Cell Surface Galectin-9 Expressing Th Cells Regulate Th17 and Foxp3+ Treg Development by Galectin-9 Secretion
et al. (2012) Cell Surface Galectin-9 Expressing Th Cells Regulate Th17 and Foxp3+ Treg Development
by Galectin-9 Secretion. PLoS ONE 7(11): e48574. doi:10.1371/journal.pone.0048574
Cell Surface Galectin-9 Expressing Th Cells Regulate Th17 + and Foxp3 Treg Development by Galectin-9 Secretion
Souichi Oomizu 0
Tomohiro Arikawa 0
Toshiro Niki 0
Takeshi Kadowaki 0
Masaki Ueno 0
Nozomu Nishi 0
Akira Yamauchi 0
Toshio Hattori 0
Tsutomu Masaki 0
Mitsuomi Hirashima 0
Alexandre Salgado Basso, Escola Paulista de Medicina - UNIFESP, Brazil
0 1 Department of Immunology and Immunopathology, Faculty of Medicine, Kagawa University , Kagawa , Japan , 2 Department of Biology, Kanazawa Medical University , Ishikawa, Japan, 3 GalPharma Co. , Ltd., Kagawa, Japan, 4 Department of Holistic Immunology, Kagawa University , Kagawa , Japan , 5 Department of Inflammation Pathology, Faculty of Medicine, Kagawa University , Kagawa , Japan , 6 Life Science Research Center, Kagawa University , Kagawa , Japan , 7 Department of Breast Surgery, Kitano Hospital , Osaka , Japan , 8 Laboratory of Disaster-related Infectious Disease, International Research Institute of Disaster Science, Tohoku University , Sendai , Japan , 9 Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University , Kagawa , Japan
Galectin-9 (Gal-9), a b-galactoside binding mammalian lectin, regulates immune responses by reducing pro-inflammatory IL17-producing Th cells (Th17) and increasing anti-inflammatory Foxp3+ regulatory T cells (Treg) in vitro and in vivo. These functions of Gal-9 are thought to be exerted by binding to receptor molecules on the cell surface. However, Gal-9 lacks a signal peptide for secretion and is predominantly located in the cytoplasm, which raises questions regarding how and which cells secrete Gal-9 in vivo. Since Gal-9 expression does not necessarily correlate with its secretion, Gal-9-secreting cells in vivo have been elusive. We report here that CD4 T cells expressing Gal-9 on the cell surface (Gal-9+ Th cells) secrete Gal-9 upon T cell receptor (TCR) stimulation, but other CD4 T cells do not, although they express an equivalent amount of intracellular Gal-9. Gal-9+ Th cells expressed interleukin (IL)-10 and transforming growth factor (TGF)-b but did not express Foxp3. In a co-culture experiment, Gal-9+ Th cells regulated Th17/Treg development in a manner similar to that by exogenous Gal-9, during which the regulation by Gal-9+ Th cells was shown to be sensitive to a Gal-9 antagonist but insensitive to IL-10 and TGF-b blockades. Further elucidation of Gal-9+ Th cells in humans indicates a conserved role of these cells through evolution and implies the possible utility of these cells for diagnosis or treatment of immunological diseases.
Funding: This work was supported by a Grant-In-Aid for young scientists (B) 20082009 (20790570) from the Japan Society for Promotion of Science (JSPS),
Scientific Research (C) 2010- (22590360) from JSPS, Scientific Research (C) 2011- (23591438) from JSPS, Scientific Research (A) 2011- (23256004) from JSPS. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. (JSPS HP: http://www.jsps.go.jp/). No
additional external funding was received for this study.
Competing Interests: The authors have read the journals policy and have the following conflicts: Drs. Niki and Hirashima are board members of GalPharma Co.,
Ltd. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials.
. These authors contributed equally to this work.
Galectin-9 (Gal-9) is a member of the galectin family of
mammalian lectins and is characterized by its ability to bind
bgalactoside. Gal-9 is expressed by the epithelium of the
gastrointestinal tract, endothelial cells and several types of immune
cells including T cells, B cells, macrophages and mast cells [1,2].
