of June 13, 2013.
This information is current as
Mice Reveal Distinct Populations of
Cutting Edge: IL-23 Receptor GFP Reporter
K. Kuchroo and Mohamed Oukka
Korn, Caroline Pot, George Galileos, Estelle Bettelli, Vijay
Amit Awasthi, Lorena Riol-Blanco, Anneli Jäger, Thomas
2009; 182:5904-5908; ;
, 4 of which you can access for free at:
cites 10 articles
is online at:
The Journal of Immunology
Information about subscribing to
Submit copyright permission requests at:
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists, Inc. All rights reserved.
Copyright © 2009 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
Cutting Edge: IL-23 Receptor GFP Reporter Mice Reveal
Distinct Populations of IL-17-Producing Cells1
Amit Awasthi,2* Lorena Riol-Blanco,2* Anneli Ja ¨ger,2†Thomas Korn,‡Caroline Pot,†
George Galileos,* Estelle Bettelli,3†Vijay K. Kuchroo,3†and Mohamed Oukka3*
IL-23, an IL-12 family member, has been implicated in
the development of Th17 cells and the progression of
autoimmune diseases. However, due to the lack of avail-
ability of sensitive Ab reagents specific for the IL-23 re-
ceptor (IL-23R), it has been difficult to characterize the
cell types that express the IL-23R and are responsive to
IL-23 in vivo. To address the role of IL-23 in vivo, we
have generated a novel “knock-in” mouse in which we
have replaced the intracellular domain of the IL-23R
with the GFP. We show that in addition to Th17 cells,
a subset of myeloid cells express IL-23R and respond
to IL-23 by producing IL-17 and IL-22. Our studies
further demonstrate that IL-23R expression is crucial
for generation of encephalitogenic Th17 cells, but its
expression on the innate immune system is dispen-
sible in the development of experimental autoim-
mune encephalomyelitis. The Journal of Immunol-
ogy, 2009, 182: 5904–5908.
p19 or the p40 chain makes mice resistant to a number of au-
toimmune diseases (1). Similarly, polymorphisms in IL-23R
autoimmune diseases including psoriasis and Crohn’s disease
(2, 3). These data suggest an important role of the IL-23:IL-
23R pathway in the development of many autoimmune dis-
eases, but the mechanism by which IL-23/IL-23R pathway op-
erates in vivo is not clear. Progress in this area is further
Ab reagents to identify and track IL-23R-bearing cells in vivo.
nterleukin-23, a heterodimeric cytokine composed of p19
and p40 chains, plays a critical role in the development of
autoimmune diseases in that the genetic loss of either the
Recent studies have suggested that besides its role as a
growth/stabilization factor for Th17 cells, IL-23 might have
an important role in innate immunity; however, innate im-
mune cells that are responsive to IL-23 have not been char-
acterized (4). To identify the cell types (innate or adaptive)
responding to IL-23 and their effector functions under var-
ious inflammatory conditions (infection or autoimmunity),
we generated an IL-23R reporter mouse. In this mouse, IL-
23R-expressing cells can be followed by their expression of
GFP, and their responsiveness to IL-23 can be eliminated
when this mouse is bred as homozygote.
Materials and Methods
C57BL/6 wild-type (WT)4mice were from The Jackson Laboratory. WT and
heterozygous IL-23R-KI mice were housed in a conventional pathogen-free fa-
of Medicine in Boston, MA. All experiments were performed in accordance
with guidelines prescribed by the Harvard Medical Area Standing Committee
on Animals at Harvard Medical School (Boston, MA).
Generation of the IL-23R-KI mice
A BAC clone (RP23-204M15) containing the IL-23R gene was used as a tem-
plate for PCR amplification to generate 5.1- and 1.8-kb arms that were sub-
cloned into enhanced GFP containing a TKPbs-LoxP-Neo cassette. The tar-
geting construct was electroporated into Bruce4 embryonic stem (ES) cells.
bred with female C57BL/6 mice.
Preparation of CNS mononuclear cells
CNS tissue was cut into pieces and digested with collagenase D (2.5 mg/ml;
Mononuclear cells were isolated by Percoll gradient (70/37%) centrifugation.
in culture medium for further analysis.
*Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical
School, Cambridge, MA 02139;†Center for Neurologic Diseases, Brigham and Women’s
Hospital, Harvard Medical School, Boston, MA 02115; and‡Technische Universita ¨t
Mu ¨nchen, Department of Neurology, Munich, Germany
Received for publication March 6, 2009. Accepted for publication March 24, 2009.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
V.K.K.) and RG-3882-A-1 (to M.O. and A.A.) and NMSS Transition Award TA
3014A1/1 (to E.B.). L.R.B. is supported by a postdoctoral fellowship from the European
Molecular Biology Organization. A.J. is the recipient of a Ph.D. scholarship by the Boehr-
inger Ingelheim Fonds. T.K. is supported by the Deutsche Forschungsgemeinschaft. C.P.
is supported by the Swiss National Science Foundation.
2A.A., L.R.-B., and A.J. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Mohamed Oukka, Center for Neu-
rologic Diseases, Brigham and Women’s Hospital, Harvard Medical School 65 Lands-
downe Street, Cambridge, MA 02139. E-mail address: email@example.com or
Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115. E-
mail addresses: firstname.lastname@example.org and email@example.com
4Abbreviations used in this paper: WT, wild type; DC, dendritic cell; EAE, experimental
autoimmune encephalomyelitis; ES, embryonic stem; LN, lymph node; LP, lamina pro-
pria; MOG, myelin oligodendrocyte glycoprotein.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
by guest on June 13, 2013
Recall response with myelin oligodendrocyte glycoprotein (MOG)35–55
WT and IL-23R-KI mice were immunized with 100 ?g MOG35–55peptide
cells were prepared and cultured with 20 ?g/ml MOG35–55and 25 ng/ml rIL-
23. After 4 days of in vitro stimulation, cells were analyzed for IL-23R(GFP)
Intracellular cytokine staining
Cells were re-stimulated with PMA (50 ng/ml; Sigma-Aldrich), ionomycin (1
?g/ml; Sigma-Aldrich), and GolgiStop (1 ?l/ml, BD Biosciences) at 37°C in
manufacturer’s instructions (BD Bioscience). Cells were analyzed by FACS
Cytokine analysis and real time PCR analysis
Cytokines from culture supernatants were determined by either ELISA or cy-
tometric bead array (BD Bioscience). RNA was extracted after 48 h of in vitro
stimulation using RNAeasy columns (Qiagen) and subjected to quantitative
RT-PCR according to the manufacturer’s instructions (Applied Biosystems).
Primer/probe mixtures of mouse IL-17A, IL-23R, IFN-?, T-bet, and ROR-?t
were obtained from Applied Biosystems.
Results and Discussion
Generation of IL-23R-GFP.KI reporter mice
To identify the cell types responding to IL-23 in vivo, we
generated a knock-in “reporter” mouse in which an IRES-
GFP cassette (where IRES is internal ribosome entry site)
was introduced after exon 8 in the endogenous IL-23R gene
(Fig. 1A). We obtained three independent ES cell clones that
were appropriately targeted as shown by Southern blotting,
and germline transmission was achieved with two of the tar-
geted clones (Fig. 1B).
by their expression of GFP, and when bred as homozygotes the
deletion of the IL-23R abrogates their responsiveness to IL-23.
IL-23R-GFP.KI and IL-23R?/?mice were healthy and fertile
and had similar numbers of CD4 cells, CD8 cells, B cells, ?? T
cells, CD11c?cells, and CD11b?cells as WT control mice
(data not shown).
IL-23R-expressing cells are present in the LN and are enriched in the
lamina propria (LP)
To determine the expression of IL-23R at the single cell level,
we examined the expression of GFP in the lymphoid tissues of
naive IL-23R-KI and WT mice. We did not find any IL-
23R(GFP) expression on CD8?T cells, B cells, or NK cells
(data not shown). However, a small percentage of IL-23R-ex-
pressing cells (?1.4%) were CD3?T cells and were detected
mainly in the LNs but not in the spleen (Fig. 2A). Only a mi-
nority of the GFP-expressing T cells were CD4?T cells
(?0.2%; data not shown), whereas the majority of them were
?? T cells. In fact, ?40% of the ?? T cells present in the LN
and targeting strategy. An IRES-EGFP cassette (where IRES is internal ribo-
kinase. B, Southern blotting was used to identify the properly targeted ES cells.
DNA purified from ES cells was digested by NheI and probed with a 1.1-kb
specific probe generated by PCR. The hybridized 15.7-kb fragment corre-
targeted allele. KO, Knockout.
Generation of IL-23R reporter mice. A, IL-23 genomic locus
Cells from LNs, spleen, and LP were prepared from naive WT and IL-23R-
GFP.KI mice and stained and analyzed by flow cytometry. A, LN cells were
stained for CD3, TCR??, CD11c, and CD11b. B, IL-23R(GFP) expression
was also analyzed in LP cells. LP cells were stained for CD45 and TCR?? (left
panel). The expressions of CD3, CD4, and TCR?? were analyzed in the
CD45?IL-23R(GFP)?compartment of LP cells (right panel).
