The Journal of Experimental Medicine
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J. Exp. Med. Vol. 206 No. 1 35-41
BRIEF DEFINITIVE REPORT
Naive T cells undergo diff erentiation on signals
received from the TCR and cytokine receptors
and diff erentiate to specialized subsets character-
ized by their production of signature cytokines.
Th17 cells produce IL-17 or IL-17A and IL-17F,
which are major mediators of infl ammation and
are critical for host defense against extracellular
bacteria and fungi ( 1, 2 ). IL-6, IL-21, and TGF-
? 1 are critical factors that promote Th17 cell dif-
ferentiation ( 1, 3 ). Although IL-23 was originally
thought to be important for inducing naive
CD4 + T cells to become Th17 cells, naive CD4 +
T cells do not express IL-23Rs ( 4 ). Rather, IL-
23 is now thought to aff ect the expansion, main-
tenance, and pathogenicity of Th17 cells ( 5 ).
IL-23 also induces IL-17 production from ? ?
T cells and invariant NKT (iNKT) cells ( 6, 7 ).
Regardless of exactly how IL-23 works, current
evidence clearly argues that IL-23 – mediated
IL-17 production is crucial in host defense and in
the pathogenesis of autoimmune diseases ( 8 – 10 ).
IL-6, IL-21, and IL-23 share the ability to
activate Stat3, which was shown to be critical
for Th17 cell diff erentiation in mouse and man
( 3, 11, 12 ). Stat3 directly regulates the Il17 and
Il21 genes but also regulates IL-23R expression
( 3, 12 ). Furthermore, the aforementioned cyto-
kines acting via Stat3 induce the retinoic acid –
related orphan receptor ? t (ROR ? t), the master
regulator of Th17 cell diff erentiation ( 13 ).
In contrast to T cells, much less is known
about the ability of innate cell subpopulations
to produce IL-17. We report that splenic lym-
phoid tissue inducer – like cells (LTi-like cells)
constitutively express ROR ? t, IL-23R, CCR6,
and aryl hydrocarbon receptor (AHR) ( 14 ),
and produce IL-17 and IL-22. Interestingly,
C.M. Tato ’ s present address is Dept. of Discovery Research,
DNAX Research Inc., Palo Alto, CA 94304.
G. Weiss ’ s present address is National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Rockville,
Lymphoid tissue inducer – like cells are
an innate source of IL-17 and IL-22
Hiroaki Takatori , 1 Yuka Kanno , 1 Wendy T. Watford , 1 Cristina M. Tato , 1
Greta Weiss , 2 Ivaylo I. Ivanov , 3 Dan R. Littman , 3 and John J. O ’ Shea 1
1 Lymphocyte Cell Biology Section, Molecular Immunology and Infl ammation Branch, National Institute of Arthritis
and Musculoskeletal and Skin Diseases, Bethesda, MD 20892
2 University of Pennsylvania – National Institutes of Health Graduate Program, Bethesda, MD 20892
3 Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University School of Medicine,
New York, NY 10016
The interleukin (IL) 17 family of cytokines has emerged to be critical for host defense as
well as the pathogenesis of autoimmune and autoinfl ammatory disorders, and serves to
link adaptive and innate responses. Recent studies have identifi ed a new subset of T cells
that selectively produce IL-17 (Th17 cells; Bettelli, E., T. Korn, and V.K. Kuchroo. 2007.
Curr. Opin. Immunol. 19:652 – 657; Kolls, J.K., and A. Linden. 2004. Immunity. 21:467 –
476), but the regulation of IL-17 production by innate immune cells is less well under-
stood. We report that in vitro stimulation with IL-23 induced IL-17 production by
recombination activating gene (Rag) 2 ? / ? splenocytes but not Rag2 ? / ? common ? chain ? / ?
splenocytes. We found that a major source of IL-17 was CD4 + CD3 ? NK1.1 ? CD11b ? Gr1 ? CD
11c ? B220 ? cells, a phenotype that corresponds to lymphoid tissue inducer – like cells (LTi-
like cells), which constitutively expressed the IL-23 receptor, aryl hydrocarbon receptor,
and CCR6. In vivo challenge with the yeast cell wall product zymosan rapidly induced IL-
17 production in these cells. Genetic deletion of signal transducer and activator of tran-
scription 3 reduced but did not abrogate IL-17 production in LTi-like cells. Thus, it appears
that splenic LTi-like cells are a rapid source of IL-17 and IL-22, which might contribute to
dynamic organization of secondary lymphoid organ structure or host defense.