Recently, the regulatory role of Gal-9 in excessive immunity has
become evident. Gal-9 suppresses interleukin (IL)-17-producing
effector T helper cells (Th)17 and Th1 ; these cells play an
exacerbating role in the pathogenesis of various autoimmune
diseases, whereas Gal-9 augments Foxp3+ regulatory T cells
(Treg), an essential suppressor of excessive immunity . In
addition to increasing Treg, Gal-9 expands the population of
monocytic myeloid-derived suppressor cells (MDSCs) ,
granulocytic MDSCs [5,6], and plasmacytoid dendritic cell-like
macrophages . Induction of these regulatory cells appears to be a
critical regulatory function of Gal-9.
The function of Gal-9 is thought to be exerted by binding to
particular sets of carbohydrate moieties in receptor molecules
expressed on the surface of target cells. Among several identified
receptors of Gal-9, the T-cell immunoglobulin- and
mucindomain-containing molecule-3 (Tim-3) has been studied most
extensively. Binding of Gal-9 to Tim-3 expressed by activated Th1
and/or Th17 triggers cellular apoptosis and terminates Th1/
Th17-skewed immunity [10,11]. Gal-9 must be secreted by some
types of cells to initiate this response. However, Gal-9 lacks a signal
sequence essential for secretion via the canonical endoplasmic
reticulum (ER)-Golgi pathway and is located predominantly in the
cytoplasm where it plays roles in protein sorting and
transcriptional regulation of cytokine genes [12,13]. Gal-9 secretion has
been demonstrated in several cell lines [14,15], but the mechanism
of Gal-9 translocation through the lipid bilayer, as well as the
identification of Gal-9-secreting cells in vivo has yet to be
elucidated. Gal-9 expression and secretion are not always
correlated. Recently, Wang et. al. suggested through indirect
observation that Treg secretes Gal-9 and ameliorates Th1
responses . From a functional perspective of the protein,
Gal-9 may be secreted by regulatory cells, including Treg.
We hypothesize that Gal-9-secreting cells might express Gal-9
on the cell surface as a translocation intermediate and may be
identified by staining the cells with specific antibodies. The goal of
this study was to identify Gal-9-secreting cells. We identified CD4
T cells expressing Gal-9 on their surfaces (Gal-9+ Th cells). Gal-9+
Th cells secrete Gal-9 upon T cell receptor (TCR) stimulation, but
other CD4 T cells lacking Gal-9 on the surface do not, although
they express indistinguishable amounts of Gal-9 intracellularly.
The characteristics of Gal-9+ Th cells and the significance of these
cells in immunoregulation are discussed.
Gal-9-secreting Cells Emerge from Nave CD4 T cells by
We recently found that exogenous Gal-9 suppresses Th17
development and simultaneously enhances Treg development in
vitro, even under Th17-skewing conditions, in an IL-2-dependent
but Tim-3-independent manner . These results suggest that
endogenous Gal-9 plays a role in Th17/Treg development and
that Gal-9 production and/or secretion may be suppressed under
Th17-skewing conditions. To confirm this, we examined Gal-9
secretion from nave CD4 T cells cultured under neutral and
Th17-skewing conditions. As expected, Gal-9 was secreted under
neutral conditions, whereas secretion was suppressed under
Th17skewing conditions (Figure 1A) largely because of the presence of
IL-6 in the culture (Figure 1B). Despite differences in secretion
capability, Gal-9 mRNA expression level did not differ between
neutral and Th17-skewing conditions (Figure 1C). We further
examined whether exogenous Gal-9 stimulates endogenous Gal-9
secretion. For this experiment, 30 nM of recombinant human
stable Gal-9 was added to the culture, and the secretion of mouse
Gal-9 was monitored using a specific enzyme-linked
immunosorbent assay (ELISA) for mouse Gal-9. Human recombinant Gal-9
cross-reacts with mouse cells biologically and has been used in
various rodent studies, but it was not detected in our mouse Gal-9
ELISA (Figure S1). As seen in Figure 1D, exogenous Gal-9
enhanced Gal-9 secretion even under Th17-skewing conditions.
These results suggest that (1) Gal-9-secreting cells are present
under neutral conditions, but the secretion capability and/or the
number of Gal-9-secreting cells is reduced under Th17-skewing
conditions, largely by IL-6, and (2) exogenous Gal-9 counteracts
the inhibition of Gal-9-secretion even under Th17-skewing
Surface Gal-9-expressing Th cells Secrete Gal-9
We hypothesized that Gal-9 could be detected on the surface of
Gal-9-secreting cells as an intermediate during translocation
through the lipid bilayer. We thus performed flow cytometry to
measure surface Gal-9 expression using cells stimulated as
described in Figure 1A. During secretion, Gal-9 likely binds to
adjacent cells via their cell surface glycoproteins or glycolipids,
which may complicate the identification of Gal-9-secreting cells.