IL-23R-expressing cells are enriched in the LP of naive mice.
5905The Journal of Immunology
by guest on June 13, 2013
were positive for IL-23R(GFP) expression and thus constitute
the major IL-23R-expressing T cell subset in the lymphoid or-
gans of naive animals. The other subsets of cells expressing IL-
23R were CD11b?macrophages (?6%) and CD11c?DCs
(?4%) (Fig. 2A). Notably, the IL-23R-positive subsets are
mainly located in the LN but not in the spleen of naive animals
In contrast to the small frequency of IL-23R(GFP)-express-
ing cells in secondary lymphoid organs, IL-23R(GFP)-positive
cells were prominently found in the LP. There, ?3% of all
CD45?cells expressed IL-23R(GFP) while the majority of ??
T cells (?65%) expressed IL-23R(GFP) (Fig. 2B). Interest-
ingly, LP DCs did not show any expression of IL-23R(GFP).
Similarly as in the LN, only a small fraction of the macrophage
subsets expressed IL-23R(GFP) (data not shown). When fur-
ther defining the IL-23R(GFP)-positive cells in the intestinal
LP, we identified a fraction of ?13% CD4?IL-23R(GFP)-
expressing cells that did not express CD3 (Fig. 2B, right panel).
they expressed CD45 and were reminiscent of previously de-
scribed lymphoid tissue inducer cells, which also express the
transcription factor ROR-?t and produce IL-17 (5, 6).
IL-23R?/?mice have a defective Th17 response and are resistant to
experimental autoimmune encephalomyelitis (EAE)
Because IL-23 plays an important role in EAE, we first assessed
tant to EAE by immunizing WT and IL-23R?/?mice with
MOG35–55emulsified in CFA. Whereas WT mice developed
signs of EAE on day 14 and reached the peak of the disease on
day 20 after immunization, IL-23R?/?mice were completely
GFP.KI mice were as susceptible to EAE as WT mice (data not
shown), we took advantage of the GFP reporter to track IL-
23R-bearing cells during active EAE. Approximately 25% of
the CD4?T cells infiltrating the inflamed CNS were positive
for IL-23R(GFP) expression, and the vast majority (68%) of
cells predominantly produced IFN-? but not IL-17 (Fig. 3B).
These data establish that IL-23R is expressed on T cells at the
site of inflammation and that the expression correlates with
IL-17 production, suggesting that IL-23R defines a population
of infiltrating proinflammatory Th17 cells in the target tissue.
The dynamics of IL-23R expression and its in vivo functions in
activated/memory T cells are not clear. To understand its func-
tional consequences, WT and IL-23R?/?mice were immu-
nized with MOG35–55and CFA LN cells and splenocytes were
restimulated in vitro with MOG35–55peptide alone or in the
presence of IL-23. The relative lack of Ag-specific Th17 cells in
the spleen but not in the draining LN of IL-23R?/?animals
naling to stably develop into Th17 cells or live long enough to
leave the LN to populate the spleen and infiltrate tissues. Alter-
natively, IL-23 might also endow developing Th17 cells with
trafficking properties essential for their egress from LN, migra-
tion into the spleen, and infiltration into the target tissues. IL-
17-producing CD4?T cells were markedly decreased in the
spleens of IL-23R?/?mice as compared with MOG recall cul-
tant to EAE. A, EAE was induced in WT and IL-23R?/?mice by immuniza-
tion with MOG35–55emulsified in CFA. The course of EAE in these mice was
monitored and is shown as a mean clinical score. B, IL-23R-GFP.KI mice were
immunized with MOG35–55/CFA. On day 16, at the peak of disease, mono-
Intracellular cytokine staining for IFN-? and IL-17 was performed. The histo-
grams represent the percentages of cytokine-positive cells in the CD4?IL-
23R(GFP)?and CD4?IL-23R(GFP)?gates, respectively. C, WT and IL-
23R?/?mice were immunized with MOG35–55/CFA. On day 8, spleens (right
in the presence of rIL-23 for 6–8 days. Cells were re-stimulated with PMA/
ionomycin before intracellular cytokine staining for IL-17 and IFN-? was per-
gate. D, CD4?CD62L?memory T cells from WT or IL-23R?/?mice were
activated with anti-CD3 in the presence rIL-23 (30 ng/ml) with irradiated syn-
genic APCs. Four days later, cells were restimulated with PMA/ionomycin and
intracellular cytokine staining was performed for IL-17 and IFN-?. E, For pas-
sive transfer of EAE, MOG35–55-specific T cells from immunized WT or IL-
23R?/?mice were cultured in the presence of MOG35–55with rIL-23 (30 ng/
ml). On day 8, CD4?T cells were reactivated with plate-bound anti-CD3 and
anti-CD28 for 48 h. Equal numbers of MOG35–55-specific CD4?T cells from
either WT or IL-23?/?mice were transferred into a WT recipient. The course
of the EAE was monitored and is shown as the mean clinical score.
