Art i cl e

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J. Exp. Med. 2014 Vol. 211 No. 7 1421-1431
www.jem.org/cgi/doi/10.1084/jem.20131501
1421
NK cells play a critical role in host defense against
some pathogens and play an essential role in clear-
ing tumor cells (Biron et al., 1999; Cerwenka
et al., 2001; Vivier et al., 2012). The BM is the key
site for multiple stages of NK development, but
the precise mechanisms that regulate the transi-
tion between various stages of NK development
remain elusive. Currently, it is established that
NK cells develop from common lymphoid pro-
genitors (CLPs), which possess precursor poten-
tial for T, B, and NK cells (Ramirez and Kee,
2010; Vosshenrich and Di Santo, 2013). CLPs
lack the markers of hematopoietic lineages but
are distinguished based on their expression of
low levels of c-Kit, Sca1, and IL7R (Kondo
et al., 1997). Under support from stromal cells,
CLPs are directed toward the NK fate through
several stages defned by patterns of expression of
CD122 (IL-2 and IL-15 receptor chain), NK1.1
(an activating NKR), and DX5 (integrin 2
and CD49b; Kim et al., 2002; Lian and Kumar,
2002; Ramirez and Kee, 2010). As CLPs develop
into NK progenitors, they begin to express
CD122 while remaining negative for other lin-
eage markers (Ter119, CD3, CD19, and Gr1;
Di Santo, 2006). Acquisition of NK1.1 occurs
at the immature NK (iNK) cell stage, charac-
terized by expression of multiple NKRs and
IL-15 dependence (Vosshenrich et al., 2005).
Transient expression of integrin

(CD51) and
TRAIL also occurs at this stage (Kim et al.,
2002). Further maturation into mature NK
(mNK) cells is accompanied by increased ex-
pression of DX5, CD11b, and CD43 and the
loss of CD51 and TRAIL (Kim et al., 2002;
Vosshenrich et al., 2005; Chiossone et al., 2009).
Although distinct stages in the progression of
CLPs to the development of mNK cells have
been identifed, how those key developmental
programs are regulated is currently unappreciated.
Lymphotoxin (LT), in its trimeric form
(LT
1

2
), is expressed by activated lympho-
cytes and binds to LTR expressed primarily
on myeloid, parenchymal, and stromal cell pop-
ulations (Fu et al., 1998; Murphy et al., 1998;
CORRESPONDENCE
Kyung-Mi Lee:
kyunglee@korea.ac.kr
OR
Yang-Xin Fu:
yxfu@uchicago.edu
Abbreviations used: CLP, com-
mon lymphoid progenitor; ILC,
innate lymphoid cell; iNK,
immature NK; LPL, lamina
propria lymphocyte; LT, lym-
photoxin; mNK, mature NK.
Innate lymphoid cells facilitate NK cell
development through a lymphotoxin-
mediated stromal microenvironment
Tae-Jin Kim,
1,2
Vaibhav Upadhyay,
1
Vinay Kumar,
1
Kyung-Mi Lee,
1,2

and Yang-Xin Fu
1
1
Department of Pathology, The University of Chicago, Chicago, IL 60637
2
Global Research Lab, Department of Biochemistry and Molecular Biology, Korea University College of Medicine,
Seoul 136-705, South Korea
Natural killer (NK) cell development relies on signals provided from the bone marrow (BM)
microenvironment. It is thought that lymphotoxin (LT)
1

2
expressed by the NK cell
lineage interacts with BM stromal cells to promote NK cell development. However, we now
report that a small number of RORt
+
innate lymphoid cells (ILCs), and not CD3

NK1.1
+

cells, express LT to drive NK development. Similar to LT
/
or RORt
/
mice, the mice
conditionally lacking LT
1

2
on RORt
+
ILCs experience a developmental arrest at the
immature NK stages, between stages of NK development to the mature NK cell stage. This
developmental block results in a functional defciency in the clearance of NK-sensitive
tumor cells. Reconstitution of Thy1
+
ILCs from BM or purifed RORt
+
ILCs from lamina
propria lymphocytes into LT-defcient RORt
+
BM cultures rescues NK cell development.
These data highlight a previously undiscovered role of RORt
+
ILCs for NK cell development
and defne LT from ILCs as an essential molecule for the stromal microenvironment sup-
porting NK cell development.
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Published June 9, 2014
1422 Innate lymphoid cells regulate NK cell development | Kim et al.
never express RORt throughout their life and that IL-15
defcient mice have defective NK cells but normal numbers
for RORt
+
ILCs (Sawa et al., 2010; Pandiyan et al., 2012).
Therefore, it is thought that NK cells are a completely dis-
tinct lineage from RORt
+
ILCs.
Upon profling the BM of wild-type mice, we unexpect-
edly have found that RORt
+
ILCs express a signifcant level
of surface LT, whereas NK cells expressed virtually undetect-
able levels of LT, casting doubt on the model in which NK
cells directed their own development through LT expression.
We therefore explored the possibility that RORt
+
ILCs,
and not NK cells, provided the LT signal to LTR-expressing
stromal cells necessary for NK cell development. Using vari-
ous RORt or LT-defcient animal models as well as in vitro
BM cell culture systems, we now report that LT
1