Therefore, Gal-9 staining was performed in the presence of
30 mM lactose, because this concentration of lactose is sufficient
for removing exogenously added Gal-9 bound on the cell surface
without affecting Gal-9 staining (Figure S2A).
Gal-9 staining revealed the existence of surface Gal-9-expressing
CD4 T cells (Gal-9+ Th cells). The frequency of Gal-9+ Th cells
was approximately 1.5% without TCR stimulation and was
increased to approximately 4% after TCR stimulation under
neutral conditions (Figure 2A). Interestingly, the frequency of
Gal-9+ Th cells as well as CD25+ CD4 T cells was reduced under
Th17-skewing conditions (Figure 2A). Surface Gal-9 stably
adhered to the cell surface during staining at 4uC even in
100 mM lactose (Figure S2B). Gal-9 secretion (Figure 1A)
correlates well with the emergence of Gal-9+ Th cells (Figure 2A).
To confirm our hypothesis that these Gal-9+ Th cells are the
primary source of secreted Gal-9, Gal-9+ and Gal-92 nave Th
cells were isolated using a cell sorter (Figure S3), cultured under
neutral conditions and examined for Gal-9 secretion by ELISA.
Consistent with our hypothesis, Gal-9+ Th cells, but not Gal-92
Th cells, secreted Gal-9 upon TCR stimulation (Figure 2B). This
is the first report of identification of Gal-9-secreting Th cells and
demonstrates a useful technique for detecting cells with Gal-9
secretion capability. Enigmatically, expression levels of Gal-9
mRNA and intracellular Gal-9 protein did not show any apparent
differences between Gal-9+ and Gal-92 Th cells stimulated under
neutral conditions (Figure 2C). Cytokine mRNA measurement
demonstrated that compared to Gal-92 Th cells Gal-9+ Th cells
induced significantly higher levels of IL-10 and TGF-b upon TCR
stimulation (Figure 2D). Induction of IFN-c was similar to
Gal92 Th cells, while IL-2, IL-4 and IL-17 were not induced by
Gal9+ Th cells (Figure 2D).
The frequency of Gal-9+ CD252 Th cells in various lymphoid
organs of normal mice was determined using flow cytometry.
Approximately 25% of CD4 single-positive cells in thymus cells
expressed surface Gal-9. Among CD252 CD4 T cells,
approximately 4% in lymph nodes, 7% in spleen and peripheral blood
mononuclear cells and 15% in Peyers patches expressed surface
Gal-9 (Table 1 and Figure S5).
Gal-9+ Th cells are Different from Treg
Recently, Treg was suggested to secrete Gal-9 to suppress Th1
immunity . As shown in Figure 2D, the expression of IL-10
and TGF-b by Gal-9+ Th cells appears to further support the
identity between Treg and Gal-9+ Th cells. However, Gal-9+ Th
cells were devoid of Foxp3 expression while Foxp3+ Th cells were
devoid of surface Gal-9 expression (Figure 3A). Therefore, Gal-9+
Th cells were clearly a different population from Foxp3+ Treg and
rarely co-expressed Tim-3 (Figure 3B). In Gal-9 knockout mice,
the frequency of IL-10+ cells was significantly lower in CD4 T cells
compared to those from wild type mice (Figure 3C). As
demonstrated in Figure 2A, the number of Gal-9+ Th cells
increased under neutral conditions but decreased under
Th17skewing conditions. Similarly, IL-10 expression decreased under
Th17-skewing conditions and was further decreased by Gal-9
deficiency (Figure 3D). These observations suggest a close
relationship between Gal-9+ Th cells and IL-10-producing CD4
Gal-9+ Th cells Regulates Th17/Treg Development by
When nave CD4 T cells committed to Th17 development were
co-cultured with Gal-9+ Th cells (1:1), IL-17A production was
significantly suppressed, whereas Foxp3 expression was
reciprocally induced (Figure 4A). Suppression of IL-17A production by
Gal-9+ Th cells was abrogated by lactose, an antagonist of Gal-9,
but not by sucrose (Figure 4B). The induction and function of
Th17 are known to be regulated by cytokines secreted by other
major T cell subsets, including IFN-c, IL-4, IL-10, and TGF-b
. Since IL-10 and TGF-b are highly expressed by Gal-9+ Th
cells, we examined the effect of blocking antibodies against IL-10
and TGF-b. However, these cytokine blockades did not affect the
regulatory activity of Gal-9+ Th cells (Figure 4C). Furthermore,
the addition of recombinant IL-10 did not suppress IL-17A
production in our assay system (Figure 4D).