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IL-17 AND IL-22 PRODUCTION BY SPLENIC LT I -LIKE CELLS | Takatori et al.
with IL-23 had no eff ect on IL-17A production; however,
IL-4 suppressed IL-23 – induced IL-17A production by WT
and Rag2 ? / ? splenocytes (Fig. S1 A, available at http://www
.jem.org/cgi/content/full/jem.20072713/DC1). In addition
to memory CD4 + T cells, iNKT cells are another important
source of IL-17 ( 7 ), but such cells are lacking in Rag2 ? / ?
mice. Accordingly, anti-CD3/28 or ? -galactosylceramide
( ? -GalCer) stimulation induced IL-17A production by WT
splenocytes but not Rag2 ? / ? splenocytes (Fig. S1, B and C).
Collectively, these data suggest that although T and NKT
cells are both major producers of IL-17, populations of non – T,
non – B cells can also produce IL-17.
To identify which population of cells might be responsible
for IL-23 – dependent IL-17 production by Rag2 ? / ? spleno-
cytes, we examined another genetic model, namely Rag2 ? / ?
? c ? / ? mice. In addition to lacking T and B cells, Rag2 ? / ? ? c ? / ?
mice also lack NK cells. We therefore cultured WT, Rag2 ? / ? ,
or Rag2 ? / ? ? c ? / ? splenocytes in the presence of IL-23. Con-
sistent with Fig. 1 A , IL-17A production was present in WT
splenocytes and reduced but not absent in Rag2 ? / ? splenocytes
( Fig. 1 B ). In contrast, IL-17A production by Rag2 ? / ? ? c ? / ?
splenocytes was essentially abrogated ( Fig. 1 B ). These results
indicate that ? c-dependent, non – T, non – B cell populations in
the spleen can produce IL-17 in vitro.
IL-23 has been shown to be induced by certain microbial
products ( 15 ). Rag2 ? / ? mice were therefore injected with
zymosan to induce IL-23 production by DCs, which resulted
in rapid IL-17A appearance in sera and spleens ( Fig. 1 C ).
Thus, these data indicate that innate immune cells can gener-
ate IL-17 in response to the fungal product in vivo.
IL-17 production by CD4 + CD3 ? CD11c ? B220 ? LTi-like cells
To delineate the population of IL-17 – producing cells, we
next used intracellular cytokine staining. We used markers
for a variety of innate immune cells expected to be present
in Rag2 ? / ? spleens but failed to detect IL-23 – mediated
IL-17A production in granulocytes, macrophages, or DCs
(Fig. S2, available at http://www.jem.org/cgi/content/full/jem
.20072713/DC1). A prominent ? c-dependent lineage that
rapidly produces cytokines upon stimulation is the NK cell.
Based on the response to IL-23 in Rag2 ? / ? splenocytes and
loss in Rag2 ? / ? ? c ? / ? splenocytes, we thought that NK cells
could be IL-17 producers. However, contrary to our expec-
tations, the majority of IL-17A – producing CD3 ? cells did
not express NK1.1 ( Fig. 2 A ). Thus far, our results have indi-
cated that neither T, NKT, NK cells (all ? c dependent)
nor ? c-independent myeloid cells were a major source of
IL-17 production in Rag2 ? / ? spleens. We did observe that in
Rag2 ? / ? splenocytes stimulated with IL-23 ( Fig. 2 B , left), a
considerable proportion of the IL-17A – producing non – T
cells expressed CD4 ( Fig. 2 B , right).
CD4 + CD3 ? cells in the Rag2 ? / ? spleen comprise three
populations, including CD11C low B220 + plasmacytoid DCs,
CD11C + B220 ? conventional DCs, and CD4 + CD3 ? CD11c ?