2
expressed
on RORt
+
ILCs in BM plays a crucial role in promoting a
microenvironment for the development of iNK into mNK
cells and therefore have uncovered a close interaction between
NK cell development and RORt
+
ILCs.
RESULTS
LT from RORt
+
cells plays a critical role
in NK cell development
NK cell development occurs independently of adaptive im-
munity, and therefore, it has been speculated that LT from
Fu and Chaplin, 1999). LT is thought to be essential for the
development of secondary lymphoid tissues (Fu and Chaplin,
1999). We and others have reported that the loss of LT (LT or
LT gene) causes a dramatic reduction of the number of NK
cells in the spleen and BM and impairment of antitumor activity
caused by defective NK cell activities (Iizuka et al., 1999; Ito et al.,
1999; Smyth et al., 1999; Wu et al., 2001). Therefore, it is possible
that LT delivers an essential signal to the LTR-expressing
stromal cells to promote NK cell development and maturation
(Iizuka et al., 1999; Wu et al., 2001; Lian et al., 2004). We have
further observed that NK cell development of RAG1
/
mice is
also reduced after prolonged blockade of LT signaling (Wu et al.,
2001). These data have supported a model in which LT from NK
lineage cells is required for optimal NK cell development.
NK cells are considered to be the founding members of
the innate lymphoid cell (ILC) family, having shared immuno-
logical and developmental characteristics. However, recent
studies have unearthed the existence of ILCs, which is a het-
erogeneous family of innate efector cells that have critical roles
in the generation and maintenance of innate immune responses.
One subset of ILCs expressing retinoic acid receptorrelated
orphan receptor t (RORt) is essential in lymphoid tissue
formation and immune defense in an LT-dependent fashion
(Cherrier and Eberl, 2012; Spits and Cupedo, 2012; Upadhyay
and Fu, 2013). Studies argue that NK cells (NK1.1
+
, CD3

)
Figure 1. RORt
+
ILCs but not NK
cells express LT. (A and B) Splenocytes
from wild-type (A) and CD19
Cre+
LT
f/f
,
CD4/CD19
Cre+
LT
f/f
, Rorc
Cre+
LT
f/f
, LT
/
,
and RORt
/
(B) mice were stimulated with
20 ng/ml PMA for 20 h. Stimulated cells
were then labeled with biotinylated LTR-Ig
for LT staining and assessed by flow cytom-
etry to detect LT expression. Data are repre-
sentative of two independent experiments.

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Published June 9, 2014
JEM Vol. 211, No. 7
Ar t i cl e
1423
conventional CD3

NK1.1
+
NK cells, RORt
+
ILCs appeared
to express LT
1

2
, which is capable of binding to LTR
(Fig. 1 A). To further determine the precise role of specifc
LT-positive lymphocyte subsets in NK cell development,
we crossed mice containing a foxed allele of LT (LT
f/f
) to
mice expressing Cre recombinase in B cells (CD19
Cre+
), T cells
(CD4
Cre+
), both B and T cells (CD19
Cre+
CD4
Cre+
), or Rorc
Cre+
.
As seen in Fig. 1 B, specifc deletion of LT was observed
in B (CD19
Cre+
) or both B and T (CD19
Cre+
CD4
Cre+
) cells.
As expected, LT expression was lacking on RORt
+
ILCs
in Rorc
Cre+
LT
f/f
mice. As additional controls, LT-defcient
NK cells is essential for NK cell development (Iizuka et al.,
1999; Smyth et al., 1999; Wu et al., 2001). LT was originally
described as expressed on T, B, and NK cells (Ware et al., 1995),
and these fndings raised doubts over the ability of NK cells to
regulate their own development using the LT pathway. How-
ever, using the biotinylated decoy receptor LTR-Ig, we have
only been able to detect membrane LT on T and B cells but
not NK cells (Fig. 1 A). Inspired by recent fndings demon-
strating LT expression on the RORt
+
ILCs (Finke, 2005), we
compared LT expression in RORt
+
ILCs and CD3

NK1.1
+

NK cells. In contrast to the absence of expression of LT on
Figure 2. LT on RORt
+
cells regulates NK cells homeostasis. (AC) Cells from spleen of 612-wk-old wild-type (n = 32), LT
/
(n = 11), RORt
/

(n = 8), CD19
Cre+
LT
f/f
(n = 5), CD19/CD4
Cre+
LT
f/f
(n = 3), and Rorc
Cre+
LT
f/f
(n = 20) mice were isolated and stained for NK1.1
+
, CD3