Figure 1. Gal-9 secretion in nave CD4 T cell culture upon TCR stimulation. (A) Nave CD4 T cells were cultured in Th17-skewing conditions,
neutral conditions (TCR stimulation), or without stimulation for 96 h before released Gal-9 was measured using ELISA. (B) Same as (A), except that the
cells were cultured under Th17-skewing conditions, neutral conditions, neutral conditions plus TGF-b1 (TGF-b1 alone), or neutral conditions plus IL-6
(IL-6 alone). (C) Gal-9 mRNA was quantified using real-time RT-PCR. (D) Gal-9 secretion in the presence of human stable Gal-9 (30 nM) was measured.
The human protein was not detected by mouse Gal-9 ELISA (see text for details). All results are shown as the mean 6 SEM of quadruplicates. p,0.001
(***), p,0.05 (*), not significant (NS). Representative data out of at least 2 experiments are shown.
Expansion of Gal-9+ Th cells by Exogenous Gal-9
As demonstrated in Figure 1D, the addition of recombinant
Gal-9 to nave CD4 T cell cultures augmented Gal-9 secretion.
This observation indicates that exogenous Gal-9 either activates or
increases Gal-9+ Th cells (or both). To address the question, nave
CD4 T cells were cultured for 4 days with no stimulation, or under
neutral- or Th17-skewing conditions in the presence or absence of
recombinant human stable Gal-9, and the frequency of Gal-9+ Th
cells was monitored using flow cytometry. As shown in Figure S1,
our anti-mouse Gal-9 antibody did not cross-react with the
recombinant human protein and could detect Gal-9+ Th cells.
Exogenous Gal-9 increased the numbers of Gal-9+ CD252 Th
cells irrespective of TCR stimulation (Figure 5A). Upon TCR
stimulation, exogenous Gal-9 increased the numbers of both
Gal9+ CD25+ and Gal-92 CD25+ Th cells under neutral and
Th17skewing conditions (Figure 5A).
Two major cytokines expressed by Gal-9+ Th cells, IL-10 and
TGF-b, were tested to determine whether they affect the
expansion of Gal-9+ Th cells. Blocking antibodies specific against
IL-10 and TGF-b added to nave CD4 T cell cultures under
neutral conditions for 4 days did not affect the number of Gal-9+
Th cells (Figure 5B). IL-10 has been shown to expand type-1
regulatory T cells (Tr1), which are known to strongly express IL-10
and may therefore be related to Gal-9+ Th cells .
However, recombinant IL-10 at 10 ng/mL did not increase
Gal9+ Th cells in 4-day cultures, whereas exogenous Gal-9 increased
the number of cells (Figure 5C).
Gal-9+ Th cells in Humans
Recombinant Gal-9 functions in human peripheral blood CD4
T cells to augment Foxp3+ Treg development while suppressing
Th17 development (Figure S4). These observations demonstrate
that the function of Gal-9 in terms of Th17/Treg development is
equivalent between humans and mice, and implies the existence of
Gal-9+ Th cells in humans. We examined peripheral CD4 T cells
from normal subjects to determine whether a surface
Gal-9expressing population could be observed. In accordance with the
findings in mouse studies, flow cytometric analysis revealed the
presence of Gal-9+ Th cells in peripheral blood mononuclear cells
(PBMC) and the expansion of the cells by TCR stimulation
(Figure 6A). To examine the characteristics of human Gal-9+ Th
cells, peripheral CD4 T cells were cultured under neutral
conditions for 4 days to allow the expansion of Gal-9+ Th cells,
were sorted into Gal-9+ and Gal-92 Th cells according to surface
Gal-9 expression, and were then cultured for another 4 days under
neutral conditions before analysis. Human Gal-9+ Th cells
secreted higher amounts of Gal-9 and expressed higher levels of
IL-10 and TGF-b mRNA compared to Gal-92 Th cells.