B220 ? cells (Fig. S3, available at http://www.jem.org/cgi/
content/full/jem.20072713/DC1). As indicated in Fig. S2,
the yeast wall product zymosan elicited IL-17 production by
LTi-like cells in vivo. Whether the production of IL-17 and
IL-22 infl uences the architecture of secondary lymphoid or-
gans (SLOs) and contributes to host defense will be important
issues to examine in the future.
RESULTS AND DISCUSSION
IL-23 induces IL-17 production by a population
of common ? chain ( ? c) – dependent non – T, non – B cells
To determine if there are considerable proportions of innate
immune cells that produce IL-17, we examined Rag2 ? / ?
mice that have few T and B cells. We fi rst assessed whether
IL-17 production occurred in Rag2 ? / ? splenocytes after
stimulation with various cytokines. As shown in Fig. 1 A , IL-
23 alone induced IL-17A production by WT splenocytes,
presumably indicative of an eff ect on memory T cells. Con-
sistent with the idea that memory T cells are lacking in
Rag2 ? / ? mice, the eff ect of IL-23 was reduced in Rag2 ? / ?
splenocytes ( Fig. 1 A ). However, Rag2 ? / ? splenocytes still
produced about one third as much IL-17A as WT splenocytes
( Fig. 1 A ). Other cytokines used separately or in combination
Figure 1. IL-23 and zymosan induce IL-17 production in a popu-
lation of non – B, non – T cells. (A) WT or Rag2 ? / ? splenocytes were cul-
tured in the presence of the indicated cytokines for 48 h, and IL-17A in
culture supernatants was measured by ELISA. The data are means ± SD
from duplicate cultures and are representative of four independent ex-
periments ( n = 8). **, P < 0.01. (B) WT, Rag2 ? / ? , or Rag2 ? / ? ? c ? / ? spleno-
cytes were cultured in the presence of IL-23 alone, and IL-17A in culture
supernatants was measured by ELISA. The data are means ± SD from
duplicate cultures and are representative of three independent experi-
ments ( n = 6). *, P < 0.05; **, P < 0.01. (C) Sera and splenocytes were har-
vested from Rag2 ? / ? mice after injection of 5 mg zymosan or PBS
(Control). (left) IL-17A in sera was measured by ELISA. (right) The relative
expression levels (/18S) of IL-17A mRNA were analyzed by quantitative
PCR (q-PCR) and are depicted as fold induction relative to cells 1 h after
treatment with zymosan. The data are means ± SD of results from two
mice per group per time point and are representative of two independent
experiments. ND, not detected.
JEM VOL. 206, January 19, 2009
BRIEF DEFINITIVE REPORT
to maintain cells for the initial 48 h, followed by IL-23 for an-
other 6 h. As shown in Fig. S7 A, IL-17A and IL-17F mRNA
expression was potently induced by IL-23 in the presence of
IL-7. These results suggest that LTi-like cells quickly enhance
the production of IL-17 in response to IL-23, and this cyto-
kine does not simply serve to keep these cells alive.
It is well known that Stat3 is activated by IL-23 and other
inducers of Th17 cell diff erentiation, and the absence of Stat3
abrogates IL-17 production by T cells ( 12 ). We therefore as-
sessed the proportion of IL-17A – producing LTi-like cells in
Stat3-defi cient mice. Not surprisingly, we found that IL-23 –
mediated IL-17A production was decreased in Stat3-defi cient
LTi-like cells of Stat3 fl /fl ; MMTV-Cre or Stat3 fl /fl ; CD4-Cre
splenocytes compared with those of Stat3 fl /fl splenocytes, al-
though IL-17A production was not completely abrogated
( Fig. 3 B and not depicted). Because Cre-mediated deletion
of STAT3 is > 90% in both systems (unpublished data), there
seems to be both STAT3-dependent and -independent path-
ways for the production of IL-17 in LTi-like cells. We also
observed that IL-23 up-regulated mRNA expression of Stat3
and Stat4 but not GATA3 in LTi-like cells (Fig. S7 B). These
results highlight the notion that the IL-17 production by
LTi-like cells might be dependent on these factors.
LTi-like cells constitutively express IL-23R, AHR, and CCR6
The complete receptor for IL-23 comprises the IL-12R ? 1
associated with the ligand-specifi c subunit IL-23R, and both
subunits are required for the action of this cytokine ( 4 ). As
memory CD4 + T cells respond to IL-23 because of constitu-
tive expression of its receptors ( 4 ), we evaluated mRNA
expression of IL-23R and IL-12R ? 1 in LTi-like cells.
