as measured by RT-PCR
and ELISA, respectively. Intracellular Ab staining for IL-21 also
showed that there were a reduced percentage of IL-21–expressing
Th17 cells within the rel2/2CD4+ T cells (Fig. 4C, 4D).
To determine the importance of c-Rel in the expression of IL-21
in TFH cells, rel+/+ and rel2/2 mice were immunized in vivo with
the MOG35–55 Ag plus CFA. CD4+ cells were isolated from the
immunized mice after 2 wk, and the levels of IL-21 protein expression in CD4+CXCR5+ICOS+ TFH cells were measured using
intracellular Ab staining for IL-21. Compared with TFH cells in
rel+/+ mice, IL-21 protein was reduced in rel2/2 TFH cells both in
the percentage of cells that were IL-21 positive and the amount of
IL-21 per cell (Fig. 4E). Thus, c-Rel appears to play a role in the
regulation of il21 gene expression in at least two specialized types
of Th cells, Th17 and TFH.
To determine whether the defect of IL-21 expression in these Th
subsets was a consequence of the impaired T cells proliferation previously observed in rel2/2 mice, CFSE-labeled CD4+ T cells were
stimulated under Th17 condition in vitro, and IL-21 expression was
detected by flow cytometry. First, the proliferation of CD4+ T cells
in rel2/2 mice was confirmed to be impaired under Th17 polarization conditions (Fig. 5A–C). Then, analysis of the cell population
that had undergone several rounds of division showed that the
percentage of IL-21–positive cells present in this proliferated
population of CD4+ cells was still reduced (Fig. 5D). Thus, the
decrease of IL-21 in CD4+ T cells cannot be completely attributed
to the T cell proliferation defect in rel2/2 mice.
Th17, TFH, and GC B cells were reduced in the rel2/2 mouse
As discussed above, IL-21 is necessary for Th17 polarization and is
essential for TFH development and GC formation. To determine
whether the immune response associated with IL-21 was affected
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Th17 and TFH cells from rel2/2 mice produce decreased levels
of IL-21
The Journal of Immunology
by the impaired production of IL-21 in rel2/2 mice, the MOG35–55immunized EAE model was performed, and the in vivo production
of Th17 cells, TFH cells, and GC B cells was compared in rel+/+ and
rel2/2 EAE mice. Following immunization with MOG35–55, the
EAE could not be induced in rel2/2 mice (Fig. 6A), which confirmed
a previous report (51). The percentages of both Th17 (CD4+ IFN-g2
IL-17+) and Th1 (CD4+IFN-g+IL-172) cells in the CD4+ population
were reduced significantly in rel2/2 cells compared with rel+/+ cells
(Fig. 6B, 6C).
In the MOG35–55-immunized mouse, the other IL-21–associated
Th subset, TFH (CD4+CXCR5+ICOS+) cells were also reduced
significantly in rel2/2 cells compared with rel+/+ cells (Fig. 7A,
7B). The IL-21–dependent GC B cells, B220+CD95+GL7+, were
almost completely absent in rel2/2 mice (Fig. 7D, 7E). Altogether, these data suggest that c-Rel is an important factor for the
development of IL-21–dependent T and B cell subsets in vivo.
FIGURE 5. IL-21 deficiency was not directly associated with the defect
of proliferation in rel2/2 mouse. A–C, CD4+ cell proliferation under Th17
polarization condition was defective in rel2/2 mice. CD4+ cells from rel+/+
or rel2/2 mice were labeled with CFSE and left unstimulated (B) or stimulated under anti-CD3, TGF-b, and IL-6 for 3.5 d (C). The total CFSE
distribution (as indicated by the gray line in A) was broken up into the
component populations based on the number of predicted divisions. The
undivided cell population is represented with orange shading, whereas the
divided populations are represented with pink shading (A). The proliferation
profiles were calculated in FlowJo and converted to normal distributions for
cells in each division. D, CD4+ cells from rel+/+ and rel2/2 mice were
labeled with CFSE and cultured under Th17 polarization conditions
in vitro. The expression of IL-21 in the divided CD4+ cells (with low CFSE)
was measured and the distribution of IL-21 is shown. All the experiments
were performed in triplicate.