Expression of IL-2 and IFN-c did not differ between the 2
populations, whereas the levels of IL-4 and IL-17 were
significantly lower in Gal-9+ Th cells (Figure 6B). These characteristics
of human Gal-9+ Th cells are identical to those observed in mouse
Gal-9+ Th cells (Figure 2).
Figure 2. Identification of Gal-9+ Th cells. (A) Nave CD4 T cells were cultured as in Figure 1A, and cell-surface Gal-9 expression was monitored
using flow cytometry. (B) Nave CD4 T cells were sorted into Gal-9+ Th and Gal-92 Th cells with a cell sorter and cultured under TCR stimulation or left
unstimulated for 4 days before Gal-9 secretion into the culture media was measured. (C) Gal-9+ Th and Gal-92 Th cells cultured under TCR stimulation
for 4 days were examined for Gal-9 mRNA expression (left) and intracellular Gal-9 protein expression (right). (D) Cytokine mRNA expression in Gal-9+
Th and Gal-92 Th cells cultured with or without TCR stimulation for 4 days. All the results are shown as the mean 6 SEM of quadruplicates. p,0.001
(***), p,0.05 (*), not significant (NS). Representative data out of at least 2 experiments are shown.
Table 1. Frequency of Gal-9+ Th cells in various organs in
% in Gal-9+ CD252 cells
Lymphocytes from indicated organs (n = 3) were stained with anti-CD3,
antiCD4, anti-CD25, and anti-Gal-9 antibodies and analyzed using flow cytometry.
SP: single positive, LN: lymph node.
Gal-9 has been demonstrated to suppress hyper immune
reactions through several modes of action, including the induction
of apoptosis in Tim-3+ Th1 and Th17, and suppression of Th17
development with concomitant induction of Treg [3,10,11]. These
regulatory functions of Gal-9 have been elucidated primarily by
pharmacological studies in vitro and in vivo during which
recombinant Gal-9 was administered. However, the cells
responsible for Gal-9 secretion have not been resolved. Here, we
identified Gal-9+ Th cells that express Gal-9 on their surfaces
secrete Gal-9 upon TCR stimulation, and regulate Th17/Treg
development. Recently, Treg was reported to secrete Gal-9 and
suppress Th1. We clarified that Gal-9+ Th cells are devoid of
Foxp3 expression and are the predominant CD4 T cells secreting
Gal-9. The discovery of Gal-9+ Th cells will improve the
understanding of complex immunoregulation by Gal-9, provide
a tool to study expression control and secretion mechanisms of
Gal-9, and may provide clinical utilities for diagnosis or cell-based
therapy in the future.
It is known that the administration of recombinant Gal-9
modulates immunity bidirectionally, not only suppressing
excessive immunity and inflammation but also enhancing these
functions in the context of compromised immunity [21,22]. It is
not clear whether Gal-9+ Th cells boost immunity or further
suppresses it under hypo immune conditions; this can be examined
using animal models such as tumor-bearing mice.
We showed that Gal-9 derived from Gal-9+ Th cells play a vital
role in regulating Th17/Treg development because the effect of
Gal-9+ Th cells was abrogated by a Gal-9 antagonist. However,
the concentration of Gal-9 secreted into the culture media was
500800 pg/mL, which was significantly lower than the effective
concentration for recombinant Gal-9 to elicit the same effect
(30 nM: approximately 1 mg/mL). We hypothesize that Gal-9+ Th
cells secrete Gal-9 in close proximity to target cells via a paracrine
mechanism that may involve cell-cell contact to identify and
confirm the target cells and achieve efficient and secure regulation
by Gal-9. It cannot be ruled out that cell-surface Gal-9 may also be
involved for the regulation by directly interacting with target
Contrary to expectation, both Gal-9+ Th cells and other CD4 T
cells express Gal-9 in the cytoplasm at comparable levels.
Therefore, it is plausible that Gal-9+ Th cells possess secretion
machinery that is absent in Gal-92 Th cells. Gal-9, like other
galectins, does not contain a signal sequence, and the secretion
mechanism has remained obscure. In one study examining Gal-1,
secretion was found to require a counter-receptor, and
translocation through the plasma membrane was found to be
energyindependent . In this case, a membrane pore must be present
to enable Gal-1 translocation. Conversely, Gal-9 is secreted as a
component of the exosome in the case of Epstein-Barr virus
infected nasopharyngeal carcinoma . Whether this mechanism
is true for Gal-9 secretion from Gal-9+ Th cells or whether there is
a third mechanism remains to be determined.