Consistent with their rapid responsiveness to IL-23, LTi-like
cells constitutively expressed IL-23R mRNA at levels sig-
nifi cantly greater than T cells ( Fig. 3 C ). LTi-like cells also
expressed slightly higher levels of IL-12R ? 1 mRNA com-
pared with those of memory CD4 + T cells ( Fig. 3 C ). In
contrast, expression levels of IL-12R ? 2 mRNA in LTi-like
cells were low compared with those in memory CD4 + T
cells ( Fig. 3 C ). Also consistent with previous studies ( 16, 18 ),
we noted higher expression levels of ROR ? t mRNA in
LTi-like cells compared with T cells ( Fig. 3 C ). In contrast,
expression of IL-6R ? and IL-17RA mRNA in LTi-like
cells was much lower compared with that in T and B cells
(unpublished data). Recently, the AHR has been reported to
be expressed in Th17 cells ( 14 ). Interestingly, LTi-like cells
also expressed AHR mRNA at levels equivalent to those in
Th17 cells polarized for 72 h ( Fig. 3 C ). Furthermore, IL-23
up-regulated mRNA expression levels of IL-23R, ROR ? t,
and AHR (Fig. S7 C).
Consistent with previous studies ( 16, 18 ), splenic LTi-like
cells were found to be CD30L + OX40L + ? c + IL-7R ? + Thy1.2 high
CD44 high CD62L low (Fig. S6). LTi-like cells are also known
to express the chemokine receptors CXCR5 + and CCR7 +
( 16, 18 ), and we again confi rmed expression of these in the
splenic LTi-like cells (Fig. S6). Additionally, it has recently
been shown that CCR6 identifi es a population of human
we did not detect IL-23 – mediated IL-17A production by
CD11C + DC populations. The latter, CD4 + CD3 ? CD11c ?
B220 ? cells, have been shown to be present in SLOs of fetal
and adult mice and are termed LTi ’ s and LTi-like cells, re-
spectively ( 16 ). Importantly, LTi-like cells are greatly reduced
in ? c ? / ? mice ( 17 ).
We next determined whether isolated CD4 + CD3 ?
CD11c ? B220 ? LTi-like subsets produced IL-17 ( Fig. 3 A ,
top). In fact, as shown in Fig. 3 A (bottom), we observed IL-17A
production by isolated LTi-like cells in response to IL-23.
Moreover, directly isolated LTi-like cells produced more
IL-17A in the presence of PMA/ionomycin (Iono), which
was dramatically enhanced by IL-23 ( Fig. 3 A , bottom). In
contrast, LTi-negative subsets produced minimal IL-17A
(Fig. S4, available at http://www.jem.org/cgi/content/full/
jem.20072713/DC1). Consistent with Fig. S2, isolated CD11c +
cells did not produce IL-17A with IL-23 stimulation (unpub-
lished data). Another source of LTi-like cells in an adult
mouse is the intestinal lamina propria ( 16 ). Interestingly,
LTi-like cells in the lamina propria from WT mice produced
IL-17A after stimulation with PMA/Iono (Fig. S5). These
results suggest that the ability to produce IL-17 is not unique
to the splenic population.
One possible explanation for our observations is that IL-23
promoted in vitro survival of LTi-like cells rather than en-
hancing IL-17 production by itself. LTi-like cells have been
shown to express IL-7Rs (Fig. S6, available at http://www
.jem.org/cgi/content/full/jem.20072713/DC1) ( 16 ). There-
fore, we modifi ed the cell-culture conditions by adding IL-7
Figure 2. CD4 + CD3 ? lineage ? cells produce IL-17. Rag2 ? / ? spleno-
cytes were cultured in the absence (Control) or presence of IL-23 for 24 h.
The proportion of IL-17A – producing CD3 ? cells was evaluated by intra-
cellular staining. (A) NK1.1 + versus IL-17A + cells gated on CD3 ? cells are
shown. (B) IL-17A + versus CD3 + cells (nongated; left) and IL-17A + versus
CD4 + cells gated on CD3 ? cells (right) are shown. Data are representative
of three independent experiments.