. (A) Dot plot represents

NK cells in spleen. (B and C) Frequency of splenic NK (NK1.1
+
, CD3

) and mNK (DX5

+
, NK1.1
+
) cell populations as assessed by fow cytometry. (D) Frequency of
splenic CD3
+
and CD19
+
cell populations from wild-type and Rorc
Cre+
LT
f/f
mice as assessed by fow cytometry (each group, n = 4). (E and F) Cells from BM
(each group, n = 13) or blood (each group, n = 6) of 612-wk-old wild-type and Rorc
Cre+
LT
f/f
mice were assessed by fow cytometry for the percentage and
total cell numbers of NK cells. (G) Cells from spleen of 3-wk-old wild-type and Rorc
Cre+
LT
f/f
mice were assessed for the frequency and cell numbers (each group,
n = 3 or 4). Data are from at least three independent experiments. All data are presented as the mean SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Published June 9, 2014
1424 Innate lymphoid cells regulate NK cell development | Kim et al.
knockout). Interestingly, the analysis of Rorc
Cre+
LT
f/f
mice
revealed a severe reduction of both CD3

NK1.1
+
and DX5
+

NK1.1
+
cells in the spleen, similar to that observed in LT
/

mice (Fig. 2, AC). However, splenic CD3
+
and CD19
+
cells
from Rorc
Cre+
LT
f/f
mice were not signifcantly diferent from
those in wild-type mice (Fig. 2 D). Not only the percentage
of CD3

NK1.1
+
cells but also their absolute numbers were
signifcantly reduced in Rorc
Cre+
LT
f/f
mice (Fig. 2, E and F).
Furthermore, these defects were apparent in 3-wk-old mice
(Fig. 2 G), demonstrating a true developmental defect for
NK cells in these mice. Together, these data confrm that LT
on RORt
+
cells is essential for NK cell development and
homeostasis in mice.
RORt
+
ILCs regulate NK cell development
Because RORt is expressed on both T cells and subsets of
ILCs, it is possible that RORt
+
T cells contribute to the de-
velopment of NK cells in addition to ILCs. To address this
mice did not show any visible expression of surface LT in all
subsets tested, whereas RORt
/
mice demonstrated nor-
mal LT expression in B and T cells (no RORt
+
ILCs found
in these mice). We next examined whether these mice dis-
played developmental NK cell defects. It has been shown
previously that LT is expressed on RORt
+
T cells and that
ILCs can control the generation of secondary lymphoid tis-
sues (Chiang et al., 2009; Cherrier and Eberl, 2012; Spits and
Cupedo, 2012). LT-defcient mice were shown to have se-
verely diminished NK cells (Iizuka et al., 1999; Smyth et al.,
1999), and as such, we saw dramatic reduction of CD3

NK1.1
+

NK cells in the spleen of LT
/
mice (Fig. 2 A). Similar to
LT
/
mice, NK cell frequency in RORt
/
mice were
severely reduced, raising the possibility that LT on RORt
+

cells could be essential for NK cell development. In con-
trast, no signifcant reduction of NK cell populations was
noted in CD19
Cre+
LT
f/f
, CD4
Cre+
LT
f/f
, and double CD19/
CD4
Cre+
LT
f/f
mice (Fig. 2, AC; not depicted for CD4 single
Figure 3. LT on RORt-expressing innate
cells infuences NK cells homeostasis.
(AC) Cells from spleen of 612-wk-old
RAG1
/
(n = 16), RAG1
/
LT
/
(n = 3),
and RAG1
/
Rorc
Cre+
LT
f/f
(n = 13) mice were
isolated and stained for NK cells (NK1.1
+
).
Splenocytes from RAG1
/
, RAG1
/
LT
/
,
and RAG1
/
Rorc
Cre+
LT
f/f
mice were stained
for NK cells (NK1.1
+
, CD3

) and mNK cell

marker (DX5
+
, NK1.1
+
) and analyzed by
fow cytometry. (D and E) Cells from BM (each
group, n = 11) and blood (each group,
n = 5 or 4) of 612-wk-old RAG1
/
and
Rorc
Cre+
LT
f/f
RAG1
/
mice were assessed for
the percentage and cell number of NK cells by
fow cytometry. Data are from at least three
independent experiments. All data are pre-
sented as the mean SEM. *, P < 0.05;
**, P < 0.01; ***, P < 0.001.

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Published June 9, 2014
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1425
using recently described cell surface markers: CD122, NK1.1,
and DX5 (Bezman et al., 2011). NKp cells are designated as
CD122
+
, Lin

, NK1.1

, DX5

, whereas iNK cells and mNK

cells are found to express CD122
+
, Lin

, NK1.1
+
, DX5

and
CD122
+
, Lin

, NK1.1
+
, DX5
+
, respectively (Rosmaraki et al.,
2001; Kim et al., 2002). Therefore, we exploited these surface
markers to evaluate a potential block that may have been occur-
ring in the NK developmental pathway in RAG1
/
Rorc
Cre+

LT
f/f
mice. As shown in Fig. 4, there are even increased per-
centages of NKp (CD122
+
, Lin

, NK1.1

, DX5

) and iNK
(CD122
+
, Lin

, NK1.1
+
, DX5

) cells present in the BM of mice

conditionally lacking LT on RORt
+
ILCs. Coordinately,
the absence of LT on this cell type resulted in a signifcantly
diminished mNK cell (CD122
+
, Lin