Using human peripheral T cells, we demonstrated that Gal-9+
Th cells are present in humans, and the immunoregulatory
function of Gal-9 on Th17/Treg appears to be equivalent between
humans and mice. These findings suggest clinical applications of
recombinant Gal-9 and Gal-9+ Th cells. We found that IL-6
abrogates the increase of Gal-9+ Th cells in vitro. Thus,
neutralization of IL-6 may be a strategy for increasing Gal-9+
Th cells in order to ameliorate Th1/Th17-skewed immunity. An
anti-IL-6 receptor-neutralizing antibody, tocilizumab, has been
used to treat rheumatoid arthritis. Currently, the primary
mechanism is explained by the suppression of Th17 cell
development by IL-6 ; however, an additional cryptic
mechanism of the antibody may involve efficient induction of
Gal-9+ Th cells. It would be interesting to measure Gal-9+ Th cells
in patients receiving IL-6 blockades, because this may demonstrate
the utility of Gal-9+ Th cells as surrogate markers to judge the
effectiveness of the therapy.
Gal-9+ Th cells must be further characterized to assess clinical
utilities, including cell-based therapies as have been attempted for
Tr1 and Treg, for the treatment of refractory autoimmune
diseases. Gal-9+ Th cells expand the population by exogenously
applied recombinant Gal-9, and the cells can be purified using
Gal-9 expressed on the cellular surface. These findings provide
useful techniques for obtaining a large number of Gal-9+ Th cells
and will help facilitate the study of Gal-9+ Th cells.
Materials and Methods
Human PBMCs were obtained from healthy adult volunteers
with the approval of the ethical committee at Kagawa University,
Faculty of Medicine. Written informed consent was obtained from
all participants. Mice used in this research received humane care
in accordance with international guidelines and national law. The
study protocol was approved by the Animal Care and Use
Committee of Kagawa University.
Isolation and Culture of Mouse CD4+ CD62L+ Nave T cells
C57BL/6J mice were purchased from Charles River
Laboratories Japan (Yokohama, Japan). Gal-9 knockout (Gal-9 KO) mice
were obtained from GalPharma (Takamatsu, Japan). All animals
were maintained under standard conditions with a 12-h day/night
rhythm and with ad libitum access to food and water. CD4+
CD62L+ nave T cells were isolated from splenocytes of 810
week-old mice using a CD4+CD62L+ T cell Isolation Kit (Miltenyi
Biotec, Bergisch Gladbach, Germany) according to the
manufacturers instructions. CD4+ CD62L+ purity was .94%. Expression
and purification of recombinant human stable Gal-9 has been
previously described . The recombinant protein was .95%
pure on SDS-PAGE with an endotoxin level of ,0.001 endotoxin
units/mg. Isolated nave T cells were cultured in 96-well plates at 2
6 105 cells/well in RPMI 1640 containing 10% heat-inactivated
fetal bovine serum, penicillin G (10 IU/mL, Sigma-Aldrich, St.
Louis, MO, USA), and streptomycin (100 mg/mL, Sigma-Aldrich)
for 96 h. For stimulation under neutral conditions, cells were
cultured in anti-CD3-coated plates (BD Biosciences, Franklin
Lakes, NJ, USA) in the presence of anti-CD28 (2 mg/mL, BD
Biosciences) and mouse IL-2 (5 ng/mL, R&D Systems,
Minneapolis, MN, USA). For Th17 skewing, cells were cultured in human
TGF-b1 (3 ng/mL, R&D Systems) and mouse IL-6 (20 ng/mL,
R&D Systems) under neutral conditions. Treg skewing was
conducted under the same culture conditions for Th17-skewing
but IL-6 was omitted. For some experiments, human stable Gal-9
(30 nM), lactose (30 or 100 mM), sucrose (30 mM), anti-mouse
IL-10 blocking antibody (10 mg/mL, BioLegend, San Diego, CA,
USA), anti-mouse IL-10 receptor blocking antibody (10 mg/mL,
BioLegend), anti-mouse TGF-b blocking antibody (10 mg/mL,
Abcam, Cambridge, MA, USA), or mouse IL-10 (1, 3 or 10 ng/
mL, R&D Systems) was included in the culture.