IL-23R?/?mice have a defective Th17 response and are resis-
5906CUTTING EDGE: EAE, Th17 CELLS, IL-23R MICE
by guest on June 13, 2013
IL-23 further up-regulated the frequency of Th17 cells in both
LN (?58%) and spleen (?48%) in WT but not in the IL-
23R?/?CD4?T cells from LN and spleen (Fig. 3C). Thus,
IL-23R expression on recently activated T cells is essential for
expanding/maintaining the Th17 population. If IL-23 were es-
sential for the stable development of long-lasting Th17 cells,
one would expect that IL-23R?/?mice would have a defect in
WT and IL-23R?/?mice were sorted based on their
CD4?CD62L?profile and were activated in the presence of
IL-23. The percentage of memory Th17 cells in WT mice was
close to 4%, whereas IL-23R?/?mice had about half that frac-
tion (Fig. 3D). Memory Th17 cells from WT mice responded
(from 4 to 18%) whereas IL-23?/?mice did not expand Th17
is absolutely required for the expansion/maintenance of Th17
effector T cells and innate immune cells, resistance to EAE in
IL-23R?/?mice could be due to a defect in IL-17 production
WT and IL-23R?/?mice with MOG35–55/CFA, and 7 days
after immunization we harvested draining LN cells that were
stimulated in vitro with MOG35–55in the presence of IL-23.
Equal numbers of activated T cell blasts were adoptively trans-
ferred into IL-23R-competent naive syngenic WT hosts.
Whereas recipients of WT T cells developed EAE, MOG35–55-
specific LN cells lacking IL-23R failed to transfer the disease
(Fig. 3E). We conclude that MOG-specific CD4?, TCR
?/??, and IL-23R?cells are crucial for inducing EAE because
the lack of IL-23R on MOG-specific T cells abrogates their
Our results are similar to the data recently published by Cua
and colleagues, who have also generated an IL-23R-deficient
cial for their effector function (8). However, in this study the
role of IL-23R on innate cells was not addressed.
IL-23R expression on an innate immune system is dispensable for the
development of EAE
To address which subsets of innate cells express IL-23R(GFP),
we analyzed IL-23R-GFP.KI mice crossed onto a RAG2-defi-
cient background (Fig. 4A). In the LNs of these mice we could
detect a subset of IL-23R(GFP)-expressing cells that also ex-
pressed the myeloid markers CD11b and CD11c (data not
shown). We also immunized IL-23R-GFP.KI mice with
MOG35–55/CFA and found that in the draining LNs a similar
subset of CD11b?CD11c?DCs that expressed MHC class II
(Fig. 4B). This subset of IL-23R(GFP)?myeloid cells, in con-
trast to its IL-23R(GFP)?counterparts, responded to IL-23 by
thermore, in response to IL-23, IL-23R(GFP)?CD11c?LN
cells also produced large amounts of IL-6 (Fig. 4C). Because
set of LN-derived myeloid DCs that express IL-23R might be
particularly well equipped to induce a Th17 response. Hence,
in addition to T cells, macrophages/monocytes also express IL-
producing IL-17 but may also be involved in de novo differen-
tiation of Th17 cells via their production of IL-6 and TGF-?
(9, 10). To assess whether innate cells that express IL-23R also
contribute to the development of EAE, we first transferred
MOG-specific Th17 cells into RAG2?/?or IL23R?/?
RAG2?/?recipients. Both genotypes developed EAE with a
similar clinical course (Fig. 4D), indicating that Th17 cells do
be involved in inducing pathogenic T cells. To address this is-
sue, we transferred naive T cells derived from MOG-specific,
TCR-transgenic mice into RAG or RAG IL-23R?/?recipients
of EAE. A, Single cell suspensions were prepared from LNs from either naive
RAG2?/?or IL-23R-GFP.KI RAG2?/?mice and IL-23R(GFP) expression
was analyzed on total LN cells. SSC-A, Side scatter area. B, IL-23R-GFP.KI
sion was analyzed on myeloid cell populations as indicated in the histogram. C,
LN CD11c?cells were FACS sorted based on the expression of IL-23R(GFP).