, NK1.1
+
, DX5
+
) fre-
quency and cell numbers in BM (Fig. 4, AC), demonstrat-
ing a developmental block at the NKp stage. These defects in
RAG1
/
Rorc
Cre+
LT
f/f
mice were also observed in Rorc
Cre+

LT
f/f
mice that are immunocompetent (Fig. 4, DF), sug-
gesting that adaptive immune T and B cells did not play a sig-
nifcant role in NK cell development. These data indicate that
LT signaling from RORt
+
ILCs controls a key stage of NK
cell development before fnal maturation.
possibility, we crossed Rorc
Cre+
LT
f/f
mice to the RAG1
/

background and analyzed the proportion of NK cells in the
absence of T and B cells. As can be seen in Fig. 3 A, the percent-
age of NK1.1
+
cells in the spleen of RAG1
/
Rorc
Cre+
LT
f/f

mice was signifcantly diminished to <30% of that observed in
RAG1
/
mice. Similar impairment was detected in RAG1
/

LT
/
mice (Fig. 3 A), suggesting that LT on ILCs, but not
on T or B cells, was critical for the normal development of
NK cells. Both NK1.1
+
and DX5
+
NK1.1
+
cell populations
in the spleen were reduced in RAG1
/
Rorc
Cre+
LT
f/f
and
RAG1
/
LT
/
mice (Fig. 3, B and C). Similar to Rorc
Cre+

LT
f/f
mice, reduction of the NK cell percentage and num-
bers was also observed in the BM and blood of RAG1
/

Rorc
Cre+
LT
f/f
mice (Fig. 3, D and E). These data clearly
demonstrate that LT signaling from RORt
+
ILCs but not
T cells is essential for NK cell development.
LT
1

2
on RORt
+
ILCs is important for early NK development
We next investigated whether RORt
+
ILCs infuence the
maturation stage of NK cell development. NK cell maturation
can be staged from CLP pre-NKp NKp iNK
mNK cells (Kim et al., 2002), and we monitored these stages
Figure 4. LT on RORt
+
ILCs is important for the transitional stage of NK cells. (AC) Flow cytometric analysis of RAG1
/
(n = 5) and
RAG1
/
Rorc
Cre+
LT
f/f
(n = 5) mice in BM. (A) Dot plots indicate the frequency of NKp (CD122
+
, Lin

, NK1.1

, DX5

), iNK (CD122
+
, Lin

, NK1.1
+
, DX5

),
and mNK cells (CD122
+
, Lin

, NK1.1
+
, DX5
+
). (B and C) Bar graphs indicate the percentage and number of NKp (NK1.1

, DX5

), iNK (NK1.1
+
, DX5

), and
mNK (NK1.1
+
, DX5
+
) cell populations. (DF) Flow cytometric analysis of BM from LT
f/f
(n = 5) and Rorc
Cre+
LT
f/f
(n = 5) mice. (D) Dot plots indicate the
frequency of NKp (CD122
+
, Lin

, NK1.1

, DX5

), iNK (CD122
+
, Lin

, NK1.1
+
, DX5

), and mNK cells (CD122

+
, Lin

, NK1.1
+
, DX5
+
). (E and F) Bar graphs indi-
cate the percentage and number of NKp (NK1.1

, DX5

), iNK (NK1.1
+
, DX5

), and mNK (NK1.1

+
, DX5
+
) cell populations. Data are from at least fve inde-
pendent experiments. All data are presented as the mean SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Published June 9, 2014
1426 Innate lymphoid cells regulate NK cell development | Kim et al.
shown to be signifcantly increased in the BM of Rorc
Cre+
LT
f/f

mice. However, the CD51

, DX5
+
NK cell (mature) fre-
quency and cell number were signifcantly decreased (Fig. 5,
E, G, and H). The ratio of mNK and iNK cells was signif-
cantly reduced in Rorc
Cre+
LT
f/f
mice in the immunocompe-
tent host (Fig. 5 F). Together, these results highlight the role of
LT
1

2
signaling from ILCs facilitating NK cell development.
LT
1

2
from RORt
+
ILCs controls
NK cell development in vitro
ILCs from mucosal tissues are thought to play various roles in
host defense (Wang et al., 2010; Tumanov et al., 2011), but
To confrm our fndings, we compared the NK maturation
status in these mice according to the expression of DX5 and
CD51, as markers for mNK and iNK cells, respectively (Kim
et al., 2002). As shown in Fig. 5 (AD), the CD51
+
, DX5

(iNK) frequency and cell number are shown to be signifcantly
increased in the BM of RAG1
/
Rorc
Cre+
LT
f/f
mice, whereas
the CD51

, DX5
+
population (mNK) is largely decreased
(Fig. 5, A, C, and D). The ratio of mNK and iNK cells was sig-
nifcantly decreased in Rorc
Cre+
LT
f/f
mice in the immuno-
compromised host, supporting developmental arrest at this
stage (Fig. 5 B). Like in RAG1
/
background host, the CD51
+
,
DX5