The TFH defect in rel2/2 mice is extrinsic and can be rescued
by IL-21 administration
To determine whether the defect in IL-21–associated lymphocyte
development was caused by impaired IL-21 in rel2/2 mice, bone
marrow chimera EAE experiments and IL-21 protein rescue
assays were performed. In the mixed chimera EAE mice, GC
B cells did not develop normally when the rel2/2 donor cells were
provided to wild-type recipients and, furthermore, was not rescued
when the 50:50 mixed wild-type: rel2/2 donors were provided to
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FIGURE 4. IL-21 mRNA and protein levels were reduced in Th17 and
TFH cells. A–D, CD4+ cells from rel+/+ and rel2/2 mice (n = 6) were
stimulated with anti-CD3, TGF-b, and IL-6 for 3.5 d to generate Th17
cells. Th17-polarized cells were analyzed for IL-21 mRNA expression by
quantitative RT-PCR (A) and for IL-21 protein level by ELISA (B) or by
intracellular Ab staining and flow cytometry (C and summary in D). All
experiments were performed in triplicate. E, IL-21 intracellular staining in
TFH cells. rel+/+ or rel2/2 mice (n = 5) were immunized with MOG35–55
for 2 wk, following which, CD4+ cells were isolated and stained for
CXCR5+ICOS+ surface expression and IL-21 expression. Shadowed area,
rel+/+; solid line, rel2/2; and dashed line, IgG control. Experiments were
performed in duplicate.
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wild-type recipients (Fig. 8A, 8B). The development of TFH, however, did not appear to be significantly decreased under any of the
conditions tested (Fig. 8C, 8D). Thus, the development defect of
GC B cells in rel2/2 mouse is intrinsic, but the TFH cell defect
appears to be extrinsic because TFH cells could develop normally
even when 100% of the donor cells were from rel2/2 animals.
In the IL-21 rescue assay, the defect of GC B cells in rel2/2 mice
was not rescued by rIL-21 administration (Fig. 8E, 8F), but the
TFH defect was rescued to a large degree by the exogenous IL-21
(Fig. 8E, 8G).
All these data together suggest that c-Rel is critical for the IL-21
depended on TFH development.
Discussion
In this study, we have investigated a new role for the NF-kB TF, cRel, in the regulation of the gc family cytokine, IL-21. We have
shown that c-Rel directly controls IL-21 expression in activated
CD4+ T cells and that loss of c-Rel has a direct impact on the
development of the IL-21–dependent TFH cells development
in vivo.
NF-kB proteins regulate immune system development and inflammatory processes by controlling the expression of many cytokine genes. Among the five members of the NF-kB family, c-Rel has
been found to have a distinct and specific function in lymphoid cell
survival and in the generation of T cell-mediated immune responses
(44, 51). The majority of studies performed in rel2/2 mice to date
have revealed that a large number of inducible cytokine genes, including IL-2, GM-CSF, IL-3, IL-12, and IFN-g, are key targets of cRel and may directly contribute the downstream effects of c-Rel
on lymphocyte development and function (44).
FIGURE 7. TFH and GC B cells were reduced in the rel2/2 mouse. rel+/+
or rel2/2 mice (n = 6) were immunized with MOG35–55 for 2 wk. Splenocytes
were isolated from the immunized mice and stained for TFH and GC using
appropriate Abs. TFH cells were detected with CD4+CXCR5+ICOS+ (A, B).
GC B cells were detected with B220+GL7+CD95+ (C, D). A and C are representative dot plots of the data, which are summarized in B and D, respectively. All experiments were performed three times.
In this study, we have shown that expression of the gc family
cytokine, IL-21, is significantly reduced in CD4+ T cells of the
rel2/2 mouse. Importantly, c-Rel appears to be generally critical
for inducible il21 expression, because levels of il21 mRNA were
reduced in cells activated by a variety of stimuli, including mitogen and calcium (P/I) stimulation, as well as more physiological
stimuli, such as signaling via the TCR with CD28 or ICOS costimulation (anti-CD3/CD28 and anti-CD3/ICOS) and IL-21 signaling. Similar effects have previously been reported for IL-2
expression in the rel2/2 mouse model in response to CD3/CD28
and mitogen stimulation (39, 40).
Interestingly, IL-21 expression in response to IL-6 stimulation
was not affected by c-Rel loss. This suggests that although cRel is involved in several different signal transduction pathways
leading to inducible IL-21 expression, IL-6–dependent expression of IL-21 is independent of the NF-kB/Rel pathway, at least
in the rel2/2 mouse model. Another possibility is that c-Rel might
be involved in the IL-6–induced suppressor of cytokine signaling
(SOCS) expression (59), which inhibits the STAT pathway signal
(60), and the consequently STAT3-dependent IL-21 expression.
Our recent unpublished data shows that c-Rel binding was detected on the socs3 promoter (ChIP-chip; S. Lee and M.F. Shannon,
unpublished data), and SOCS3 expression was reduced in rel2/2
T cells (quantitative PCR; G. Chen, unpublished data). Accordingly, the expression of IL-21 under IL-6 stimulation was low in
wild-type CD4+ cells because of the normal SOCS3 induction, but
the increase observed in rel2/2 mice could be a result of the
impairment of SOCS3 expression in the rel2/2 mice.