IL-17 AND IL-22 PRODUCTION BY SPLENIC LT I -LIKE CELLS | Takatori et al.
tion of IL-17A – producing LTi-like cells was determined by
FACS. As shown in Fig. 4 (top) the proportion of IL-17 –
producing LTi-like cells was signifi cantly greater in zymo-
san-treated mice compared with control mice. To further
establish the ability of LTi-like cells to produce IL-17 in
vivo, we treated Rag2 ? / ? mice with BFA i.v. ( 21 ) and chal-
lenged them with zymosan i.p. for 6 h. We observed few IL-
17A – producing LTi-like cells in control mice, whereas
zymosan-treated mice had a signifi cant increase in IL-17A –
producing cells ( Fig. 4 , bottom). These results establish that
LTi-like cells can rapidly produce IL-17 in vivo when chal-
lenged with the product of fungal pathogens.
LTi-like cells also produce IL-22
It has been argued that Th17 cells also preferentially produce
IL-22 ( 22 ). Interestingly, we found that treatment with zymo-
san induced not only IL-17A but also IL-22 in the sera and
spleens of Rag2 ? / ? mice ( Fig. 5 A ). In vitro stimulation with
memory CD4 + T cells that selectively produce IL-17 ( 19 ), we
found that most of the splenic LTi-like cells express CCR6
( Fig. 3 D ). Approximately half of the CCR6 + LTi-like cells
coexpressed CXCR5 and CCR7 (unpublished data), suggest-
ing that the expression of those chemokine receptors on LTi-
like cells is heterogeneous, as previously reported ( 20 ). On the
other hand, the expression of CXCR3, CCR5, or CCR4,
which are preferentially expressed on Th1 or Th2 cells ( 19 ),
was low on splenic LTi-like cells (Fig. S6 and not depicted).
LTi-like cells produce IL-17 during zymosan-induced
infl ammation in Rag2 ? / ? mice
We next determined whether LTi - like cells produced IL - 17
in vivo in an infl ammatory setting. We approached this
problem in two ways: fi rst, we injected Rag2 ? / ? mice with
zymosan i.p. to induce infl ammation and harvested spleno-
cytes 2 h later. The cells were then stimulated ex vivo with
PMA/Iono and Brefeldin A (BFA) for 2 h, and the propor-
Figure 3. Isolated CD4 + CD3 ? CD11c ? B220 ? LTi-like cells produce IL-17 and constitutively express IL-23R, ROR ? t, AHR, and CCR6. (A) Iso-
lated CD4 + CD3 ? CD11c ? B220 ? LTi-like subsets from Rag2 ? / ? splenocytes (top) were cultured in the absence (Control) or presence of IL-23 (or with PMA/
Iono) for 24 h, and IL-17A in culture supernatants was measured by ELISA (bottom). Data are means ± SD from duplicate cultures and are representative
of two independent experiments. (B) Stat3 fl /fl or Stat3 fl /fl ; MMTV-Cre splenocytes were cultured in the absence (Control) or presence of IL-23 for 24 h. The
proportion of IL-17A – producing LTi-like cells was evaluated by intracellular staining. Data are means ± SD from duplicate cultures and are representative
of four independent experiments ( n = 8). *, P < 0.05. (C) Total RNA was prepared from LTi-like cells isolated from Rag2 ? / ? spleens. The relative expression
levels (/18S) of the indicated mRNA was analyzed by q-PCR and are depicted as fold induction relative to fresh B cells (or fresh naive T cells for AHR
mRNA expression). Data are representative of three independent experiments. M-T, memory T cells; N-T, naive T cells. (D) The expression of CCR6 on LTi-
like cells of WT splenocytes was analyzed by FACS. The shaded histogram indicates staining with isotype-matched control antibodies. The continuous line
histogram indicates the surface expression level of CCR6 on LTi-like cells. Data are representative of three independent experiments. ND, not detected.
JEM VOL. 206, January 19, 2009
BRIEF DEFINITIVE REPORT
it is becoming increasingly clear that they are not the only
source of IL-17. Clearly, ? ? T and iNKT cells produce IL-17
to protect against bacterial infection ( 6, 7 ). In addition, a re-
cent study provides evidence that IL-23 can drive IL-17
production by innate immune cells in animal models of in-
fl ammatory bowel disease ( 23 ).