NK cell (immature) frequency and cell number are

Figure 5. Absence of LT on RORt
+
ILCs leads
to accumulation of the CD51
+
DX5

iNK cell
population. (A) Dot plots indicate the percentage of
iNK (CD51
+
, DX5

) and mNK (CD51

, DX5
+
) cell pop-
ulations in BM of RAG1
/
or RAG1
/
Rorc
Cre+
LT
f/f

mice. (B) Bar graph indicates ratio of iNK (CD51
+
,
DX5

) to mNK (CD51

, DX5
+
) cells in the indicated
mouse strains (each group, n = 10). (C and D) Bar
graphs represent the mean percentage SEM of
frequency or cell number of iNK (CD51
+
, DX5

) and
mNK (CD51

, DX5
+
) cells. (E) Dot plot indicates the
percentage of iNK (CD51
+
, DX5

) and mNK (CD51

,
DX5
+
) cells in BM of the indicated mouse strains on
the B6 background. (F) Bar graph indicates the ratio
of iNK (CD51
+
, DX5

) to mNK (CD51

, DX5
+
) cells in
the indicated mouse strains on the B6 background
(each group, n = 9). (G and H) Bar graphs represent
percentage and cell number of iNK (CD51
+
, DX5

) and
mNK (CD51

, DX5
+
) cells. Data are from at least nine
independent experiments. All data are presented as the
mean SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Published June 9, 2014
JEM Vol. 211, No. 7
Ar t i cl e
1427
Fig. 6 (B and C), the number of NK cells generated from
Rorc
Cre+
LT
f/f
BM cultures was signifcantly reduced compared
with those obtained from wild-type BM cultures, regardless
of the presence or absence of adaptive immune cells (Fig. 6,
B and C). These data strongly suggest the pivotal role of LT on
RORt
+
ILCs in the early stage of NK cell development.
Our in vivo data demonstrated that LT signaling on RORt
+

ILCs is essential for NK cell development, and therefore, we
next investigated whether ILCs from wild-type mice could
rescue the NK cell defect in Rorc
Cre+
LT
f/f
mice. To investigate
whether Lin

NK1.1

Thy1
+
ILCs could contribute to the NK
cell development, we isolated Lin

NK1.1

Thy1
+
ILCs from
wild-type BM cells using a magnetic beads system. We then
supplemented the BM cultures of Rorc
Cre+
LT
f/f
with these
cells at the beginning of the culture. As depicted in Fig. 6 D,
addition of wild-type ILCs from BM completely rescued the
ability of Rorc
Cre+
LT
f/f
BM cultures to generate NK cells.
Therefore, Lin

NK1.1

Thy1
+
ILCs rescue NK cell develop-
ment in an LT-dependent fashion in the in vitro BM culture
system. To address the possibility that IL-15 was being supple-
mented from the Lin

NK1.1

Thy1
+
subset, Lin

NK1.1

Thy1

cell populations were cultured in the presence of IL-15;
supplementing IL-15 to the Lin

NK1.1

Thy1

cells failed to
rescue the NK cell defect observed from Rorc
Cre+
LT
f/f
BM
the role of ILCs within the BM is poorly defned. To study
whether ILCs from BM cells can regulate NK cell develop-
ment and avoid NK cell tracking between the periphery and
BM, a BM cell culture system was developed to model NK
cell development inside BM without interference of their traf-
fcking. We frst tested whether blockade of LT signaling using
the decoy receptor LTR-Ig impairs NK development in wild-
type BM. BM cells were initially cultured in the absence of
IL-15 for 1014 d to eliminate all mNK cells. IL-15 was then
added into the BM culture system to drive NKp cells for their
maturation and development. After an additional 10 d of cul-
ture following IL-15 treatment, we assessed NK cell number
using the markers described above.
As previously demonstrated, in vitro BM culture success-
fully generated mNK cells (Fig. 6) in both immunocompetent
and immunocompromised mice. However, blocking LTR
signaling by the addition of LTR-Ig to the culture signif-
cantly decreased NK cell recovery compared with the human-Ig
(hIg) control group (Fig. 6 A). These data indicate that LT
signaling within the cultured BM microenvironment is nec-
essary and sufcient for NK cell development. To determine
whether LT
1

2
on RORt
+
ILCs is important for NK cell
development, we compared the number of NK cells generated
from wild-type and Rorc
Cre+
LT
f/f
BM cultures. As seen in
Figure 6. ILCs could restore defective LT-mediated
early NK cell development. Fresh isolated BM cells
were cultured without IL-15 cytokine for 10 d and
then with 20 ng/ml IL-15 cytokine for an additional
10 d. NK cells were harvested and counted. (A) hIg
(n = 4) and LTR-Ig (n = 4) were added to fresh
isolated BM cells from B6 mice at 0, 3, and 6 d.
(B and C) BM cells from wild-type, Rorc
Cre+
LT
f/f
, and
CD4/CD19
Cre+
LT
f/f
mice from the B6 (B) or RAG1
/