Isolation of Gal-9+ Th cells and Co-culture with
Th17skewed T cells
Nave CD4+ T cells were isolated from splenocytes as described
above. Gal-9+ and Gal-92 Th cells were sorted by positive or
negative surface Gal-9 expression, respectively, using an
antimouse Gal-9 antibody (clone 108A2, BioLegend) and a FACSAria
cell sorter (BD Biosciences). Cell purity was .97%. For co-culture
experiments, nave T cells (5 6 104) were cultured under
Th17skewing conditions for 6 h, and then co-cultured with Gal-9+ or
Gal-92 Th cells (5 6 104) for an additional 90 h.
Quantification of mouse Gal-9 was carried out using ELISA as
described previously with minor modifications . Briefly,
96well plates were coated with an anti-mouse Gal-9 antibody (Clone
108A2), blocked with 3% fetal bovine serum in
phosphatebuffered saline, and then incubated with culture supernatant.
Gal9 was detected using polyclonal anti-mouse Gal-9 antibody
conjugated with biotin (GalPharma) and streptavidin-conjugated
horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA,
USA). After color development with tetramethyl benzidine (KPL,
Gaithersburg, MD, USA), Gal-9 was quantified using a standard
curve constructed with a recombinant mouse Gal-9. Mouse Gal-9
ELISA cannot be used to detect human stable Gal-9. Human
9 ELISA was reported previously . IL-17 was measured using a specific ELISA kit from R&D Systems according to the manufacturers instructions.
Flow Cytometric Analysis
CD4 T cells were evaluated by flow cytometry using the
following antibodies: anti-mouse CD3-PerCP (BD Biosciences),
anti-mouse CD4-FITC (BD Biosciences or eBioscience, San
Diego, CA, USA), anti-mouse Tim-3-PE (eBioscience), anti-mouse
Gal-9-PE (clone 108A2, BioLegend) or biotinylated anti-mouse
Gal-9 (clone 108A2, GalPharma), anti-mouse CD25-APC
(BioLegend), anti-mouse IL-10-PE (BioLegend), anti-human
Gal-9Alexa488 (clone 9M1-3, GalPharma), anti-human CD3-PerCP
(BD Biosciences), anti-human CD4-FITC (BioLegend),
antihuman CD4-PE (BioLegend), anti-human CD25-APC
(BioLegend), and anti-human/mouse Foxp3-PE (BioLegend). All data
were acquired using a FACSCalibur cytometer (BD Biosciences)
and analyzed with FlowJo software (Tree Star, Ashland, OR,
Figure 5. Expansion of Gal-9+ Th cells by exogenous Gal-9. (A) Nave CD4 T cells were cultured under unstimulated, neutral, or Th17-skewing
conditions for 4 days in the presence or absence of 30 nM human stable Gal-9 before surface Gal-9 expression was monitored by flow cytometry
using an anti-mouse Gal-9 antibody. The antibody does not cross-react with the added human Gal-9. Dot plots are representative results obtained
from neutral conditions in the presence or absence of exogenous Gal-9. (B and C) Flow cytometric analysis of Gal-9+ Th cell frequency after 4-day
culture of nave CD4 T cells under neutral conditions in the presence of blocking anti-IL-10 or anti-TGF-b antibody (B) or in the presence of the
indicated concentration of IL-10 or 30 nM human stable Gal-9 (C). Results are means 6 SEMs of quadruplicates. p,0.001 (***), p,0.01 (**), p,0.05 (*),
not significant (NS). Representative data out of 2 experiments are shown.
Isolation and Culture of Human Peripheral Blood CD4 T
PBMCs from healthy donors were prepared using a
Lymphocyte Separation Kit (Nakalai, Kyoto, Japan). CD4 T cells were
isolated using a CD4 T Cell Isolation Kit II (Miltenyi Biotec)
according to the manufacturers instructions. The CD4 T cells (2
6 105 cells/well) were cultured under TCR stimulation
(antiCD28 (2 mg/mL, BD Biosciences) and IL-2 (5 ng/mL, R&D
Systems) in anti-CD3-coated plates) or left unstimulated for 96 h,
and cell-surface Gal-9 expression or released Gal-9 in the culture
media was measured. To determine cytokine mRNA expression,
CD4 T cells cultured under TCR stimulation for 4 days were
sorted into Gal-9+ CD25+ cells and Gal-92 CD25+ cells using a
FACSAria cell sorter at a purity of .97%. Sorted cells were
further cultured for 4 days under TCR stimulation. Human Th17
cell development was performed as reported previously .