After ex vivo stimulation with LPS for 12 h in the presence of IL-23, quantita-
tive real-time PCR was performed for IL-6. The graph represents one of two
experiments. D, MOG-specific Th17 cells were i.v transferred into RAG2?/?
and RAG2?/?IL-23R?/?mice. The course of EAE in these mice was moni-
tored and shown as a mean clinical score. E, Naive T cells derived from MOG-
IL-23R expression on APCs is dispensable for the development
5907The Journal of Immunology
by guest on June 13, 2013
and immunized these mice with MOG/CFA for the develop- Download full-text
ilar kinetics and disease severity (Fig. 4E), suggesting that the
major effect of IL-23 on EAE is to expand and stabilize proin-
flammatory Th17 cells, whereas its effect on innate cells is dis-
pensible for the development of EAE. This raises the issue of
One can speculate that responsiveness to IL-23 in innate im-
make the target tissues responsive to tissue inflammation. Fur-
thermore, IL-23R expression may define a subset of APCs that
are particularly equipped to mediate epitope spreading by in-
ducing pathogenic Th responses within the target tissue that
thus further propagate autoimmunity and tissue inflammation
in the CNS. Therefore, it will be interesting to study the role of
IL-23R in the model of proteolipid protein-induced EAE in
which epitope spreading is crucial for the relapsing-remitting
course of the disease.
In summary, our studies demonstrate in vivo that IL-23R is
not only expressed on T cells but is expressed on various innate
immune cells. The innate immune cells respond to IL-23 by
producing IL-17 and IL-22; however, their IL-23R is dispens-
able for the induction of acute EAE, whereas the loss of IL-23R
is absolutely required for the myelin-specific T cells to induce
EAE. This is surprising, because recent studies suggest that
emphasize, however, that the loss of tissue inflammation and
resistance to autoimmunity observed in the IL-23R mice may
not just be due to loss of IL-17 but also to the loss of other
cytokines like IL-22 produced by Th17 upon activation with
We thank D. Kozoriz for cell sorting and technical assistance.
The authors have no financial conflict of interest.
1. Kastelein, R. A., C. A. Hunter, and D. J. Cua. 2007. Discovery and biology of IL-23
and IL-27: related but functionally distinct regulators of inflammation. Annu. Rev.
Immunol. 25: 221–224.
2. Duerr, R. H., K. D. Taylor, S. R. Brant, J. D. Rioux, M. S. Silverberg, M. J. Daly,
A. H. Steinhart, C. Abraham, M. Regueiro, A. Griffiths, et al. 2006. A genome-wide
3. Cargill, M., S. J. Schrodi, M. Chang, V. E. Garcia, R. Brandon, K. P. Callis,
N. Matsunami, K. G. Ardlie, D. Civello, J. J. Catanese, et al. 2007. A large-scale ge-
netic association study confirms IL12B and leads to the identification of IL23R as
psoriasis-risk genes. Am. J. Hum. Genet. 80: 273–290.
4. Hue, S., P. Ahern, S. Buonocore, M. C. Kullberg, D. J. Cua, B. S. McKenzie,
F. Powrie, and K. J. Maloy. 2006. Interleukin-23 drives innate and T cell-mediated
intestinal inflammation. J. Exp. Med. 203: 2473–2483.
fate mapping of ROR?t?cells. Science 305: 248–251.
6. Takatori, H., Y. Kanno, W. T. Watford, C. M. Tato, G. Weiss, I. I. Ivanov,
source of IL-17 and IL-22. J. Exp. Med. 206: 35–43.
7. Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian,
is the critical cytokine for autoimmune inflammation of the brain. Nature 421:
8. McGeachy, M. J., Y. Chen, C. M. Tato, A. Laurence, B. Joyce-Shaikh,
W. M. Blumenschein, T. K. McClanahan, J. J. O’Shea, and D. J. Cua. 2009. The
interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-
producing effector T helper cells in vivo. Nat. Immunol. 10: 314–324.
9. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, and
V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of
pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238.
10. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. 2006.
TGF? in the context of an inflammatory cytokine milieu supports de novo differen-
tiation of IL-17-producing T cells. Immunity 24: 179–189.
5908 CUTTING EDGE: EAE, Th17 CELLS, IL-23R MICE
by guest on June 13, 2013