(C) backgrounds were cultured for 20 d (each group,
n = 4). (D) Isolated Lin

, NK1.1

, Thy1
+
or Lin

, NK1.1

,
Thy1

ILCs from wild-type BM cells were added into

the Rorc
Cre+
LT
f/f
BM culture group at 0 d (each
group, n = 4). (E, left) Flow cytometry gating strategy.
RORt
+
ILCs from LPLs were isolated from RAG1
/

mice. (right) Isolated RORt
+
ILCs from RAG1
/

intestine LPLs were added into Rorc
Cre+
LT
f/f
BM
cultures at day 0, and recovered NK cells were enu-
merated on day 20 (each group, n = 3 or 4). Data are
representative of three (E) or at least four (AD) inde-
pendent experiments. All data are presented as the
mean SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Published June 9, 2014
1428 Innate lymphoid cells regulate NK cell development | Kim et al.
ILCs with RMA-S and RMA, which difer by expression of
MHC class I and subsequent sensitivity to NK killing. As de-
picted in Fig. 7 (C and D), Rorc
Cre+
LT
f/f
mice were suscep-
tible to the RMA-S tumor line, whereas RMA tumor growth
was similar between WT and Rorc
Cre+
LT
f/f
mice (Fig. 7,
C and D). These data confrm that LT signaling is critical for
the clearance of MHC class Idefcient tumor cells and im-
mune surveillance provided by NK cells in vivo. Furthermore,
it suggests that LT from RORt
+
ILCs creates a unique micro-
environment for NK cell development that renders the host
resistant to some tumors.
DISCUSSION
Although the developmental stages of NK cells within the BM
have been identifed, how the BM stromal microenvironment
supports NK cell development is not well defned. It has been
shown that LT-defcient mice exhibit a developmental arrest of
NK cells (Iizuka et al., 1999; Ito et al., 1999; Smyth et al., 1999;
Wu et al., 2001). Furthermore, LTR signaling on BM stromal
cells is required for optimal NK development in the absence
of T and B cells (Wu et al., 2001). It is thought that membrane-
bound LT expressed on NK celllike precursors is required for
NK development. In this study, we revisit this hypothesis and
refne this concept by discovering a new population of LT-
expressing ILCs within BM, which control the development
and maturation of NK cells: (a) RORt
/
mice as well as
Rorc
Cre+
LT
f/f
mice yield reduced NK cells, providing com-
pelling evidence that LTs on RORt
+
ILCs are important
players in NK cell development; (b) depletion of ILCs blocked
the development of NK cell; (c) adoptive transfer of LT-
expressing ILCs rescued NK cell development in LT-defcient
BM cells, suggesting that ILCs, not NK precursors, are the pop-
ulation essential for NK cell development.
The ILC population can be broadly divided into classical
NK cells and three newly defned distinct populations of ILCs.
(Fig. 6 D). To rule out the potential contamination of NKps
during the cell transfer, RORt
+
ILC populations were isolated
from intestinal lamina propria lymphocytes (LPLs) using cell
sorting and added to the BM cultures of Rorc
Cre+
LT
f/f
. As
shown in Fig. 6 E, addition of RAG1
/
ILC3 from LPLs could
successfully restore NK cell generation in the Rorc
Cre+
LT
f/f

BM culture group. Together, these data confrm that NK cells
generated from the addition of wild-type ILCs in Rorc
Cre+
LT
f/f

BM cultures were not likely from the contaminated wild-type
CLP or NKp, but rather from the LT signaling provided by
wild-type ILCs themselves.
Lack of LT on ILCs signifcantly impairs
NK-mediated tumor clearance
NK cells have been observed to play a critical role in the scav-
enging of hematopoietic cells that have undergone malignant
transformation (Dong et al., 2009). Therefore, we hypothe-
sized that impaired LT signaling on ILCs could afect the abil-
ity of NK cells in providing the host protection from malignant
hematopoietic cells. To test this, we injected an NK-sensitive
tumor line, RMA-S (MHC class Idefcient T cell lymphoma;
van den Broek et al., 1995; Smyth et al., 1998), into the right
fank of LT
/
, LTR
/
, and wild-type mice and moni-
tored tumor growth every 34 d. Although wild-type mice
controlled RMA-S tumors almost completely, LT
/
and
LTR
/
mice failed to control tumor growth (Fig. 7, A and B).
To examine whether LT signaling directly afects tumor clear-
ance, we injected LTR-Ig (150 g/each, one time, 0 d) into
wild-type mice to block LT signaling in mice. No signifcant
diference in tumor growth was obtained between hIg- and
the LTR-Igtreated group, arguing that the defect seen in
mice lacking the LT pathway occurred indirectly through a
developmental defect present in these mice (not depicted).
Given that active LT signaling did not protect mice from MHC
Idefcient tumor cells, we challenged mice lacking LT from
Figure 7. Absence of LT on RORt
+
ILCs
signifcantly impaired NK-mediated tumor
surveillance. (AC) RMA-S cells (5 10
5
) were in-
jected s.c. into wild-type (n = 4), LT
/
(n = 4),
LTR
/
(n = 3), or Rorc
Cre+
LT
f/f
(n = 5) mice. (D) RMA
cells (5 10
5
) were injected s.c. into wild-type and
Rorc
Cre+
LT
f/f
mice (each group, n = 3). Tumor volume
was assessed at the indicated time points. Data are
representative of two (B and D) or at least three
(A and C) independent experiments. All data are
presented as the mean SEM.