We were very impressed by the loss of IL-17 production
in Rag2 ? / ? ? c ? / ? mice ( Fig. 1 B ). We initially suspected that
this pointed to production of IL-17 by NK cells but were sur-
prised by the failure to see IL-17 production by NK1.1 + CD3 ?
NK cells ( Fig. 2 A ). However, LTi-like cells are also depen-
dent on ? c cytokines for development ( 17 ). Before our stud-
ies, it was known that these cells expressed ROR ? t, which
was required for their function in LNs ( 16, 18 ). ROR ? t has
several important functions, but it is now clear that one func-
tion is the regulation of Th17 cell diff erentiation ( 13 ). We
confi rmed that splenic LTi-like cells constitutively express
ROR ? t ( Fig. 3 C ) and that IL-23 further up-regulated its ex-
pression (Fig. S7 C), arguing that this factor is essential in
controlling IL-17 and IL-22 production by non – T cells. In-
terestingly, fresh LTi-like cells also constitutively expressed
more AHR than polarized Th17 cells ( Fig. 3 C ). LTi-like
cells also constitutively expressed IL-23R and CCR6 ( Fig. 3,
C and D ), similar to CCR6 + IL-23R + human memory CD4 +
T cells, which are major producers of IL-17 ( 19, 24 ). Thus,
the expression of ROR ? t, AHR, CCR6, and IL-23R seems
to be the signature to defi ne IL-17 – and IL-22 – producing
cells in both adaptive and innate immune cells.
LTi-like cells are involved in the proper formation of pe-
ripheral LNs, the spleen, and gut-associated lymphoid tissue
by highly expressing lymphotoxin ? , lymphotoxin ? , and
TNF- ? ( 16, 25 ). Our data indicate that splenic and gut LTi-
like cells also produce IL-17 and IL-22 ( Figs. 3 A and 5 B ;
and Fig. S7, A and D) ( 26 ). It has been recently shown that
blocking IL-17 results in disruption of the germinal center –
like formation in the spleen, and restoration of the lymphoid
microanatomy is dependent on the proliferative accumula-
tion of LTi-like cells in SLOs during viral infection ( 27, 28 ).
In addition, the cross talk between bacteria, LTi cells, stromal
cells, DCs, and B cells seems to be essential for isolated lym-
phoid follicle formation in the gut ( 29 ). LTi-like cells might
IL-23 induced more IL-22 production by Rag2 ? / ? spleno-
cytes than WT splenocytes ( Fig. 5 B ). Furthermore, isolated
LTi-like cells also produced IL-22 ( Fig. 5 B and Fig. S7 D).
Although we observed that NK1.1 + CD3 ? NK cells failed to
produce IL-17A ( Fig. 2 A ), isolated NK cells produced small
amounts of IL-22 in response to IL-23 (Fig. S8, available at
These fi ndings illustrate the potentially distinct modes of reg-
ulation for IL-17 and IL-22.
The IL-23 – IL-17 axis has emerged to be important in
host defense and in models of autoimmunity such as experi-
mental autoimmune encephalomyelitis and infl ammatory
bowel disease ( 10, 23 ). Despite the importance of Th17 cells,
Figure 4. Zymosan induces IL-17 production in splenic LTi-like
cells in vivo. (left) Intracellular staining of IL-17A was performed with in
vitro conventional staining (top) or in vivo staining (bottom). For conven-
tional staining, Rag2 ? / ? mice were challenged with PBS (Control) or 12.5
mg zymosan i.p. Splenocytes were isolated 2 h later and stimulated with
PMA/Iono and BFA for 2 h in vitro. The proportion of IL-17A – producing
LTi-like cells was evaluated by intracellular staining. For the in vivo stain-
ing, Rag2 ? / ? mice were injected with PBS (Control) or 12.5 mg zymosan
i.p. together with 0.25 mg BFA i.v. (reference 22 ). The proportion of IL-17A –
producing LTi-like cells in spleens was directly evaluated by intracellular
staining. (right) The mean values (horizontal bars) for IL-17A – producing
LTi-like cells were calculated for in vitro staining (top, n = 6) and in vivo
staining (bottom, n = 6). Data are representative of three (top) or two
(bottom) independent experiments. ***, P < 0.001.