Briefly, CD4 T cells were cultured under TCR stimulation as
described above in the presence or absence of IL-1b (50 ng/mL,
R&D Systems), IL-6 (20 ng/mL, R&D Systems) and IL-23
(50 ng/mL, R&D Systems) for 9 days.
Figure 6. Gal-9+ Th cells in humans. (A) CD4 T cells from human peripheral blood were cultured with or without TCR stimulation for 4 days.
Surface Gal-9 and CD25 expression was analyzed using flow cytometry. Results are shown as the mean 6 SEM from 4 healthy donors. (B) CD4 T cells
were cultured under neutral conditions for 4 days and then sorted into Gal-9+ CD25+ Th cells and Gal-92 CD25+ Th cells. Sorted cells were cultured
under neutral conditions for 4 days before the measurement of secreted Gal-9 by ELISA or cytokine mRNA using real-time RT-PCR. Results are shown
as the mean 6 SEM from 8 healthy donors. * p,0.05. Data from 2 representative experiments are shown.
mRNA levels were evaluated using the SYBR Green I-based
real-time RT-PCR with an ABI PRISM 7000 sequence detector
(Applied Biosystems, Foster City, CA, USA) as previously
described . All gene primers were obtained from Takara Bio
(Otsu, Japan). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA levels were used as an internal standard for
For statistical comparisons, non-parametric two-tailed
MannWhitney U-tests and one- or two-way analysis of variance were
used. All statistical analyses were performed with Prism 5 software
(Graphpad Software). A p-value of ,0.05 was considered
Figure S1 Specificity of anti-Gal-9 antibody. (A)
Schematic drawing of wild-type Gal-9 and stable Gal-9. Gal-9 consists of 2
carbohydrate-recognition domains (CRD) at the N- and
Ctermini, tethered by a linker peptide. Stable Gal-9 is a
geneengineered linker-less Gal-9, which retains biological activity of
wild-type Gal-9. (B) Anti-mouse Gal-9 antibody 108A2 recognizes
linker peptide of mouse Gal-9 and does not cross-react with stable
Gal-9. The indicated proteins were coated in ELISA plates and
detected using the 108A2 antibody. Mean 6 SD (n = 3). (C)
Mouse Gal-9 ELISA is constructed by 108A2 antibody as the
coating antibody and polyclonal anti-mouse Gal-9 antibody as the
detection antibody. The ELISA is highly specific to mouse Gal-9
and does not cross-react to human stable Gal-9 at 0.37 mg/mL.
Figure S2 Elimination of exogenously added Gal-9
bound on the cell surface by 30 mM lactose. (A) Nave
CD4 T cells were incubated with biotinylated human stable Gal-9
(30 nM) for 30 min on ice followed by incubation with lactose or
sucrose (30 mM) for 30 min on ice. Human stable Gal-9 bound on
the cells was stained with streptavidin- APC and analyzed using
flow cytometry. (B) Nave CD4 T cells were cultured under neutral
conditions for 4 days to allow expansion of Gal-9+ CD25+ Th cells.
The cells were incubated in the presence or absence of 100 mM
lactose for 30 min on ice before staining of surface Gal-9 and
analysis by flow cytometry.
Figure S4 Regulation of human Th17/Treg
development by Gal-9. (A and B) CD4 T cells were isolated from
human peripheral blood (4 healthy donors) by magnetic sorting
and were cultured with or without 4 days of TCR stimulation in
the presence or absence of 30 nM human stable Gal-9. CD25+
CD4 T cells (A) or CD25+ Foxp3+ CD4 T cells (B) were
determined using flow cytometry. (C) Human CD4 T cells from 4
healthy donors were cultured under TCR stimulation in the
presence of indicated cytokines and in the presence or absence of
30 nM human stable Gal-9 for 9 days before IL-17 secretion was
measured by ELISA. Results are shown as the mean 6 SEM of
quadruplicate experiments. ***, p,0.001. Data representative of 2
experiments are shown.
We thank Drs. Yokota and Iwata (Faculty of Pharmaceutical Sciences,
Kagawa Campus, Tokushima Bunri University, Japan) for technical
assistance with cell sorting.
Conceived and designed the experiments: SO TA TN TH TM MH.
Performed the experiments: SO TA TN TK MU NN. Analyzed the data:
TK MU AY MH. Contributed reagents/materials/analysis tools: TN NN
AY MH. Wrote the paper: TN TH TM MH.
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