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Published June 9, 2014
JEM Vol. 211, No. 7
Ar t i cl e
1429
Collectively, our data present a new paradigm of NK cell
developmental process by incorporating a critical role of ILCs
expressing LT; upon generation of iNK cells, membrane-bound
LT on RORt
+
ILCs provides signal LTR on BM stromal
cells, which stimulate NF-B signaling for chemokines and
cytokines necessary for iNK maturation within the BM mi-
croenvironment. In the absence of LT-dependent signaling,
NK cell development was arrested at the iNK stage express-
ing CD51 and resulted in functional impairment. However,
interactions among newly defned ILCs and NK cells for their
development and function have not been well defned and
should be important aspects of future studies. Thus, it is fasci-
nating that such a small number of RORt
+
ILCs can play
a critical role for NK cell development and that we have un-
covered a close interaction between NK cell development and
RORt
+
ILCs. Our study further defnes membrane LT as an
essential molecule from RORt
+
ILCs in controlling the de-
velopment of NK cells within the BM microenvironment.
MATERIALS AND METHODS
Mice. C57BL/6 mice were purchased from Harlan Laboratories, Inc. RAG1
/
,
LT
/
, LTR
/
, and RORt
/
mice were bred in our animal facility at
the University of Chicago. CD4, CD19
Cre+
LT
f/f
mice were intercrossed as
previously described (Tumanov et al., 2002, 2003). Rorc
Cre+
LT
f/f
mice were
generated by crossing LT foxed with Rorc-Cre transgenic mice (Eberl and
Littman, 2004). Animal care and use were in accordance with institutional and
National Institutes of Health guidelines, and all experiments were approved
by the Animal Care and Use Committee of the University of Chicago.
Flow cytometric analysis and antibodies. The cells were treated with
anti-CD16/CD32 antibody (clone 2.4G2) before surface staining. Antibod-
ies conjugated to FITC, PE, Percp-cy5.5, PE-cy7, APC, APC-cy7, Pacifc
blue, and biotin for the following antigens were used for this study: NK1.1
(PK136), CD3e (145-2C11), CD19 (1D3), CD49b (DX5), CD122 (TM1),
CD51 (RMV-7), TER-119 (TER-119), Ly-6G/Ly-6C (RB6-8C5), CD45R/
B220 (RA3-6B2), CD45 (30-F11), and CD90.2 (30-H12). These antibodies
were purchased from BioLegend and eBioscience. Live cells were gated based
on the 7AAD-negative cell population. For RORt staining, cells were fxed
and permeabilized with the Foxp3 staining bufer set (eBioscience). For de-
tection of LT, splenocytes from mice were stimulated with 20 ng/ml PMA
for 20 h. After stimulation, cells were stained with LTR-biotin antibodies,
and then we stained streptavidin-conjugated APC antibodies. Cells were ana-
lyzed with an LSR Fortessa (BD). Acquired data were analyzed with FlowJo
software (Tree Star).
BM in vitro culture. BM cells were obtained from the tibias and femurs of
mice. Fresh isolated BM cells were harvested from wild-type or Rorc
cre+
LT
f/f

mice and cultured in vitro for 10 d without exogenous cytokines, and 10 or
20 ng/ml IL-15 (BioLegend) was then added for the last 10 d. LTR-Ig was
added into each well at day 0 for neutralizing LT. Thy1
+
Lin

NK1.1

cells were
isolated from BM using MACS magnetic beads (Miltenyi Biotec). RORt
+

ILCs were isolated from intestinal LPLs using cell sorting. Absolute NK cell
(NK1.1
+
, CD3