Figure 5. LTi-like cells produce high levels of IL-22. (A) Sera and splenocytes were harvested from Rag2 ? / ? mice 3 h after injection of PBS (Control)
or 5 mg zymosan. (left) IL-22 in sera was measured by ELISA. (right) The relative expression levels (/18S) of IL-22 mRNA were determined by q-PCR and
are depicted as fold induction relative to control cells. The data are means ± SD from two mice and are representative of two independent experiments.
(B) WT or Rag2 ? / ? splenocytes or isolated LTi-like cells were cultured in the presence of IL-23 for 24 h, and IL-22 in culture supernatants was measured
by ELISA. The data are means ± SD from duplicate cultures and are representative of two independent experiments.
IL-17 AND IL-22 PRODUCTION BY SPLENIC LT I -LIKE CELLS | Takatori et al.
CD4 + CD3 + CD62L ? CD44 + memory T cells; CD4 + CD3 + CD62L high CD44 low
naive T cells; CD19 + B cells, Th17 cells polarized with IL-6, TGF ? -1, and
anti-CD3/-CD28 for 3 d; and CD4 + CD3 ? CD11c ? B220 ? LTi-like cells.
cDNA was synthesized with the TaqMan Reverse Transcription kit (Ap-
plied Biosystems). TaqMan primers and probes for mouse IL-17A, IL-17F,
IL-22, IL-23R, IL-12R ? 1, IL-12R ? 2, RORc (for ROR ? t), IL-17RA,
IL-6R ? , CCR6, AHR, GATA3, and 18SrRNA (as endogenous control) were
purchased from Applied Biosystems. Samples were analyzed by using a se-
quence detection system (ABI PRISM 7700; Applied Biosystems).
The amounts of cytokines in the culture supernatant were measured us-
ing mouse IL-17 Quantikine assay kits (for IL-17A; R & D Systems) and the
mouse IL-22 ELISA construction kit (Antigenix America Inc.) according to
the manufacturers ’ instructions. IL-23 – mediated cell stimulations were per-
formed at cell concentrations of 4 × 10 6 cells/ml. Samples were measured in
duplicate against the standard curve of the assay.
Data analysis. Statistical signifi cance was determined by the Student ’ s t test.
P < 0.05 was considered to indicate a signifi cant diff erence.
Online supplemental material. Fig. S1 shows IL-17A production by WT
or Rag2 ? / ? splenocytes in the presence of IL-23 with ? c-dependent cyto-
kines, anti-CD3/-CD28, or ? -GalCer. Fig. S2 shows that IL-17A – producing
CD3 ? cells do not express myeloid markers. Fig. S3 shows three populations
in CD4 + CD3 ? cells from Rag2 ? / ? spleens. Fig. S4 shows that LTi-negative
subsets produce minimal IL-17A. Fig. S5 shows IL-17A production by LTi-
like cells in the gut. Fig. S6 shows the surface expression of various markers
on LTi-like cells. Fig. S7 shows mRNA expression of various factors in LTi-
like cells after IL-23 stimulation with IL-7. Fig. S8 shows IL-22 and IL-17A
production by isolated NK cells. Online supplemental material is available at
We thank J. Simone and the fl ow cytometry core, and the animal facility of the
NIAMS for excellent advice and technical service. We thank Drs. H. Young and D.
McVicar for helpful discussions. We thank Drs. L. Hennighhausen and D. Levy for
providing Stat3 fl /fl ; MMTV-Cre mice.
This research was supported by the Intramural Research Program, NIAMS, NIH.
The authors have no confl icting fi nancial interests.