Evidence presented in this study suggested that c-Rel played a direct role in controlling IL-21 expression. First, we identified a novel c-Rel binding site in the il21 proximal promoter, which not only
bound recombinant c-Rel protein in in vitro binding assays but
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FIGURE 6. Th17 and Th1 cells were impaired in an EAE-resistant
rel2/2 mouse. A, rel+/+ or rel2/2 mice (n = 6) were immunized with
MOG35–55, and the clinical scores were monitored every day until day
35 after immunization. B and C, rel+/+ or rel2/2 mice were immunized
with MOG35–55 for 2 wk. The splenocytes were isolated from the immunized mice and stained using CD4, IL-17, and IFN-g. The Th17 (CD4+IL17a+IFN-g2; B, C) and Th1 (CD4+IFN-g +IL-17a2; B, C) percentages
were shown in a representative dot plot in B and summarized in C. All
experiments were performed three times.
c-Rel REGULATES IL-21 EXPRESSION
The Journal of Immunology
was also bound by endogenous c-Rel in vivo. Second, we used
reporter assays to show that the proximal promoter region of il21
was sufficient for IL-21 gene activity in response to an inducible
stimulus, P/I, and was also responsive to c-Rel overexpression.
Third, the DNA binding ability of c-Rel appears to be critical
for il21 gene expression, because mutation of the c-Rel site in
the il21 proximal promoter abolished c-Rel binding in vitro as
well as c-Rel–dependent activation of the promoter region in
reporter assays. Finally, the mutated c-Rel binding site also affected the inducibility of the IL-21 proximal promoter region in
response to P/I activation, suggesting that this site is also important in the context of endogenous binding of c-Rel and other inducible factors potentially recruited to this site in activated T cells.
Taken together, these experiments argue that c-Rel plays a direct
and positive role in the regulation of the IL-21 gene.
IL-21 was previously reported to be regulated by NFATc2
through the calcium pathway (37, 38). However, IL-21 expression
is not reduced in the NFATc22/2 mouse (37). In this study, we
show evidence, both in the rel2/2 mouse and in experiments that
directly test the requirement for c-Rel in il21 gene transcription,
that c-Rel is a critical factor for inducible il21 expression. Because
in rel2/2 mice, IL-21 expression is reduced but not eliminated, as
has been shown for many other direct targets of c-Rel (52), it is
likely that other inducible TFs, including NFATc2, or other NF-kB
family members such as RelA work cooperatively with c-Rel to
initiate and enhance il21 gene expression. Other possible cooperative factors include Bcl-6, which is essential for TFH development
(61), STAT3, which is critical in the IL-21 autocrine loop in Th17
development (62), and tax-1, which was recently shown to play
a role in IL-21–dependent human T cell leukemia virus type 1–
infected T cell dysregulation (63). The generation of doubleknockout mice or small interfering RNA knockdown experiments
will be useful in deciphering the individual and combined contributions of these factors to c-Rel–mediated il21 gene expression.
Despite sharing a common receptor unit and using a common
signal transduction pathway, each of the gc-chain family cytokines
is known to play a specific function in immune system development and immune responses. Unlike other family members, IL-21
has a specific role in the differentiation of the specialty Th cell
subsets, TFH and Th17, but not in Th1 or Th2 cells. Consistent
with the expression pattern and role of IL-21 in these specific
Th cell subsets, we have shown in this study that in the rel2/2
mouse, IL-21 levels are specifically reduced in Th17 cells and
in TFH cells generated by in vitro-polarizing conditions or in vivo
immunization with MOG peptide, respectively. The results of the
CFSE proliferation assay suggest that the decrease in IL-21 was
not simply a result of decreased T cell proliferation, because there
was still a decrease in the percentage of IL-21–positive cells in the
proliferated cell population. These results now implicate c-Rel in
Th17 and TFH cell-specific IL-21 production. In addition, we
showed that the percentage of CD4+IFN-g2IL-17+ (Th17 cells)
and CD4+CXCR5+ICOS+ (TFH cells) were reduced in rel2/2
mice, suggesting that c-Rel–dependent expression of IL-21 might
be required for the differentiation and amplification of these Th
cell subsets.
Th17 cells are a novel Th subset that mainly expresses IL-17
during inflammatory responses. Th17 differentiation can be induced by IL-6 and TGF-b through the TFs RAR-related orphan
receptor-gt, RORa, STAT3, IFN regulatory factor 4, and Batf (64),
leading to the production of IL-17a, IL-17F, IL-22, and IL-21.