) numbers were counted by fow cytometry based cell bead

assay (Invitrogen), and acquired data were analyzed with FlowJo software.
Isolation of RORt
+
ILCs from intestinal LPLs. ROR
+
ILCs from
LPLs were isolated by modifcation of a previous study (Guo et al., 2014).
Mice were sacrifced, and small and large intestines were removed. These in-
testines were cut open longitudinally, thoroughly washed in PBS, and cut into
small pieces. Intestines were incubated in 1 HBSS supplemented with 5%
FBS, 10 mM Hepes, 5 mM EDTA, and 1 mM DTT at 37C for 20 min. After
washing, the intestines were digested in RPMI 1640 supplemented with 0.05%
Group 1 ILCs are T-betexpressing ILCs producing IFN-
and are associated with cell-mediated immunity, whereas group 2
ILCs are dependent on the transcription factor ROR, ex-
press the transcription factor GATA3, and produce the Th2-
associated cytokines IL-5 and IL-13. Group 3 ILCs consist of
RORt
+
ILCs that include LTi-like, ILC17, and NCR22 cells.
All ILCs derive from an Id2-dependent lymphoid precursor
and respond to
c
cytokines, which play important roles in lym-
phoid cell development and function (Cherrier and Eberl, 2012;
Mjsberg et al., 2012; Spits and Cupedo, 2012). RORt
+
group
3 ILCs partially depend on Aryl hydrocarbon receptor (AhR)
signaling for development and function, express the IL-23R,
and can produce IL-17A, IL-17F, and IL-22. RORt
+
ILCs
and NK cells are derived from same CLP (Mebius et al., 2001;
Yoshida et al., 2001) and develop into common precursor NK
(NKp) cells. But recent lineage mapping suggests that they are
a distinct population. NK cells (NK1.1
+
) never express RORt
through their life (Sawa et al., 2010). IL-15defcient mice
have defective NK cells but normal numbers of RORt
+

ILCs (Pandiyan et al., 2012). In various LT-defcient mice, such
as Rorc
Cre+
LT
f/f
mice with RAG1
/
or B6 background, gen-
eration of NKp appears to be normal within BM. However,
the number of mNK is signifcantly reduced, whereas that of
iNK cells, characterized by a CD51
+
NK1.1
+
CD3

DX5

cell
population, is elevated. Therefore, it appears that the NK cell
development process in the Rorc
Cre+
LT
f/f
mice is arrested at
the stage of iNK after surface CD51 expression. This increased
iNK cell population is also observed in the secondary lymphoid
organs, including spleen, blood, and lung, suggesting that the
iNK cells are able to emigrate from the BM at the immature
state. The defect in iNK development persists even after addi-
tion of IL-15 in the BM culture in vitro. These data highlight
the critical role of LT signaling from ILCs in guiding iNK
cells to further develop into the NK1.1
+
DX5
+
stage before the
IL-15dependent phase.
NK cells can be divided into four diferent stages accord-
ing to the expression patterns of cell surface CD27 and CD11b
(Chiossone et al., 2009; Vosshenrich and Di Santo, 2013). Upon
analysis of mNK cells in the spleen, no signifcant diference was
observed in the percentage of CD27- and CD11b-expressing
NK cells between wild-type and Rorc
Cre+
LT
f/f
mice. Therefore,
the remaining small numbers of mNK cells developing inde-
pendently of LT signaling are normal in LT-defcient mice.
Experiments are in progress to identify the mechanism con-
trolling LT-independent developmental pathways of NK cells.
Upon analysis of NK efector function, we found a signif-
icant defect in eliminating NK-sensitive class I
low
RMA-S
tumors in the absence of LT signaling. RMA-S tumor burdens
are signifcantly bigger in LT
/
, LTR
/
, and Rorc
Cre+

LT
f/f
mice than wild-type mice, indicating that NK cell func-
tion is heavily suppressed in vivo. This defect is largely caused
by the defect in NK cells because NK-insensitive class I
+
RMA
tumor burden was not statistically diferent between WT and
Rorc
Cre+
LT
f/f
mice. Therefore, these data demonstrate that
LT is essential, not only in the maturation process within BM
but also the antitumor efector functions.

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Published June 9, 2014
1430 Innate lymphoid cells regulate NK cell development | Kim et al.
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DNase I (Sigma-Aldrich) and 0.1 mg/ml Liberase (Roche) at 37C for 20 min.
The digested intestines were homogenized by a gentleMACS dissociator
(Miltenyi Biotec) and passed through a 70-m strainer. Mononuclear cells
were obtained from a Percoll gradient. After washing, RORt
+
ILCs were
obtained by cell sorting using a FACSAria cell sorter (BD).
Tumor cell lines and tumor model. RMA-S (MHC class I negative) and
RMA (MHC class I positive) cell lines were cultured in RPMI 1640 supple-
mented with 10% FBS, 1% nonessential amino acid, 1% Hepes, and 1% pen-
icillin/streptomycin. RMA-S or RMA cells were injected s.c. into the lateral
fank of mice. Tumor volumes were determined along three orthogonal axes
(A, B, and C) and calculated as tumor volume = ABC/2. Tumor volume
was measured every 34 d.
Statistical analysis. The statistical signifcances were determined by Stu-
dents t test. The p-values were defned as follows: *, P < 0.05; **, P < 0.01;
***, P < 0.001. Statistical analyses were performed using Prism version 5.01
(GraphPad Software).
This work was supported by the Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of Science,
ICT, and Future Planning (grants NRF-2007-00107 and NRF-2013M3A9D3045719)
and the Converging Research Center Program (grant 2013K000268) awarded
to K.-M. Lee. T.-J. Kim is additionally supported by a grant from the NRF (NRF-
2013R1A1A2013308). This research was supported in part by the US National
Institutes of Health (grants CA141975 and CA134563-01).
The authors declare no competing fnancial interests.
Submitted: 16 July 2013
Accepted: 30 April 2014
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