Submitted: 20 December 2007
Accepted: 2 December 2008
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help organize the development of lymphoid tissues through
the production of IL-17. In addition, IL-17 and IL-22 act on
epidermal cells, producing antimicrobial products that pro-
tect the host from extracellular bacteria and fungi ( 22 ). Our
in vivo experiments in which Rag2 ? / ? mice challenged with
zymosan produced IL-17 ( Fig. 4 ) are consistent with the no-
tion that splenic LTi-like cells might participate in host de-
fense. It is therefore possible that splenic LTi-like cells exist
not only for the development of SLOs but also for the rapid
response to pathogens that elicit IL-17 and IL-22. Thus, our
report brings to light new potential roles of LTi-like cells in
host defense in addition to their known roles in regulating
MATERIALS AND METHODS
Recombinant cytokines and antibodies. Mouse IL-6, IL-12, IL-21, IL-
23, and TGF ? -1 were purchased from R & D Systems. Mouse IL-2, IL-4,
IL-7, and IL-15 were purchased from PeproTech. Antibodies to CD4
(GK1.5), CD3 (145-2C11), CD28 (37.51), CD11c (HL3), B220 (RA3-
6B2), ? c (4G3), IL-7R ? (SB199), Thy1.2 (30-H12), CD44 (IM7), CD62L
(MEL-14), CCR5 (C34-3448), CXCR5 (2G8), and IL-17 (TC11-18H10;
for IL-17A) were purchased from BD. Antibodies to CD30L (RM153),
OX40L (RM134L), and CCR7 (4B12) were purchased from eBioscience.
Anti-CCR6 antibody (clone 140706) was obtained from R & D Systems.
Both anti-CXCR3 and -CCR4 antibodies were purchased from Abcam.
? -GalCer was obtained from AXXORA, LLC.
Mice. C57BL/6J WT mice (The Jackson Laboratory), Rag2 ? / ? mice, and
Rag2 ? / ? ? c ? / ? mice (Taconic) were purchased as indicated. Stat3 fl /fl mice
were bred with mice expressing Cre under the control of the MMTV-LTR
( MMTV-Cre ) to produce Stat3 fl /fl ; MMTV-Cre mice (provided by L.
Hennighhausen [National Institute of Diabetes and Digestive and Kidney
Diseases] and D. Levy [New York University, New York, NY]) ( 30 ). All animal
experiments were performed according to the National Institutes of Health
(NIH) guidelines for laboratory animals and were approved by the National
Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) Ani-
mal Care and Use Committee.
Isolation of cells and cell culture. Single-cell suspensions were prepared
from spleens of healthy 8 – 10-wk-old mice. All cell cultures were performed
in RPMI 1640 supplemented with 10% FBS, 2 mM l -glutamine, 5 mM
Hepes, 100 U/ml Pen-Strep, and 2.5 μ M 2-ME at 37 ° C for 4, 24, or 48 h.
Cells stained with the appropriate antibodies were isolated by fl ow cytomet-
ric cell sorting using a Mo-Flo cell sorter (Dako). Whole splenocytes or iso-
lated cells were cultured in the presence of 20 ng/ml IL-23, 20 ng/ml IL-4,
10 ng/ml IL-6, 20 ng/ml IL-7, 20 ng/ml IL-15, 20 ng/ml IL-2, 20 ng/ml
IL-12, 100 ng/ml IL-21, or 5 ng/ml TGF ? -1.
Flow cytometric analysis and intracellular cytokine staining. Cells
were stimulated for 2 h with 50 ng/ml PMA and 1 μ g/ml Iono, followed by
incubation with BFA (GolgiPlug; BD) for an additional 2 h. Cells were fi xed
in 4% formyl saline and permeabilized with 0.1% saponin permeabilization
buff er after surface staining. PE-conjugated anti – IL-17 antibody was used to
detect intracellular cytokine levels (BD). Stained cells with the appropriate
antibodies were analyzed on a fl ow cytometer (FACSCalibur; BD). Events
were collected and analyzed with FlowJo software (Tree Star, Inc.). To
evaluate production of IL-17A in vivo, Rag2 ? / ? mice were injected i.p.
with 5 or 12.5 mg zymosan (Sigma-Aldrich). Control animals received PBS.
To assess in vivo intracellular cytokine levels, 0.25 mg BFA (Sigma-Aldrich)
was simultaneously injected i.v., as previously described ( 21 ).
RNA isolation and measurement of cytokines. Total RNA was
isolated using TRI zol reagent (Invitrogen) from freshly isolated
JEM VOL. 206, January 19, 2009 Download full-text
BRIEF DEFINITIVE REPORT
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