Differentiated Th17 cells are further stabilized and amplified by
the actions of IL-21 and IL-23. Th17 cells have frequently been
implicated in the EAE model of autoimmune disease. Using this
model of autoimmunity, we have shown that IL-17+IFN-g2
Th17 cells development was decreased significantly in rel2/2
mice when compared with wild type. Impaired IFN-g and Th1
responses have previously been implicated in the resistance of
rel2/2 mice to EAE (51). We have also previously identified
a large cohort of chemokines and cytokines that are targets of cRel in T cells and have been implicated in Th1-mediated immune
responses and in various models of autoimmune disease (44, 52).
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FIGURE 8. TFH cell, but not GC B cell, development was extrinsic and
could be rescued by IL-21 administration to rel2/2 mice. A–D, The
CD45.1 congenic C57BL/6 mice (rel+/+) were irradiated, following by
i.v. injection of bone marrow cells from CD45.2 congenic rel+/+, rel2/2,
or mixture of CD45.1 rel+/+ and CD45.2 rel2/2 at a 1:1 ratio. The chimeric
mice were immunized with MOG35–55 on week 4 after the reconstruction
of immune system. The splenocytes were isolated for flow cytometry
analysis of GC B cells (A, B) and TFH cells (C, D) gating from the corresponding donor B220+CD45.2+ and CD4+CD45.2+, respectively, at day 14
after immunization. The experiments were repeated three times. E and F,
CD45.2 rel+/+ (n = 10) or rel2/2 (n = 8) mice were injected i.v. with rIL-21
protein at days 0, 4, 7, 10, and 13 after MOG35–55 immunization. The
splenocytes were isolated and analyzed at day 14 after immunization. E
shows a representative dot plot, and the combined data for GC B cells (F)
and TFH cells (G) are also shown.
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Acknowledgments
We thank Drs. Carola Garcia de Vinuesa and Di Yu for advice, Dr. Harpreet
Vohra for flow cytometry expertise, Dr. Debbie Howard for the irradiation
work, and Dr. David Liñares for advice on EAE.
Disclosures
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
The authors have no financial conflicts of interest.
26.
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Thus, c-Rel plays a role in the differentiation of at least two T cell
subsets, Th1 and Th17, involved in these inflammatory responses.
Our recent data showed that c-Rel might affect the development of
Th17 cells in an indirect manner, via regulating the expression of
other TFs (G. Chen, unpublished data).
TFH cells play a specific and prominent role in the GC and
provide help to GC B cells during Ig affinity maturation in B cell
follicles. These cells have a high expression of CXCR5, ICOS,
programmed cell death (PD)1, and IL-21 and have recently been
shown to require Bcl-6 for their development (61, 65, 66). IL-21
has also been reported to be essential for TFH development and GC
formation (10, 28). In this study, we have shown that both TFH cells
and GC B cells are reduced in rel2/2 mice. The bone marrow
chimera experiments showed that the defect of GC B cells was
intrinsic in rel2/2 mouse but that the TFH defect was extrinsic.
Furthermore, rIL-21 protein administration could rescue the TFH
defect but not GC B cell defect in rel2/2 mouse. Thus, the reduced
level of IL-21, caused by loss of c-Rel, is likely to be responsible
for the deficiency of TFH cells in rel2/2 mice.
We previously reported that c-Rel was an important modulator
of, but was not essential for, the inducible T cell gene expression
program (52). Previous studies have also shown that c-Rel is required for the proliferation and function of mature lymphoid and
myeloid cells (44) but is not essential during the early stages of
hematopoietic cell development. In this study, we have identified
IL-21 as a novel and direct target of c-Rel in activated CD4+
T cells. Combined with our compelling evidence showing the
impairment of Th17, absence of GC B cells, and deficiency of
IL-21–dependent TFH cells in rel2/2 mice, c-Rel, indeed, plays an
important role in the late maturation/differentiation stages of lymphocyte development. The requirement for c-Rel in defining
Th cell subsets is also supported by recent work from our laboratory showing a requirement for c-Rel in Treg cell development in
the thymus (67).
In summary, we have shown that c-Rel regulates IL-21 gene expression and is important for the downstream development of
TFH cells. These novel findings help us to not only clarify the specific
and nonredundant roles of NF-kB family members in lymphocyte
development and T cell-dependent immune responses but also provide important information with regard to the use of IL-21 as a therapeutic target in the treatment of autoimmune disease and cancer.
c-Rel REGULATES IL-21 EXPRESSION
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